PRODUCTION OF SUGAR FROM SOYA BEAN WASTE BY ENZYMATIC HYDROLYSIS
AMY LIM JIA HUI
177485
PROJECT REPORT SUBMITTED IN PARTIALLY FULFILLMENT OF THE REQUIREMENT FOR THE
BACHELOR OF ENGINEERING (PROCESS AND FOOD)
DEPARTMENT OF PROCESS AND FOOD ENGINEERING
FACULTY OF ENGINEEERING
UNIVERSITI PUTRA MALAYSIA
2018
APPROVAL SHEETThis project report attached is entitled “Production of Sugar from Soya Bean Waste by Enzymatic Hydrolysis” submitted by AMY LIM JIA HUI (177485) to the Faculty of Engineering in partial fulfillment of the requirement for the degree of Bachelor of Engineering (Food and Processing), is hereby approved and accepted.

Associate Professor Ir. Dr. Siti Mazlina Binti Mustapa Kamal
Department of Food and Processing Engineering,
Faculty of Engineering, Universiti Putra Malaysia
(Supervisor)
Associate Professor Dr. Farah Saleena Binti Taip
Department of Food and Processing Engineering,
Faculty of Engineering, Universiti Putra Malaysia
(Examiner)
Dr. Alifdalino Bin Sulaiman
Department of Food and Processing Engineering,
Faculty of Engineering, Universiti Putra Malaysia
(Examiner)
DECLARATIONI declare that this report entitled “Production of sugar from soya bean waste by enzymatic hydrolysis” is my original work except for quotations and citations which have been fully acknowledged. I also declare that this report has not been accepted previously, and is not concurrently submitted for any other degree at Universiti Putra Malaysia or at any other institution.

Signature: _______________________
Date: _______________________
Name: AMY LIM JIA HUI
The library,
Universiti Putra Malaysia,
43400 UPM Serdang,
Selangor Darul Ehsan.

Permission to Make Photocopies of Thesis
I am Amy Lim Jia Hui (940724-01-5452) hereby declare that the project report/thesis entitled “Production of Sugar from Soya Bean Waste by Enzymatic Hydrolysis” belongs to me. The content of this report may be reffered by anyone for the academic purposes of teaching, learning and research only.Universiti Putra Malaysia is permitted to make photocopies of this document for the same academic purposes.

Signature: ______________________
Date: _________________
Name: AMY LIM JIA HUI
ACKNOWLEDGEMENTForemost, I would like to forward my deepest appreciation to my thesis supervisor Associate Professor Ir. Dr. Siti Mazlina Binti Mustapa Kamal, who have always motivating and guiding me throughout the research work. I believe I had produced a best final year project based on her comments and advices.
Besides this, I would give my massive gratitude to my examiners which are Associate Professor Dr. Farah Saleena Binti Taip and Dr. Alifdalino Bin Sulaiman for checking, advised and comment on my final year project. Special gratitude towards Nurfatminah binti Mohd Thani and all of the technician in the Process and Food Engineering laboratory for helping and advised me when I conducted the experiment for the research study. I’m very grateful to University Putra Malaysia (UPM) for providing good facilities in the laboratory and in campus.

Last but not least, I am grateful to all my family members, friends others who have provided assistance at various occasions. I really gained a lot of knowledge and experience throughout this research work. I am appreciating for this valuable experience and keep it heart always.

ABSTRACTIn soya milk processing, large amount of soya bean waste known as okara can be utilize to recover sugar. The sugar can be recovered using enzymatic hydrolysis as one of the effective method due to enzyme properties which are highly specific and very sensitive. Therefore, this paper was designed to study the effect of concentration of the enzyme and the temperature on sugar production using enzymatic hydrolysis. Soya bean waste, a residue from soya milk production will act as the substrate was hydrolyzed by enzyme amylase to produce sugar. The amount of sugar production, which is either monosaccharide or oligosaccharide was determined by high performance liquid chromatography (HPLC). From the HPLC result showed that soya bean waste sample contains oligosaccharide which is sucrose and monosaccharide which is arabinose. The optimal conditions chosen by the central composite design are 59°C with enzyme concentration of 1.35% and 2%. From the HPLC result, the sucrose concentration was produced at the temperature 59°C with enzyme concentration of 1.35% and 2% were 172.60 mg/L and 140.82 mg/ml, respectively. For arabinose, the concentrations were 1243.13 mg/L and 1291.19 mg/L, respectively. By changing the concentration of enzyme and temperature will results in different sugar yield production because the enzyme lost their three-dimensional functional shape due to disruption from unstable conditions of the environment system. As a conclusion, all the requirements for complementary in the configuration of soya bean waste and enzyme must be considered seriously for the highest sugar yield production.

ABSTRAKHidrolisis enzimatik adalah salah satu kaedah yang berkesan dalam pengeluaran gula kerana sifat enzim yang sangat spesifik dan sensitif. Oleh itu, projek ini direka untuk mengkaji kesan kepekatan enzim dan suhu bagi pengeluaran gula dengan menggunakan hidrolisis enzimatik.Sisa kacang soya dari pengeluaran soya bean akan bertindak sebagai substrat dihidrolisis oleh enzim-enzim ini untuk menghasilkan gula. Jumlah pengeluaran gula yang sama ada monosakarida atau oligosakarida ditentukan oleh kromatografi cecair prestasi tinggi (HPLC). Dari hasil HPLC menunjukkan bahawa sampel sisa kacang soya mengandungi oligosakarida yang merupakan sukrosa dan monosakarida yang merupakan arabinose. Keadaan pengoptimuman yang dipilih oleh reka bentuk komposit pusat ialah 59 °C dengan kepekatan enzim sebanyak 1.35% dan 2%. Dari hasil HPLC, kepekatan sukrosa dihasilkan pada suhu 59 °C dengan kepekatan enzim sebanyak 1.35% dan 2% adalah 172.60 mg / L dan 140.82 mg / ml, masing-masing. Untuk arabinose, kepekatan arabinose masing-masing adalah 1243.13 mg / L dan 1291.19 mg / L. Dengan mengubah kepekatan enzim dan suhu akan menghasilkan pengeluaran gula yang berbeza kerana enzim itu kehilangan bentuk fungsi tiga dimensi mereka disebabkan oleh gangguan daripada keadaan tidak stabil sistem alam sekitar. Sebagai kesimpulan, semua keperluan untuk pelengkap dalam konfigurasi sisa kacang soya dan enzim mesti dianggap serius untuk pengeluaran hasil gula tertinggi.

TABLE OF CONTENTS
TOC o “1-3” h z u APPROVAL SHEET PAGEREF _Toc514063116 h 2DECLARATION PAGEREF _Toc514063117 h 3ACKNOWLEDGEMENT PAGEREF _Toc514063118 h 5ABSTRACT PAGEREF _Toc514063119 h 6ABSTRAK PAGEREF _Toc514063120 h 7CHAPTER 1 PAGEREF _Toc514063121 h 13INTRODUCTION PAGEREF _Toc514063122 h 131.1 Problem Statement PAGEREF _Toc514063123 h 151.2 Objectives PAGEREF _Toc514063124 h 151.2 Project Outline PAGEREF _Toc514063125 h 16CHAPTER 2 PAGEREF _Toc514063126 h 17LITERATURE REVIEW PAGEREF _Toc514063127 h 172.1 Soybean PAGEREF _Toc514063128 h 172.2 Soya milk processing PAGEREF _Toc514063129 h 172.2.1 Soya milk processing: Soya bean waste production PAGEREF _Toc514063130 h 192.3 Composition of soya bean waste PAGEREF _Toc514063131 h 192.4 Different methods of identifying sugar yield PAGEREF _Toc514063132 h 212.5 Potential of Enzymatic hydrolysis on determining sugar yield on soya bean waste PAGEREF _Toc514063133 h 232.5.1 Process parameter that influences enzymatic hydrolysis PAGEREF _Toc514063134 h 242.6 Proximate analysis PAGEREF _Toc514063135 h 262.7 Total carbohydrate PAGEREF _Toc514063136 h 282.7.1 Phenol Sulphuric Acid Method PAGEREF _Toc514063137 h 282.8 Total reducing sugar analysis PAGEREF _Toc514063138 h 302.8.1 Dintrosalicylic acid solution PAGEREF _Toc514063139 h 312.9 UV Spectrophotometer PAGEREF _Toc514063140 h 322.10 Design of Experiment for optimization PAGEREF _Toc514063141 h 332.11 Monosaccharide and oligosaccharide PAGEREF _Toc514063142 h 342.12 High Pressure Liquid Chromatography PAGEREF _Toc514063143 h 35CHAPTER 3 PAGEREF _Toc514063144 h 36METHODOLOGY PAGEREF _Toc514063145 h 363.1 Introduction PAGEREF _Toc514063146 h 363.2 Material, Equipment, Apparatus and chemicals PAGEREF _Toc514063149 h 383.2.1 Raw material PAGEREF _Toc514063150 h 383.2.2 Equipment and apparatus PAGEREF _Toc514063151 h 383.2.3 Chemicals used PAGEREF _Toc514063152 h 393.3 Sample preparation PAGEREF _Toc514063153 h 393.4 Proximate analysis PAGEREF _Toc514063154 h 393.4.1 Moisture content PAGEREF _Toc514063155 h 403.4.2 Ash PAGEREF _Toc514063156 h 403.4.3 Crude protein PAGEREF _Toc514063157 h 413.4.4 Crude lipid PAGEREF _Toc514063158 h 413.4.5 Carbohydrate PAGEREF _Toc514063159 h 423.5 Enzymatic hydrolysis PAGEREF _Toc514063160 h 423.5.1 Enzyme activity of enzyme alpha-amylase PAGEREF _Toc514063161 h 423.5.2 Concentration of enzyme PAGEREF _Toc514063162 h 433.5.3 Temperature PAGEREF _Toc514063163 h 433.6 Total carbohydrate PAGEREF _Toc514063164 h 443.6.1 Preparation of arabinose standard curve PAGEREF _Toc514063165 h 453.7 Total reducing sugar PAGEREF _Toc514063166 h 453.7.1 Preparation of Dinitrosalicylic acid reagent PAGEREF _Toc514063167 h 463.7.2 Preparation of arabinose standard curve PAGEREF _Toc514063168 h 463.8 Experimental design and analysis PAGEREF _Toc514063169 h 473.8.1 Central Composite Design (CCF) PAGEREF _Toc514063170 h 473.9 Analysis of monosaccharide and oligosaccharide PAGEREF _Toc514063171 h 503.10 Fourier transform infrared spectroscopy (FT-IR) analysis PAGEREF _Toc514063172 h 50CHAPTER 4 PAGEREF _Toc514063173 h 51RESULTS AND DISCUSSIONS PAGEREF _Toc514063174 h 514.1 Proximate analysis PAGEREF _Toc514063175 h 514.2 Statistical analysis of total carbohydrate and total reducing sugar by enzymatic hydrolysis PAGEREF _Toc514063176 h 534.2.1 Model fitting of total carbohydrate PAGEREF _Toc514063177 h 534.2.2 Model fitting of total reducing sugar PAGEREF _Toc514063178 h 574.2.3 Relationship of temperature towards total carbohydrate and total reducing yield. PAGEREF _Toc514063179 h 604.2.4 Relationship of enzyme concentration towards total carbohydrate and total reducing yield. PAGEREF _Toc514063180 h 614.2.5 Optimization of operating conditions on enzymatic hydrolysis on producing total carbohydrate and total reducing sugar. PAGEREF _Toc514063181 h 614.3 Analysis of monosaccharide and oligosaccharide PAGEREF _Toc514063182 h 654.4 Determination of functional group of hydrolyzate of soya bean waste by Fourier transform infrared spectroscopy PAGEREF _Toc514063183 h 68CHAPTER 5 PAGEREF _Toc514063184 h 70CONCLUSION AND RECOMMENDATION PAGEREF _Toc514063185 h 705.1 Conclusion PAGEREF _Toc514063186 h 705.2 Future recommendations PAGEREF _Toc514063187 h 71CHAPTER 6 PAGEREF _Toc514063188 h 72REFERENCES PAGEREF _Toc514063189 h 72CHAPTER 7 PAGEREF _Toc514063190 h 82APPENDIX PAGEREF _Toc514063191 h 82
LIST OF TABLE
TOC h z c “Table” Table 1: Percentage of carbohydrate, crude protein, ash and fat on a dry matter basis: PAGEREF _Toc514063033 h 21Table 2: Percentage of moisture content, carbohydrates, crude protein, ash and fat on a wet matter basis: (Adapted from Li et al., 2013). PAGEREF _Toc514063034 h 21Table 3: Advantages and disadvantages of methods of oligosaccharide production: (Adapted from de Moura et al., 2015) PAGEREF _Toc514063035 h 23Table 4: Summary of proximate analysis method PAGEREF _Toc514063036 h 28Table 5: Detailed elements of independent variables and dependent variables PAGEREF _Toc514063037 h 49Table 6: Specific factor with two levels in central composite design PAGEREF _Toc514063038 h 49Table 7: Specific process parameters for each sequenced run PAGEREF _Toc514063039 h 50Table 8: Proximate analysis of soya bean waste PAGEREF _Toc514063040 h 52Table 9: Analysis of variance (ANOVA) for optimization of total carbohydrate yield by the hydrolysis of okara PAGEREF _Toc514063041 h 56Table 10: Analysis of variance (ANOVA) for optimization of total reducing sugar yield by the hydrolysis of okara PAGEREF _Toc514063042 h 60Table 11: Optimization conditions and predicted response values of hydrolysate okara PAGEREF _Toc514063043 h 63
LIST OF FIGURES TOC h z c “Figure”
Figure 1: Soya milk production: (Adapted from Lui,1997). PAGEREF _Toc514063044 h 19Figure 2: Effect of temperature on the enzymatic reaction: (Adapted from Worthington Biochemical Corporation, 2012) PAGEREF _Toc514063045 h 25Figure 3: Effect of enzyme loading on the enzymatic reaction: (Adapted from Worthington Biochemical Corporation, 2012) PAGEREF _Toc514063046 h 27Figure 4: Dehydration reaction: (Adapted from Cui; Brummer, 2005) PAGEREF _Toc514063047 h 29Figure 5: Furan derivatives from (a) pentose and hexuronic acid, (b) hexoses, (c) 6-deoxyhexoses and (d) keto-hexoses and respectively: (Adapted from Cui ; Brummer, 2005) PAGEREF _Toc514063048 h 30Figure 6: Reaction of formation of coloured complex in phenol sulphuric acid methods: (Adapted from Toledo et al., 2012) PAGEREF _Toc514063049 h 30Figure 7: Reaction of reducing sugar with 3,5-dinitro-salycilic acid reagent: ( Adapted from Toledo et al., 2012) PAGEREF _Toc514063050 h 32Figure 8: Schematic diagram of a dual beam UV-visible spectrophotometer PAGEREF _Toc514063051 h 33Figure 9: Structural of D and L-glucose: (Adapted from Wikibooks,2015) PAGEREF _Toc514063052 h 35Figure 10: Conversion between furanose,acyclic and pyranose from D-glucose: (Adapted from Wikibooks,2015) PAGEREF _Toc514063053 h 35Figure 11: Summary details of the process flow for the studies PAGEREF _Toc514063054 h 38Figure 12: Response surface plot of temperature-enzyme concentration for the total carbohydrate yield PAGEREF _Toc514063055 h 57Figure 13: Response surface plot of temperature-enzyme concentration for the total reducing sugar yield PAGEREF _Toc514063056 h 61Figure 14: Contour surface plot (experiment 1) with total carbohydrate yield PAGEREF _Toc514063057 h 64Figure 15: Contour surface plot (experiment 2) with total carbohydrate yield PAGEREF _Toc514063058 h 64Figure 16: Contour surface plot (experiment 1) with total reducing sugar yield PAGEREF _Toc514063059 h 65Figure 17: Contour surface plot (experiment 2) with total reducing sugar yield PAGEREF _Toc514063060 h 65Figure 18: Production of sucrose in 50?, 55? and 60? PAGEREF _Toc514063061 h 66Figure 19: Production of arabinose in 50?,55? and 60? PAGEREF _Toc514063062 h 67Figure 20: Production of sucrose and arabinose in optimization point (59?) PAGEREF _Toc514063063 h 68Figure 21: Fourier-transform infrared spectroscopy PAGEREF _Toc514063064 h 69Figure 23: Calibration curve for determination of total carbohydrate by phenol sulphuric acid PAGEREF _Toc514063065 h 85Figure 24: Calibration curve for determination of total reducing sugar by DNS method PAGEREF _Toc514063066 h 85Figure 25: Calibration curve for determination of total carbohydrate by phenol sulphuric acid PAGEREF _Toc514063067 h 86Figure 26: Calibration curve for determination of total carbohydrate by DNS method PAGEREF _Toc514063068 h 86
LIST OF ABBREVIATION
WASDE – World Agricultural Supply and Demand Estimate Report
DNS – Dinitrosalicylic acid
HPLC – High performance liquid chromatography
FTIR – Fourier transform infrared spectroscopy
CHAPTER 1INTRODUCTIONSoya bean is one of the valuable crops in the world. The soya bean production has increased annually in the world agricultural crop production. In the World Agricultural Supply and Demand Estimate Report (WASDE), it stated that the soya bean production in 2016 and 2017 is estimated to be around 348 million tons which is a 10.1 % increase in 2 years (DAFF, 2013). Based on the increment on the soya bean production, it shows that the demand of the public towards a high protein meal is being increased.

Malaysia is not producing soya bean and mainly dependent on the imported soya bean for food and feed sectors. Based on the United State Department of Agriculture’s (USDA) Foreign Agricultural Service report on March 2016, it said that the import of soya bean to Malaysia is expected to be increased in 2016 and 2017 which is from 650000 tonnes (2015/2016) to 653000 tonnes (2016/2017) (DAFF, 2013).Malaysia is also one of the largest soy milk and soya food product industries in Southeast Asia. Around 15 % of the imported quantity is used to process tofu and soya milk. The production of soya bean products results in larger quantities of okara being produced.

Soya bean waste is the by-product produced from soya milk and tofu production. About 1.1 kg of soya bean waste is being produced from every kilogram of soya bean that processed into tofu or soya milk ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “ISSN” : “22317546”, “abstract” : “u00a9 All Rights Reserved. The production of soymilk and tofu results in an insoluble residue called okara that present 23% of soybean protein. The objective of this study was to optimize the protein hydrolysis conditions of okara with Alcalase @ endopeptidase. A central composite rotational design was carried out to evaluate the influence of enzymatic reaction temperature (40 – 70u00b0C), enzyme:substrate ratio (1.0 – 10.0%, g enzyme/100 g protein) and pH (7.0 – 9.0) on the degree of hydrolysis (DH). Kinetic of reaction curves (DH versus time) were characterized by high initial reaction rates followed by decreases in the reaction rate up to the stationary phase. By the response surface methodology, the process of hydrolysis was optimized in order to get higher values of DH. The results showed a quadratic dependence of DH in respect to all independent variables. The optimum condition of enzymatic hydrolysis was 55u00b0C, enzyme:substrate ratio of 8.8% and pH 9.0. Under this condition, a experimental DH of 37.3% was obtained and the predicted model was validated. In addition, the pretreatment of okara using ultrasound was evaluated, aiming to increase the DH. There was significant difference (p < 0.1) on DH value (38.8%) obtained at optimum condition, increasing 4%.”, “author” : { “dropping-particle” : “”, “family” : “Montilha”, “given” : “M. S.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Sbroggio”, “given” : “M. F.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Figueiredo”, “given” : “V. R.G.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ida”, “given” : “E. I.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kurozawa”, “given” : “L. E.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Food Research Journal”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2017” }, “title” : “Optimization of enzymatic protein hydrolysis conditions of okara with endopeptidase Alcalase”, “type” : “article-journal” }, “uris” : “http://www.mendeley.com/documents/?uuid=ca3b1738-4d1f-35ad-8e7a-8524fa35f440” } , “mendeley” : { “formattedCitation” : “(Montilha, Sbroggio, Figueiredo, Ida, & Kurozawa, 2017)”, “plainTextFormattedCitation” : “(Montilha, Sbroggio, Figueiredo, Ida, & Kurozawa, 2017)”, “previouslyFormattedCitation” : “(Montilha, Sbroggio, Figueiredo, Ida, & Kurozawa, 2017)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Montilha, Sbroggio, Figueiredo, Ida, & Kurozawa, 2017). It is a nutritional product which is high in carbohydrate, protein, fiber, vitamins, mineral and fat. Soya bean waste can be directly used to make a salad and soup or fermented to be tempeh. Small production of soya bean waste can be observed in home cooking. However, in food industry, soya bean waste is commonly become the factory waste. The discarding of soya bean waste as waste had raised the environmental issue. The presence of monosaccharide and oligosaccharide make soya bean waste able to be recognized as the potential food material in the food industry. So, soya bean waste can develop as a useful food product rather than dispose as waste.

Monosaccharide are the simple sugars containing three to eight carbon atoms. It is classified by the number of carbon atoms and their functional group. The important monosaccharide in soya bean waste are arabinose. Arabinose is the sugar that activates the production of green fluorescent protein.

Oligosaccharide is the carbohydrate that containing twenty monosaccharide which is linked by glycosidic bonds. The prebiotic such as oligosaccharide in soya bean waste can increase the growth of beneficial bacteria and inhibit the growth of harmful bacteria in the small intestine. By taking the soya bean waste product, it is believed that the health of the human can be improved.

There are many methods that can be used to recover the sugar yield on soya bean waste such as acid hydrolysis, enzymatic hydrolysis and physical hydrolysis. Acid hydrolysis is the reaction of decomposition and splitting of a compound that is catalyzed by an acid. The common acid used in acid hydrolysis are sulphuric acid and hydrochloric acid. Enzymatic hydrolysis is the process of converting the pre-treated substrate into sugars by using an enzyme. The common enzyme that is used to identify the monosaccharide and oligosaccharides are ?- amylase and glycoamylase. Physical hydrolysis is the auto-hydrolysis with water in high temperature between 130 ? and 230 ? ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1111/ijfs.12681”, “ISBN” : “0950-5423”, “ISSN” : “13652621”, “abstract” : “Oligosaccharides are carbohydrates with a low molecular weight, which, when nondigestible, can produce bifidogenic activity and several other effects to human health. Oligosaccharides can be found naturally in foods or are produced by the synthesis from disaccharide substrates or by the hydrolysis of polysaccharides. Although it has yet to be improved, the hydrolysis of polysaccharides is the best choice for oligosaccharide production on a large scale, due to its reproducibility and smaller cost. This review concisely presents the main processes for the production of oligosaccharides by depolymerisation of polysaccharides (enzymatic, acid, and physical hydrolysis), taking into account their advantages, disadvantages, and perspectives.”, “author” : { “dropping-particle” : “”, “family” : “Moura”, “given” : “Fernanda A.”, “non-dropping-particle” : “de”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Macagnan”, “given” : “Fernanda T.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Silva”, “given” : “Leila P.”, “non-dropping-particle” : “da”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Journal of Food Science and Technology”, “id” : “ITEM-1”, “issue” : “2”, “issued” : { “date-parts” : “2015” }, “page” : “275-281”, “title” : “Oligosaccharide production by hydrolysis of polysaccharides: A review”, “type” : “article-journal”, “volume” : “50” }, “uris” : “http://www.mendeley.com/documents/?uuid=a0319d2e-594a-4d59-b245-2db6d8960d70” } , “mendeley” : { “formattedCitation” : “(de Moura, Macagnan, & da Silva, 2015)”, “plainTextFormattedCitation” : “(de Moura, Macagnan, & da Silva, 2015)”, “previouslyFormattedCitation” : “(de Moura, Macagnan, & da Silva, 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(de Moura, Macagnan, & da Silva, 2015).

Enzymatic hydrolysis is a possible method that will be used to recover the sugar from soya bean waste. The factor that will affect the enzymatic hydrolysis are the concentration of enzyme used, the temperature during the experiment and the time of the experiment. Different parameters will affect the total yield which are the total carbohydrate and total reducing sugar that present in the soya bean waste.

1.1 Problem StatementMalaysia is one of the largest soya product production in Asia. The utilization of soya bean waste in Malaysia is limited to animal feedstock and some food production such as tempeh in small scale of processing. In the factory, the soya bean waste considered as food waste. The discarding of soya bean waste as waste had raised the environmental issue. The presence of monosaccharide and oligosaccharide make soya bean waste able to be recognized as the potential food material in the food industry.

1.2 ObjectivesThe objective of this project are:
To investigate the effect of operating process parameter (concentration and temperature) on sugar yield using enzymatic hydrolysis and optimization of the operating conditions.

To characterize the total carbohydrate and analyze the monosaccharide and oligosaccharides.

1.2 Project OutlineThe thesis consists of 5 chapters.

Chapter 1 focuses on defining the introduction of production on soya bean in the world, the usage of soya bean in producing soya milk and tofu in Malaysia and by product produced from the processing. In this chapter, the problem statement and the purpose of this project is briefly explained.

Chapter 2 is the literature review on the concept of processing of soya milk, the composition of the soya bean waste and the enzymatic hydrolysis on determining the sugar yield of soya bean waste.

Chapter 3 depicts the methodology of the projects where the method and approaches are defined in this chapter.

Chapter 4 explains about the results obtained from the experiment regarding the effect of process parameter on sugar yield using enzymatic hydrolysis, the total carbohydrate and total sugar content on soya bean waste. The monosaccharide and oligosaccharide also been analyzed.

Chapter 5 concludes the project based on the results obtained and also proposed the suggestions for future improvement of this project.

CHAPTER 2LITERATURE REVIEW2.1 Soybean
Soya bean Glycine max (L.) Meir, (Lui 1997) is one of the most important agricultural crops in the world that used in food and oil production (Lui 1997). United States is currently the largest producer and exporter of soybean in the world with around twenty-six percent of land acres being utilized for soybean cultivation. However, the Asian countries are the largest market in using soya bean in food industry (Lui, 1997; Setchell, 1998). Most of the Asian countries including Malaysia import the soya bean from Unites States to produce traditional soya food products such as soya milk, tofu, soya sauce and tempeh.

2.2 Soya milk processingThere are different kind of soya milk product exist in the market. The method on soya milk processing is depend on the manufacturer and the requirement of the product. Generally, the soya bean will soak in the water and ground into slurry. Soya milk is produced by extracting the aqueous portion of the soya bean slurry. The soya milk production is depend on the soaking water temperature, separation of the soya bean waste from the soymilk pre or post cooking, blanching the beans before grinding, adding chemicals to the blanching or grinding step, grinding size and the number of times the okara is washed (Cai, 1999: Riaz 2006). Figure 1 shows the step to produce soya milk.

3080719350983244426234974Soybean
0Soybean

4152900255905Water
Water
37287204108452433955249555Soak overnight
Soak overnight

3086735149860
309054535687041694106985Water
Water
372745015176524466557620Rinse
0Rinse

4191635295910Water
Water
2444750211455Grinds soya bean into slurry
0Grinds soya bean into slurry

3104515302895374269024765
726440144145Soya bean waste
Soya bean waste
20129502971802472055147320Filter
0Filter

2463800273050Heat soya milk
0Heat soya milk
309562512065
3090545168910
245173538100Rinse
0Rinse

Figure SEQ Figure * ARABIC 1: Soya milk production: (Adapted from Lui,1997).2.2.1 Soya milk processing: Soya bean waste productionIn soya milk processing, large amount of soya milk waste which also known as okara being produced. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “ISSN” : “22317546”, “abstract” : “u00a9 All Rights Reserved. The production of soymilk and tofu results in an insoluble residue called okara that present 23% of soybean protein. The objective of this study was to optimize the protein hydrolysis conditions of okara with Alcalase @ endopeptidase. A central composite rotational design was carried out to evaluate the influence of enzymatic reaction temperature (40 – 70u00b0C), enzyme:substrate ratio (1.0 – 10.0%, g enzyme/100 g protein) and pH (7.0 – 9.0) on the degree of hydrolysis (DH). Kinetic of reaction curves (DH versus time) were characterized by high initial reaction rates followed by decreases in the reaction rate up to the stationary phase. By the response surface methodology, the process of hydrolysis was optimized in order to get higher values of DH. The results showed a quadratic dependence of DH in respect to all independent variables. The optimum condition of enzymatic hydrolysis was 55u00b0C, enzyme:substrate ratio of 8.8% and pH 9.0. Under this condition, a experimental DH of 37.3% was obtained and the predicted model was validated. In addition, the pretreatment of okara using ultrasound was evaluated, aiming to increase the DH. There was significant difference (p < 0.1) on DH value (38.8%) obtained at optimum condition, increasing 4%.”, “author” : { “dropping-particle” : “”, “family” : “Montilha”, “given” : “M. S.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Sbroggio”, “given” : “M. F.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Figueiredo”, “given” : “V. R.G.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ida”, “given” : “E. I.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kurozawa”, “given” : “L. E.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Food Research Journal”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2017” }, “title” : “Optimization of enzymatic protein hydrolysis conditions of okara with endopeptidase Alcalase”, “type” : “article-journal” }, “uris” : “http://www.mendeley.com/documents/?uuid=ca3b1738-4d1f-35ad-8e7a-8524fa35f440” } , “mendeley” : { “formattedCitation” : “(Montilha et al., 2017)”, “manualFormatting” : “Montilha et al., (2017)”, “plainTextFormattedCitation” : “(Montilha et al., 2017)”, “previouslyFormattedCitation” : “(Montilha et al., 2017)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Montilha et al., (2017) stated that about one kilogram of soya bean waste is generated from every kilogram of soya bean while Riaz, (2006) stated that 1.4 – 1.8 kg of soya bean waste is produced for every kilogram of soybeans used for soymilk processing. Wu, Wu, Yang, & Wang (2012) said that about 20,000,000 tons of moist soya bean waste were produced annually from the soybean production industry in China. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1155/2013/423590”, “abstract” : “The production of soybean products has been increasing throughout the world, and there has been a corresponding increase in the quantity of soybean curd residue (SCR) being thrown out. The dumping of SCR has become a problem to be solved due to its contamination to the environment. SCR is rich in fiber, fat, protein, vitamins, and trace elements. It has potential for value-added processing and utilization; options that simultaneously hold the promise of increased economic benefit as well as decreased pollution potential for the environment. The objective of this study is to fully investigate, review, and summarize the existing literature in order to develop a comprehensive knowledge base for the composition and reuse of SCR. It is evident from the literature survey that SCR shows good potential as a functional food material. However, there are several drawbacks to the use of SCR and corresponding solutions presented in this paper.”, “author” : { “dropping-particle” : “”, “family” : “Li”, “given” : “Shuhong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhu”, “given” : “Dan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Kejuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Yingnan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lei”, “given” : “Zhongfang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhang”, “given” : “Zhenya”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bermejo”, “given” : “M D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rajabathar”, “given” : “J”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “ISRN Industrial Engineering”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2013” }, “publisher” : “Hindawi Publishing Corporation”, “title” : “Soybean Curd Residue: Composition, Utilization, and Related Limiting Factors”, “type” : “article-journal”, “volume” : “8” }, “uris” : “http://www.mendeley.com/documents/?uuid=5c761e1d-5a21-3cda-acba-4fc3eeeeaec7” } , “mendeley” : { “formattedCitation” : “(S. Li et al., 2013)”, “manualFormatting” : “S. Li et al., (2013)”, “plainTextFormattedCitation” : “(S. Li et al., 2013)”, “previouslyFormattedCitation” : “(S. Li et al., 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }S. Li et al., (2013) said that about 800,000 tons of soya bean waste are disposed of annually as byproducts of tofu production in Japan.

2.3 Composition of soya bean wasteSoya bean waste contains a high number of carbohydrate, protein, fiber, vitamin, mineral and fat. The composition of soya bean waste is mainly depending on the amount of water extracted from round soybeans, and whether the water is further added to extract residual extractable components. A summary of the proximate compositions of soya bean waste on a dry matter basis and wet matter basis is shown in table (1 and 2) below.

Table SEQ Table * ARABIC 1: Percentage of carbohydrate, crude protein, ash and fat on a dry matter basis:
(Adapted fromADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1155/2013/423590”, “abstract” : “The production of soybean products has been increasing throughout the world, and there has been a corresponding increase in the quantity of soybean curd residue (SCR) being thrown out. The dumping of SCR has become a problem to be solved due to its contamination to the environment. SCR is rich in fiber, fat, protein, vitamins, and trace elements. It has potential for value-added processing and utilization; options that simultaneously hold the promise of increased economic benefit as well as decreased pollution potential for the environment. The objective of this study is to fully investigate, review, and summarize the existing literature in order to develop a comprehensive knowledge base for the composition and reuse of SCR. It is evident from the literature survey that SCR shows good potential as a functional food material. However, there are several drawbacks to the use of SCR and corresponding solutions presented in this paper.”, “author” : { “dropping-particle” : “”, “family” : “Li”, “given” : “Shuhong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhu”, “given” : “Dan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Kejuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Yingnan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lei”, “given” : “Zhongfang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhang”, “given” : “Zhenya”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bermejo”, “given” : “M D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rajabathar”, “given” : “J”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “ISRN Industrial Engineering”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2013” }, “publisher” : “Hindawi Publishing Corporation”, “title” : “Soybean Curd Residue: Composition, Utilization, and Related Limiting Factors”, “type” : “article-journal”, “volume” : “8” }, “uris” : “http://www.mendeley.com/documents/?uuid=5c761e1d-5a21-3cda-acba-4fc3eeeeaec7” } , “mendeley” : { “formattedCitation” : “(S. Li et al., 2013)”, “manualFormatting” : ” Li et al., 2013)”, “plainTextFormattedCitation” : “(S. Li et al., 2013)”, “previouslyFormattedCitation” : “(S. Li et al., 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” } Li et al., 2013) .

Carbohydrate (%)Crude Protein (%)Ash (%)Fat (%)References
53.6
52.6
52.9

32.6

3.8-5.3 29.3
16.1
26.8
26.8±0.125.5
25.0
25.4-28 4.0
5.3


4.0

– 0.8
2.2
12.3
22.3 ± 1.512.0
20.0
9.3-10.9 Muroyama et al., 2006
Hsieh and yang, 2004
Ma et al., 1996
Guermani et al., 1992
Rashad et al., 2011
Suruga et al., 2007
Van der Riet et al., 1989
Table SEQ Table * ARABIC 2: Percentage of moisture content, carbohydrates, crude protein, ash and fat on a wet matter basis: (Adapted from Li et al., 2013).
Moisture content (%)Carbohydrate (%)Crude Protein (%)Ash (%)Fat
(%)References
85.0 – 3.6 – 1.4 Zhu et al., 2008
83.9 8.3 4.5 0.7 2.6 Turhan et al., 2007
81.0 6.4 4.8 0.8 3.6 Ohno et al., 1996
2.4 Different methods of identifying sugar yield
There are several methods that can be used to identify the sugar content in the product. Acid hydrolysis is the reaction of decomposition and splitting of a compound that is catalyzed by an acid. According to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.carres.2008.10.019”, “ISBN” : “0008-6215”, “ISSN” : “00086215”, “PMID” : “19054503”, “abstract” : “To discover drug candidates with anti-angiogenesis activity for cancer therapeutics, three galactooligosaccharides (OJ1-OJ3) were prepared by acid hydrolysis of the polysaccharides from Nerium indicum Mill. Their structures were characterized using sugar analysis, methylation analysis, and both 1D and 2D NMR spectroscopy, complemented by mass spectrometry. They were hexasaccharide (OJ1), a pentasaccharide (OJ2), and an undecasaccharide (OJ3), which was a new linear galactan. Bioactivity testing in vitro showed that OJ2 and OJ3 significantly inhibited the HMEC-1 (human microvascular endothelial cell) cell tube formation. u00a9 2008 Elsevier Ltd. All rights reserved.”, “author” : { “dropping-particle” : “”, “family” : “Hu”, “given” : “Ke”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Liu”, “given” : “Qin”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Wang”, “given” : “Shunchun”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ding”, “given” : “Kan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Carbohydrate Research”, “id” : “ITEM-1”, “issue” : “2”, “issued” : { “date-parts” : “2009” }, “page” : “198-203”, “publisher” : “Elsevier Ltd”, “title” : “New oligosaccharides prepared by acid hydrolysis of the polysaccharides from Nerium indicum Mill and their anti-angiogenesis activities”, “type” : “article-journal”, “volume” : “344” }, “uris” : “http://www.mendeley.com/documents/?uuid=3396c3ed-5ef0-432b-a722-9c661f146b6e” } , “mendeley” : { “formattedCitation” : “(Hu, Liu, Wang, & Ding, 2009)”, “manualFormatting” : “Hu et al., (2009)”, “plainTextFormattedCitation” : “(Hu, Liu, Wang, & Ding, 2009)”, “previouslyFormattedCitation” : “(Hu, Liu, Wang, & Ding, 2009)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Hu et al., (2009), dilute acid hydrolysis can be done in short time without compromising the sugars produced. It is a very simple and controllable method. This method is carried out in a very high temperature and time interval of 1 to 6 hours. The common acid used in acid hydrolysis are sulphuric acid, hydrochloric acid and trifluroacetic acid. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.ijbiomac.2017.08.028”, “ISSN” : “18790003”, “abstract” : “The effects of acid hydrolysis intensity on the physicochemical properties of starch/xanthan gum (XG) system were studied. Waxy corn starch (WCS) was subjected to different concentrations of hydrochloric acid, and crystallization and relative molecular weight analysis were performed. The results revealed that the starch granules became smaller during acid hydrolysis. X-ray diffraction pattern analysis showed that the crystal structure did not change with acid hydrolysis. Evaluation of the properties and digestibility of different acid-thinned starch/XG systems indicated that the viscosity of acid-thinned starch/XG decreased with increased acid hydrolysis intensity. Rheological property measurements indicated that the compound systems were a pseudo-plastic fluid, which is a typical weak gel structure. Finally, we show that the WCS1.0. M/XG has the highest stability of the tested mixtures. We conclude that adjusting the conditions of acid hydrolysis improves the stability and food quality-enhancing properties of starch.”, “author” : { “dropping-particle” : “”, “family” : “Jiang”, “given” : “Min”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Hong”, “given” : “Yan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Gu”, “given” : “Zhengbiao”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Cheng”, “given” : “Li”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Zhaofeng”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Caiming”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Journal of Biological Macromolecules”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2017” }, “page” : “320-329”, “publisher” : “Elsevier B.V.”, “title” : “Effects of acid hydrolysis intensity on the properties of starch/xanthan mixtures”, “type” : “article-journal”, “volume” : “106” }, “uris” : “http://www.mendeley.com/documents/?uuid=98e74792-e047-4b41-8917-52051cbbe834” } , “mendeley” : { “formattedCitation” : “(Jiang et al., 2017)”, “manualFormatting” : “Jiang et al., (2017)”, “plainTextFormattedCitation” : “(Jiang et al., 2017)”, “previouslyFormattedCitation” : “(Jiang et al., 2017)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Jiang et al., (2017) use different concentrations of hydrochloric acid to study the intensity on the wax corn starch. The result revealed that the starch granules become smaller during acid hydrolysis.

Enzymatic hydrolysis involving an enzyme to convert the pre-treatment of substrate into sugar. This method usually is carried out in the range of 40? to 60? for 24 hours. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1111/ijfs.12681”, “ISBN” : “0950-5423”, “ISSN” : “13652621”, “abstract” : “Oligosaccharides are carbohydrates with a low molecular weight, which, when nondigestible, can produce bifidogenic activity and several other effects to human health. Oligosaccharides can be found naturally in foods or are produced by the synthesis from disaccharide substrates or by the hydrolysis of polysaccharides. Although it has yet to be improved, the hydrolysis of polysaccharides is the best choice for oligosaccharide production on a large scale, due to its reproducibility and smaller cost. This review concisely presents the main processes for the production of oligosaccharides by depolymerisation of polysaccharides (enzymatic, acid, and physical hydrolysis), taking into account their advantages, disadvantages, and perspectives.”, “author” : { “dropping-particle” : “”, “family” : “Moura”, “given” : “Fernanda A.”, “non-dropping-particle” : “de”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Macagnan”, “given” : “Fernanda T.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Silva”, “given” : “Leila P.”, “non-dropping-particle” : “da”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Journal of Food Science and Technology”, “id” : “ITEM-1”, “issue” : “2”, “issued” : { “date-parts” : “2015” }, “page” : “275-281”, “title” : “Oligosaccharide production by hydrolysis of polysaccharides: A review”, “type” : “article-journal”, “volume” : “50” }, “uris” : “http://www.mendeley.com/documents/?uuid=a0319d2e-594a-4d59-b245-2db6d8960d70” } , “mendeley” : { “formattedCitation” : “(de Moura et al., 2015)”, “manualFormatting” : “De Moura et al., (2015)”, “plainTextFormattedCitation” : “(de Moura et al., 2015)”, “previouslyFormattedCitation” : “(de Moura et al., 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }De Moura et al., (2015) stated that the enzymatic hydrolysis can produce the highest yield of sugar. Enzyme amylase and glucoamylase are the common enzyme to be used to hydrolyze starch to produce sugar.
Physical autolysis involving auto-hydrolysis with water in high temperature between 130? to 230?. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.cej.2010.05.048”, “ISBN” : “1385-8947”, “ISSN” : “13858947”, “abstract” : “Fractionation in aqueous media is an environmentally friendly technology suitable for obtaining oligomeric products from pectins and hemicelluloses. Sugar beet pulp (SBP) samples were subjected to aqueous processing under non-isothermal conditions to reach maximal temperatures in the range 140-200 ??C, in order to cause the conversion of pectins into soluble compounds of lower molecular weight with potential applications as prebiotic ingredients.Mixtures of arabinooligosaccharides, oligogalacturonides and oligomers made up of other structural units were obtained by non-isothermal processing of SBP in aqueous media. Kinetic models suitable for reproducing and predicting compositional data of reaction liquors as a function of the operational conditions were developed. According to the model predictions, the maximum arabinooligosaccharides yield (15.7. g/100. g SBP) corresponded to a treatment carried out to achieve 171.5 ??C. Alternatively, the maximum oliogogalacturonide yield (14.1. g/100. g SBP) was predicted for a treatment performed to achieve 158.2 ??C. In both cases, the overall yield of oligomeric saccharides was near to 30. g/100. g dry SBP, whereas the oligosaccharide mixture of higher purity was obtained operating at 158.2 ??C. Depending on the operational conditions, SBP processing in aqueous media yielded oligomers with different compositional profiles. The developed models were suitable for a quantitative interpretation of experimental data, and provided key information for both design calculations and economic evaluation. ?? 2010 Elsevier B.V.”, “author” : { “dropping-particle” : “”, “family” : “Martu00ednez”, “given” : “Martina”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Gullu00f3n”, “given” : “Beatriz”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yu00e1u00f1ez”, “given” : “Remedios”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Alonso”, “given” : “Josu00e9 Luis”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Paraju00f3”, “given” : “Juan Carlos”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Chemical Engineering Journal”, “id” : “ITEM-1”, “issue” : “2”, “issued” : { “date-parts” : “2010” }, “page” : “480-486”, “title” : “Kinetic assessment on the autohydrolysis of pectin-rich by-products”, “type” : “article-journal”, “volume” : “162” }, “uris” : “http://www.mendeley.com/documents/?uuid=f6a78a16-3899-4c2d-9293-bbbd74ec6224” } , “mendeley” : { “formattedCitation” : “(Martu00ednez, Gullu00f3n, Yu00e1u00f1ez, Alonso, & Paraju00f3, 2010)”, “manualFormatting” : “Martu00ednez et al., (2010)”, “plainTextFormattedCitation” : “(Martu00ednez, Gullu00f3n, Yu00e1u00f1ez, Alonso, & Paraju00f3, 2010)”, “previouslyFormattedCitation” : “(Martu00ednez, Gullu00f3n, Yu00e1u00f1ez, Alonso, & Paraju00f3, 2010)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Martínez et al., (2010) studied the auto-hydrolysis of pectin-rich by-products using sugar beet pulp (SBP). The sugar beet pulp (SBP) samples were subjected to an aqueous processing under non-isothermal conditions to reach maximal temperatures in the range 140–200 ?C in order to convert pectins into soluble compounds of lower molecular weight with potential applications as prebiotic ingredients ( ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.cej.2010.05.048”, “ISBN” : “1385-8947”, “ISSN” : “13858947”, “abstract” : “Fractionation in aqueous media is an environmentally friendly technology suitable for obtaining oligomeric products from pectins and hemicelluloses. Sugar beet pulp (SBP) samples were subjected to aqueous processing under non-isothermal conditions to reach maximal temperatures in the range 140-200 ??C, in order to cause the conversion of pectins into soluble compounds of lower molecular weight with potential applications as prebiotic ingredients.Mixtures of arabinooligosaccharides, oligogalacturonides and oligomers made up of other structural units were obtained by non-isothermal processing of SBP in aqueous media. Kinetic models suitable for reproducing and predicting compositional data of reaction liquors as a function of the operational conditions were developed. According to the model predictions, the maximum arabinooligosaccharides yield (15.7. g/100. g SBP) corresponded to a treatment carried out to achieve 171.5 ??C. Alternatively, the maximum oliogogalacturonide yield (14.1. g/100. g SBP) was predicted for a treatment performed to achieve 158.2 ??C. In both cases, the overall yield of oligomeric saccharides was near to 30. g/100. g dry SBP, whereas the oligosaccharide mixture of higher purity was obtained operating at 158.2 ??C. Depending on the operational conditions, SBP processing in aqueous media yielded oligomers with different compositional profiles. The developed models were suitable for a quantitative interpretation of experimental data, and provided key information for both design calculations and economic evaluation. ?? 2010 Elsevier B.V.”, “author” : { “dropping-particle” : “”, “family” : “Martu00ednez”, “given” : “Martina”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Gullu00f3n”, “given” : “Beatriz”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yu00e1u00f1ez”, “given” : “Remedios”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Alonso”, “given” : “Josu00e9 Luis”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Paraju00f3”, “given” : “Juan Carlos”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Chemical Engineering Journal”, “id” : “ITEM-1”, “issue” : “2”, “issued” : { “date-parts” : “2010” }, “page” : “480-486”, “title” : “Kinetic assessment on the autohydrolysis of pectin-rich by-products”, “type” : “article-journal”, “volume” : “162” }, “uris” : “http://www.mendeley.com/documents/?uuid=f6a78a16-3899-4c2d-9293-bbbd74ec6224” } , “mendeley” : { “formattedCitation” : “(Martu00ednez et al., 2010)”, “manualFormatting” : “Martu00ednez, Gullu00f3n, Yu00e1u00f1ez, Alonso, & Paraju00f3, 2010)”, “plainTextFormattedCitation” : “(Martu00ednez et al., 2010)”, “previouslyFormattedCitation” : “(Martu00ednez et al., 2010)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Martínez, Gullón, Yáñez, Alonso, & Parajó, 2010).

Figure 2 shows the advantages and disadvantages of these hydrolysis method. For enzymatic hydrolysis, it is efficient and able to get a good yield of sugar. But, this method requires rigorous control of the reaction and higher cost. For acid hydrolysis, it requires a small amount of cost and it is easy to conduct the process. However, the generation of toxic substances might occur and causing low yield of oligosaccharides. For physical hydrolysis, it does not require the use of any chemical product so it generates less environmental waste among the methods. However, in physical hydrolysis, the oligomers can be hydrolyzed to monosaccharide due to high temperature effects.

Table SEQ Table * ARABIC 3: Advantages and disadvantages of methods of oligosaccharide production: (Adapted from de Moura et al., 2015)Method Production References Advantages Disadvantages
Enzymatic hydrolysis
Fructooligosaccharides
Pectioligosaccharides
Maltooligosaccharides
Xyloooligosaccharides
Chitinoligosaccharides
Cazetta et al. (2005)
Holck et al. (2011)
Barreteau et al. (2006) Menezes et al. (2009)
Ramirez-Coutino et al. (2006) Efficiency
Specific action
Good yield High cost
Microbiological contaminants
Acid hydrolysis
Fructooligosaccharides
Maltooligosaccharides
Xyloooligosaccharides
Avila-Fernandez et al. (2011)
Warrand & Janssen (2007) Reis et al. (2003)
Simplicity of the process
Small cost
Quickness
Monosaccharide and toxic substances generation
Low yield of oligosaccharide
Nonspecific action
Physical hydrolysis Pectioligosaccharides
Xyloooligosaccharides
Arabinoxyxylooligosaccharides
Mart?nez et al. (2010)
Vazquez et al. (2005) Rose & Inglett (2010) Not require usage of chemical product
Less waste generated
Oligomers can hydrolyzed to monosaccharide
High installation
Cost
Limited application in industry
2.5 Potential of Enzymatic hydrolysis on determining sugar yield on soya bean wasteEnzymes are macromolecules that catalyze biological transformations in living systems. It is a protein with catalytic properties due to its power of specific activation. It’s able to accelerate the rate of chemical reaction by lowering the activation energy. The molecules that bind to the enzymes is known as substrates where enzyme will convert the substrates into products.
Enzymatic hydrolysis rely on the ability of the enzyme to catalyze a specific reaction and employ a suitable method for monitoring the progression of the reaction. This method is highly specific, rapid and sensitive to a low sugar concentration. It can be considered as a mild process compared to other chemical process. It requires less energy than other methods. According to Barreteau et al. (2006), enzymatic hydrolysis is the best choice for sugar production.
2.5.1 Process parameter that influences enzymatic hydrolysis2.5.1.1 Temperature
Temperature has a significant effect on the enzyme to convert the substrate into products. The rate of enzyme-catalyzed reaction increases as the temperature rises. However, every enzyme has a range of temperature for optimal activity. If the enzyme is reacted within its optimal range of temperature, the activity of the enzyme will be inhibited or fully stopped.

Figure SEQ Figure * ARABIC 2: Effect of temperature on the enzymatic reaction: (Adapted from Worthington Biochemical Corporation, 2012)From the graph above (Figure 2), the enzyme activity increases with the rise of temperature until it reaches the maximum level which is known as the optimum temperature for the enzyme. The increasing of enzymatic activity is due to the breaking bond of weak hydrogen and ionic bond which cause the active site of the enzyme to change shape. The activity of enzymes started to decrease with further increasing of the temperature. The activity of enzyme declines due to the denaturation of the enzyme. Denaturation of enzyme is due to the less complementary of enzyme active sites to the substrate.

2.5.1.2 Enzyme loading
Enzyme loading also known as the concentration of the enzyme able to affect the rate of an enzyme-catalyzed reaction. The concentration of the substrate must present in an excess amount so that the reaction will be mainly dependent on the concentration of the enzyme used. From the graph below (Figure 3), it is clearly shown that the increasing of the concentration of the enzyme can increase the rate of reaction as more enzyme are able to collide with the substrate molecules.

Figure SEQ Figure * ARABIC 3: Effect of enzyme loading on the enzymatic reaction: (Adapted from Worthington Biochemical Corporation, 2012)2.6 Proximate analysis
Proximate analysis is a method to get the information about the nutritional value of the feed. This method partitioned nutrients in divided into 5 components which are water, ash, crude protein, crude lipid and carbohydrate. Table 3 shows the summary of the proximate analysis method.

Table SEQ Table * ARABIC 4: Summary of proximate analysis methodNutrient component Procedure
Moisture content The moisture content is determined by measuring the weight loss of the sample. The sample is dried in the oven until constant weight obtained.

Ash Ash content is obtained by burning off the organic matter at 400? – 600 ? in a muffle furnace.

Crude protein The crude protein is determined by using Kjedahl method where the nitrogen factor of the sample is being measured and multiply by 6.25.

Crude lipid The crude lipid is determined using Soxhlet equipment. The ether extract of the sample is the fat that presented in the sample.

Carbohydrate Carbohydrate is determined by subtracting the total percentage of weight, which is moisture content, ash, crude protein and crude lipid from the sample.

2.7 Total carbohydrateCarbohydrate is an organic compound which consists of carbon, oxygen and hydrogen. It is one of the most important ingredients in the food industry. It helps in improving the texture of the food product besides giving nutrient to the food. The carbohydrate component contains sugar, complex carbohydrate and starch. It is divided into four special chemical groups which are monosaccharide, disaccharides, polysaccharides and oligosaccharides. 
Monosaccharide is the simplest form of carbohydrate that used in metabolism. For example, glucose is small which can easily break down and absorbed by human. Polysaccharides act as structural components and energy storage area. It normally acts as the stabilizers in food industry. For example, gums, starches and pectins are used in cakes, jams, noodles, cookies and canned food as thickening agents. Oligosaccharide is normally act as the prebiotics or bio-preservatives in food industry.

2.7.1 Phenol Sulphuric Acid MethodThe Phenol-Sulphuric acid reaction method for carbohydrate analysis described by DuBois et al (1956) is used to determine sub-micro amounts of sugars and related substances. In the presence of strong acid and heat, carbohydrate will undergo a series of reaction which leads to the formation of furanaldehyde and hydroxymethyl furanaldehyde. The dehydration of the reaction and the formation of furan derivatives ,which will then condense with a phenol solution to produce dark colored ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1201/9780203485286.ch2”, “ISBN” : “978-0-8493-1574-9”, “author” : { “dropping-particle” : “”, “family” : “Cui”, “given” : “Steve”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Brummer”, “given” : “Yolanda”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Food Carbohydrates”, “id” : “ITEM-1”, “issue” : “May 2005”, “issued” : { “date-parts” : “2005” }, “title” : “Understanding Carbohydrate Analysis”, “type” : “book” }, “uris” : “http://www.mendeley.com/documents/?uuid=b7714156-3c78-42be-b46a-0d21cc08a29c” } , “mendeley” : { “formattedCitation” : “(Cui & Brummer, 2005)”, “plainTextFormattedCitation” : “(Cui & Brummer, 2005)”, “previouslyFormattedCitation” : “(Cui & Brummer, 2005)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Cui ; Brummer, 2005). Simple sugars, oligosaccharides, polysaccharides and their derivatives are stained orange-yellow when treated with phenol and concentrated sulphuric acid.  This quality can be utilized to perform quantitative analyses of sugars and their derivatives using colorimetric methods such as spectrophotometry in 490nm wavelength.

Figure SEQ Figure * ARABIC 4: Dehydration reaction: (Adapted from Cui; Brummer, 2005)
Figure SEQ Figure * ARABIC 5: Furan derivatives from (a) pentose and hexuronic acid, (b) hexoses, (c) 6-deoxyhexoses and (d) keto-hexoses and respectively: (Adapted from Cui ; Brummer, 2005)
Figure SEQ Figure * ARABIC 6: Reaction of formation of coloured complex in phenol sulphuric acid methods: (Adapted from Toledo et al., 2012)2.8 Total reducing sugar analysis
The carbohydrate, which is capable to be oxidized and cause reduction of other substances without having to be hydrolyzed will be known as reducing sugar, but other compounds that oxidized and cannot reduce other substances will be known as non-reducing sugar. The free monosaccharide which consist of the free aldehyde or a hydroxyl ketonic group are capable to be oxidized and cause reduction of other substances. Example of reducing sugar are glucose, galactose, xylose and arabinose.

2.8.1 Dintrosalicylic acid solutionDinitrosalicylic acid (DNS) method was mentioned by Summer and Sisler (1994) and modified by Miller, (1959) to determine the amount of total reducing sugars in a product. In an alkaline medium, the reducing sugars will reduce the dinitrosalicylic acid to 3, 5- dinitrosalicylic acid to 3-amino-5-nitro-salicyclic acid. The aldehyde group of sugars is oxidized to the respective carboxylic acid. The 3-amino-5-nitrosalicylic acid is an orange color product. However, the intensity of the color depends on the concentration of the reducing sugar presented in the product. This quality can be utilized to perform quantitative analyses of sugars and their derivatives using colorimetric methods such as spectrophotometry in 540 nm wavelength.

Figure SEQ Figure * ARABIC 7: Reaction of reducing sugar with 3,5-dinitro-salycilic acid reagent: ( Adapted from Toledo et al., 2012)2.9 UV SpectrophotometerA spectrophotometer is an equipment which using the light absorption to determine the concentration of a molecule. These obey the Beer-Lambert Law where it relates the absorbance of light by a sample to the concentration of the absorbing species. In the Beer-Lambert Law, the absorbance is directly proportional to the concentration of the absorbance and also the path length. It is commonly used to measure the concentration of total carbohydrate and sugar of a product because it is an easy and effective method. Spectrophotometer come in a variety of instrumentation design. The high quality of design offers the accurate and consistent data in quantitative analysis and are available for use in a variety of applications.
A diagram of the components of a typical spectrometer is shown in the following diagram (Figure 8). A beam of light from a visible and UV light source is separated into its component wavelengths by a prism or diffraction grating. A diffraction grating consists of a series of parallel grooves (lines) on a reflecting surface. Each wavelength will split into two beams which have equal intensity. The sample beam will pass through a transparent container containing a solution of the compound whereas the reference beam will pass through an identical transparent container that containing the solvent only. The intensity of both light beams will be measured and compare. The intensity of the sample beam and reference beam will be defined as I and I0 respectively.

Figure SEQ Figure * ARABIC 8: Schematic diagram of a dual beam UV-visible spectrophotometer2.10 Design of Experiment for optimizationIn mathematics, optimization is an effective tool in order to get the maximum or minimum of functions that subjected to certain restriction. In the fields of engineering, process optimization can be considered as one of the most crucial issue as any process is a combination of various sources which is linked to money and time (Sibalija ; Majstorovi?, 2015).

Design of experiment is an effective methodology on planning experiment on order to obtain the data which can be evaluated to produce conclusions that are valid. It can be considered as the most precise and explicit criterion for analyzing an assumption. It can be applied for different goals, such as optimization of a process and robust testing of products and process.

2.11 Monosaccharide and oligosaccharideMonosaccharide is the smallest carbohydrate building block which exists as ketoses or aldoses. It can be found in two different enantiometric forms, D and L which are based on the configuration in the chiral center in relation to D-glyceraldehyde. Most common monosaccharides are with five (pentose) or six (hexose) carbon atoms and they are in an equilibrium between an open form and cyclic hemiacetals, formed between the carbonyl group and the hydroxy group on carbon 4 or 5, yielding furanoses and pyranoses respectively. The hydroxy group at the anomeric center can be in either ? or ? position because the equilibrium between the open and closed forms the configurations can interchange.

Figure SEQ Figure * ARABIC 9: Structural of D and L-glucose: (Adapted from Wikibooks,2015)
Figure SEQ Figure * ARABIC 10: Conversion between furanose,acyclic and pyranose from D-glucose: (Adapted from Wikibooks,2015)Oligosaccharide is carbohydrates composed between two and ten monosaccharide residues glycosidically linked. Oligosaccharide can be divided into either N-glycosidic bonds or O-glycosidic bonds. N-linked oligosaccharides are pentasaccharides which attached to asparagine via a beta linkage to the amine nitrogen of the side chain.  O-linked oligosaccharides are generally attached to threonine or serine on the alcohol group of the side chain.

The major component of oligosaccharide in okara is sucrose ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “ISSN” : “09647058”, “PMID” : “17392083”, “abstract” : “Oligosaccharides have been credited with many health-promoting functions, which had been identified in many clinical studies, such as promoting the growth of Bifidobacterium in human intestine and balance of intestinal bacteria, modulating the immune response, inhibition of cancer and tumor, stimulation of mineral absorption. In this study the effect of processing unit operations on the levels of soybean oligosaccharides during production of soybean sheet were investigated. The concentrations of oligosaccharide in initial raw soybean were: sucrose 43.05 g/kg, raffinose 7.52 g/kg and stachyose 41.32 g/kg (in dry matter). Oligosaccharide losses in the soaking water, in the first filtrating stage, in the second filtrating stage and finally in the sheet formation stage were 0.68, 10.3, 8.15 and 47.22 g/kg (initial dry soybean) respectively, representing 0.74, 11.21, 8.87 and 51.39% of the total oligosaccharides present in the initial soybeans. The recovery of oligosaccharides in the final soybean sheet from the initial soybean was 27.92%. The loss of soybean oligosaccharides in different processing stages, especially in the by-product, the sweet slurry, was considerable. The loss of oligosaccharides was mainly associated with water/matter removal in production process. The analysis of loss profile implied possible ways to improve the technology for production of oligosaccharides-enriched soy-sheets.”, “author” : { “dropping-particle” : “”, “family” : “Wang”, “given” : “Qiushuang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ke”, “given” : “Leqin”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Dongmei”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bao”, “given” : “Bili”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Jiang”, “given” : “Jianmei”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ying”, “given” : “Tiejin”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Asia Pacific Journal of Clinical Nutrition”, “id” : “ITEM-1”, “issue” : “SUPPL.1”, “issued” : { “date-parts” : “2007” }, “page” : “89-94”, “title” : “Change in oligosaccharides during processing of soybean sheet”, “type” : “article-journal”, “volume” : “16” }, “uris” : “http://www.mendeley.com/documents/?uuid=fd26f336-688f-4f72-8bb3-77c2a577e37f” } , “mendeley” : { “formattedCitation” : “(Wang et al., 2007)”, “plainTextFormattedCitation” : “(Wang et al., 2007)”, “previouslyFormattedCitation” : “(Wang et al., 2007)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Wang et al., 2007). According to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.3390/molecules17010753”, “ISSN” : “14203049”, “PMID” : “22245942”, “abstract” : “Okara is a byproduct generated during tofu or soymilk production processes. Crude polysaccharide (yield 56.8%) was isolated by removing fat, protein and low molecular weight carbohydrates from initial okara. Crude okara polysaccharide was further divided into four soluble fractions and an insoluble residue fraction by extracting with 0.05 M EDTA + NH(4) oxalate, 0.05 M NaOH, 1 M NaOH and 4 M NaOH, with yields of 7.7%, 3.6%, 20.7%, 16.0% and 27.9%, respectively. Arabinose, galactose, galacturonic acid, xylose and glucose (only for the insoluble fraction) were the major constituent sugars. The primary sugar residues of okara polysaccharides were 1,4-linked u03b2-galactopyranose, 1,5- and 1,3-linked u03b1-arabinofuranose, 1,5-linked u03b1-xylofuranose, 1,2-linked, 1,2,4-linked and terminal u03b1-rhamnopyranose (or fucopyranose), and 1,4-linked u03b2-glucopyranose (only for the insoluble fraction), indicating okara polysaccharides might contain galactan, arabinan, arabinogalactan, xylogalacturonan, rhamnogalacturonan, xylan, xyloglucan and cellulose.”, “author” : { “dropping-particle” : “”, “family” : “Li”, “given” : “Bo”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lu”, “given” : “Fei”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Nan”, “given” : “Haijuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Liu”, “given” : “Yang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Molecules”, “id” : “ITEM-1”, “issue” : “1”, “issued” : { “date-parts” : “2012” }, “page” : “753-761”, “title” : “Isolation and structural characterisation of okara polysaccharides”, “type” : “article-journal”, “volume” : “17” }, “uris” : “http://www.mendeley.com/documents/?uuid=9d6aa294-b05b-4aec-80b2-fbad977f7763” } , “mendeley” : { “formattedCitation” : “(B. Li, Lu, Nan, & Liu, 2012)”, “manualFormatting” : “B. Li, Lu, Nan, & Liu, (2012)”, “plainTextFormattedCitation” : “(B. Li, Lu, Nan, & Liu, 2012)”, “previouslyFormattedCitation” : “(B. Li, Lu, Nan, & Liu, 2012)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }B. Li, Lu, Nan, ; Liu, (2012), authors such as Mateos-Aparicio et al. (2010) and Tsubaki et al. (2009) also reported arabinose and galacturonic acid as the major sugars.
2.12 High Pressure Liquid ChromatographyHigh Pressure Liquid Chromatography is an equipment where a mixed sample is separated into components for identification, quantification and purification of mixtures. It pumps a sample mixture of analyte in a solvent (mobile phase) at high pressure through a column with chromatographic packing material (stationary phase). A little amount of sample is being introduced to the stream of the mobile phase and then sample molecules are retained by a specific chemical and physical interactions with the materials of the stationary phase as it travels the length of the column. The mobile phase must be degassed to eliminate the formation of bubbles. The equipment consists of a high pressure solvent delivery system, a sample auto injector, a separation column, a detector and a computer to control the system and display the result.

CHAPTER 3METHODOLOGY3.1 IntroductionThe soya bean waste was obtained in a soya milk stall in Johor Bahru. The determination of sugar yield of soya bean waste was done by using enzymatic hydrolysis method. The highest sugar yield of soya bean waste can be obtained by adjusting some of the parameters of enzymatic hydrolysis such as the concentration of the enzyme used and the temperature during the experiment. The chemical properties of the soya bean waste such as proximate analysis were determined. Analytical methods such as phenol-sulphuric acid, dinitrosalicyclic acid and high performance liquid chromatography were used to determine the sugar yield obtained from the soya bean waste. The summary detail of the process flow of this study was showed in the figure below (Figure 11).

center257175Soya bean waste
( Waste from soya milk )
00Soya bean waste
( Waste from soya milk )

2838450433705
405765029083013239752863851314449294640
495300120016Proximate analysis
i) Moisture content
ii) Ash
iii) Crude Protein
iv) Crude fat and oil
v) Carbohydrates
0Proximate analysis
i) Moisture content
ii) Ash
iii) Crude Protein
iv) Crude fat and oil
v) Carbohydrates
3161665128270Enzymatic hydrolysis
(alpha-amylase)
00Enzymatic hydrolysis
(alpha-amylase)

4048125277495
248782778946Parameter used
i) Enzyme concentration (v/w)
(0.7 %, 1.35 % and 2 %)
ii) Temperature
(50?, 55?, 60?)
iii) pH: 5.5
iv) Hydrolysis time: 24 hours
00Parameter used
i) Enzyme concentration (v/w)
(0.7 %, 1.35 % and 2 %)
ii) Temperature
(50?, 55?, 60?)
iii) pH: 5.5
iv) Hydrolysis time: 24 hours

4063313290968
255373083751Analytical methods
i) Total carbohydrate
( Phenol sulphuric acid )
ii) Total reducing sugar
( Dintrosalicylic acid solutions)
iii) Analysis of monosaccharide and
oligosaccharide
( High Performance Liquid Chromatography )
iv)Determination of functional group(Fourier transform infrared spectroscopy)00Analytical methods
i) Total carbohydrate
( Phenol sulphuric acid )
ii) Total reducing sugar
( Dintrosalicylic acid solutions)
iii) Analysis of monosaccharide and
oligosaccharide
( High Performance Liquid Chromatography )
iv)Determination of functional group(Fourier transform infrared spectroscopy)

Figure SEQ Figure * ARABIC 11: Summary details of the process flow for the studies3.2 Material, Equipment, Apparatus and chemicals3.2.1 Raw materialThe soya bean waste is the main material that will be used in the study. It was obtained from the soya milk stall in pasar malam at Century Garden, Johor Bahru. The owner soaked the soya bean with water in 10 hours in the ratio of 0.8: 2 and grind into the slurry using grinding machine. The soya bean waste that being produced will be placed into the grinding machine to squeeze out all the soya milk.
3.2.2 Equipment and apparatusThe equipments that will be used in this experiment are weighing balance, drying oven, soxhlet, muffle furnace, Kjeldahl method, reflux condenser, UV spectrophotometer and high performance liquid chromatography (HPLC).

The apparatus being used in this experiment are crucible, measuring cylinder, beakers, pipette, test tube, spatula and Erlenmeyer flask.

3.2.3 Chemicals usedThe chemicals used in this experiment are sulphuric acid, phenol, Kjedahl tablet, glucose solution, dinitrosalicylic acid, sodium hydroxide, sodium potassium tartrate and enzyme alpha-amylase.

3.3 Sample preparationThe sample was frozen in the freezer (-18?). It will undergo thawing method where it will be thawed in the chiller of 4? for one day.

3.4 Proximate analysis
Proximate analysis is used to determine the composition such as moisture content, ash, crude protein, crude lipid and carbohydrate in soya bean waste. All determination method is based on the Association of Official Analytical Chemists (AOAC) standard (1995). All the proximate values will be calculated in percentage. The determination of moisture content and ash will be based on the difference of the weight of the sample. The protein is determined by the Kjeldahl method where 6.25 is the factor to use to convert nitrogen into crude protein. The crude lipid will be determined by Soxhlet. The carbohydrate of the sample is determined by difference method.

3.4.1 Moisture contentThe moisture content of the sample is determined by loss in weight where the sample is dried in the oven until a constant weight is achieved. The crucible dish is weighed and 5 g of the sample is added to the crucible dish. The crucible with the sample is dried in the oven for 105? for 24 hours. The crucible dish was taken out, cooled at the desiccator and weighted. The amount of water loss during the drying process was determined from the difference in weight. The formula of calculating the moisture content is shown as below.

Moisture content %=weight of sample+dishbefore drying-weight of sample+dishafter dryingweight of sample+dishbefore dryingx100%3.4.2 AshAsh content of the sample is determined by burning the organic matter at 400? to 600? in the muffle furnace for 2 hours. The crucible dish is weighed and 2 g of the sample is added to the crucible dish. The crucible with the sample is dried in the muffle furnace at 600? for 2 hours. The crucible dish was taken out, cooled in the desiccator and weighted.

Ash content %=weight of sample+dishbefore drying-weight loss on ashingweight of sample+dishbefore dryingx100%3.4.3 Crude proteinThe crude protein of the soya bean sample was determined using Kjeldahl method. 1 g of the sample was weighed and placed in the Kjeldahl flask. Half of the Kjeldahl tablet and 12 ml of concentrated sulphuric acid (H2SO4) were mixed into the Kjeldahl flask. Immediately connect the Kjeldahl flask to the distilling bulb of the distillation apparatus immediately. The flask was heated until all ammonia had passed over into the standard acid. The same procedure was repeated for blank. The blank contains half of the Kjeldahl tablet and 12 ml of concentrated sulphuric acid. The factor used to convert nitrogen into crude protein was 6.25.

3.4.4 Crude lipid
The crude lipid of the soya bean sample was determined using the Soxhlet extraction with the Soxhlet extraction machine. Boiling thimble was dried at 105? for an hour and cooled to room temperature. The initial weight of extraction thimble was weighed and 2.0 g of sample was put into the thimble before transferring to the thimble stand handler. 90 ml of the hexane was put into the dried boiling flask. The extraction thimble was plugged tightly and inserted into the system. The system was started automatically with the first steps of boiled the sample for 15 minutes, then extracted at 155? for 45 minutes. The sample is then cooled at 30? for 1 minutes. The thimble was removed carefully and collected the hexane from the top container. The flask was dried in the oven at 105? for 1 hour. It was weighted after cooled to room temperature.
Fat content %=weight of fatweight of samplex100%3.4.5 CarbohydrateThe carbohydrate of the soya bean sample can be determined by subtracting the total percentage of moisture content, crude protein, crude lipid and ash from the sample.

Carbohydrate content %=100%-moisture content+crude protein+crude lipid+ash % sample3.5 Enzymatic hydrolysis3.5.1 Enzyme activity of enzyme alpha-amylaseThe alpha-amylase enzyme also known as alpha amylase enzyme is a food grade enzyme complex obtained by controlled fermentation of Aspergillus orgae. The enzyme activity for enzyme amylase in this experiment is 800 FAU/g.  The activity unit (FAU) which is defined as the amount which will catalyze the transformation of 1 micromole of the substrate per minute under standard conditions. Alpha amylase are a group of enzyme that hydrolyze starch. It able to cleaves internal ?-1,4- glycosidic linkage in starch to produce glucose, maltose and dextrins. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.ab.2006.01.036”, “ISBN” : “0003-2697 (Print)\r0003-2697 (Linking)”, “ISSN” : “00032697”, “PMID” : “16500607”, “abstract” : “Alpha-amylase (EC 3.2.1.1), which cleaves internal ?-1,4- glycosidic linkages in starch to produce glucose, maltose, or dextrins, and glucoamylase (EC 3.2.1.3), which cuts ?-1,4- and ?-1,6-glycosidic linkages to release glucose from the nonreducing ends of starch, are widely used in the indus- trial conversion of starch into sugars. The characterization of ?-amylases and glucoamylases generally needs to use diVerent chromatography techniques such as paper chro- matography 1,2, high-performance liquid chromatogra- phy 3,4, and thin-layer chromatography 5,6. There are mainly two types of assays that are used to determine the activity of ?-amylase and glucoamylase. One is based on measuring the amount of reducing sugars by the dinitrosal- icylic acid (DNS)2 assay 2,4,5,7u201310 or the Nelsonu2013Somo- gyi 1,11,12 method, whereas the other is based on the decreased staining value of blue starchu2013iodine complexes 13. The second method, which was developed by Fuwa 13 and is widely used 10u201312,14u201316, is based on color development that results from iodine binding to starch polymers. However, the starchu2013iodine assays reported by diVerent researchers are quite diverse with iodine concen- trations ranging from 3?M 12 to 0.25mM 15 and with the wavelength used to measure color development varying from 550nm 15 to 700nm 13. Moreover,”, “author” : { “dropping-particle” : “”, “family” : “Xiao”, “given” : “Zhizhuang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Storms”, “given” : “Reginald”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Tsang”, “given” : “Adrian”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Analytical Biochemistry”, “id” : “ITEM-1”, “issue” : “MAY 2006”, “issued” : { “date-parts” : “2006” }, “page” : “146-148”, “title” : “A quantitative starch u2013 iodine method for measuring alpha-amylase and glucoamylase activities”, “type” : “article-journal”, “volume” : “362” }, “uris” : “http://www.mendeley.com/documents/?uuid=86367667-e0e4-4656-ba41-329f8efe0285” } , “mendeley” : { “formattedCitation” : “(Xiao, Storms, & Tsang, 2006)”, “plainTextFormattedCitation” : “(Xiao, Storms, & Tsang, 2006)”, “previouslyFormattedCitation” : “(Xiao, Storms, & Tsang, 2006)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Xiao, Storms, ; Tsang, 2006)
3.5.2 Concentration of enzyme The enzymatic hydrolysis method was modified from the method of ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.biortech.2018.02.054”, “ISSN” : “09608524”, “author” : { “dropping-particle” : “”, “family” : “Islam”, “given” : “S.M. Mahfuzul”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Loman”, “given” : “Abdullah A.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Ju”, “given” : “Lu-Kwang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Bioresource Technology”, “id” : “ITEM-1”, “issue” : “February”, “issued” : { “date-parts” : “2018” }, “page” : “438-445”, “publisher” : “Elsevier”, “title” : “High monomeric sugar yields from enzymatic hydrolysis of soybean meal and effects of mild heat pretreatments with chelators”, “type” : “article-journal”, “volume” : “256” }, “uris” : “http://www.mendeley.com/documents/?uuid=2f633984-f2ea-480f-87fd-0c250d998f09” } , “mendeley” : { “formattedCitation” : “(Islam, Loman, & Ju, 2018)”, “manualFormatting” : “Islam et al., (2018)”, “plainTextFormattedCitation” : “(Islam, Loman, & Ju, 2018)”, “previouslyFormattedCitation” : “(Islam, Loman, & Ju, 2018)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Islam et al., (2018), Montilha et al., (2017) and ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “abstract” : “The experiment was conducted to determine the optimal concentration Naphthalene Acetic Acid (NAA) on the growth and yield of two rice varieties MTL560 and IR50404. The experiments were carried out in two factors completely randomized design with four replications per treatment. The first factor included two rice varieties (MTL560 and IR50404) and the second factor included four levels of NAA (0, 50, 100, 200 ppm). The results show that NAA was not effective on plant height, number of tillers.pot-1, number of panicles.pot-1, filled grain ratio (%), 1000-grain weight. However, NAA was able to improve the number full grains.panicle-1 and grain yield. The highest grain yield (23.7 g.pot-1) was observed in 100 ppm NAA. The yield of MTL560 rice (23.4 g.pot-1) was significantly higher than IR50404 rice (23 g.pot-1).”, “author” : { “dropping-particle” : “”, “family” : “Thi Luyen”, “given” : “Cao”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Binh”, “given” : “Ho Thanh”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Journal of Science u2013 Part C: Agricultural Sciences, Fisheries and Biotechnology”, “id” : “ITEM-1”, “issue” : “3”, “issued” : { “date-parts” : “2015” }, “page” : “138-148”, “title” : “Hydrolysis of Starch Using Alpha-Amylase and Glucoamylase During the Processing of Rice Milk From Some Rice Varieties in an Giang Province”, “type” : “article-journal”, “volume” : “3” }, “uris” : “http://www.mendeley.com/documents/?uuid=c45553e9-5819-4825-a4f1-59c237eecfae” } , “mendeley” : { “formattedCitation” : “(Thi Luyen & Binh, 2015)”, “manualFormatting” : “Thi Luyen & Binh (2015)”, “plainTextFormattedCitation” : “(Thi Luyen & Binh, 2015)”, “previouslyFormattedCitation” : “(Thi Luyen & Binh, 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Thi Luyen ; Binh (2015). Enzymatic hydrolysis will be conducted by using Erlenmeyer flask. 1.5 g of soya bean sample will be mixed with 30 ml of water. The mixture was kept in the shaking incubator at hydrolysis temperature which is 55?. Enzyme amylase was added. The enzymatic hydrolysis were performed at pH 5.5, 55?, 200 rpm and 24 hours at the shaking incubator. The procedure was repeated for various of enzyme concentration which is 0.7 %, 1.35 % and 2.0 % (modified from ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “abstract” : “The experiment was conducted to determine the optimal concentration Naphthalene Acetic Acid (NAA) on the growth and yield of two rice varieties MTL560 and IR50404. The experiments were carried out in two factors completely randomized design with four replications per treatment. The first factor included two rice varieties (MTL560 and IR50404) and the second factor included four levels of NAA (0, 50, 100, 200 ppm). The results show that NAA was not effective on plant height, number of tillers.pot-1, number of panicles.pot-1, filled grain ratio (%), 1000-grain weight. However, NAA was able to improve the number full grains.panicle-1 and grain yield. The highest grain yield (23.7 g.pot-1) was observed in 100 ppm NAA. The yield of MTL560 rice (23.4 g.pot-1) was significantly higher than IR50404 rice (23 g.pot-1).”, “author” : { “dropping-particle” : “”, “family” : “Thi Luyen”, “given” : “Cao”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Binh”, “given” : “Ho Thanh”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Journal of Science u2013 Part C: Agricultural Sciences, Fisheries and Biotechnology”, “id” : “ITEM-1”, “issue” : “3”, “issued” : { “date-parts” : “2015” }, “page” : “138-148”, “title” : “Hydrolysis of Starch Using Alpha-Amylase and Glucoamylase During the Processing of Rice Milk From Some Rice Varieties in an Giang Province”, “type” : “article-journal”, “volume” : “3” }, “uris” : “http://www.mendeley.com/documents/?uuid=c45553e9-5819-4825-a4f1-59c237eecfae” } , “mendeley” : { “formattedCitation” : “(Thi Luyen & Binh, 2015)”, “manualFormatting” : “Thi Luyen & Binh (2015)”, “plainTextFormattedCitation” : “(Thi Luyen & Binh, 2015)”, “previouslyFormattedCitation” : “(Thi Luyen & Binh, 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Thi Luyen ; Binh (2015) ). After that, the hydrolysate of soya bean waste were centrifuged at 9000 rpm for 10 minutes. The supernatant was kept in frozen (-35? ) for further analysis.

3.5.3 Temperature Enzymatic hydrolysis will be conducted by using Erlenmeyer flask. 1.5 g of soya bean sample will be mixed with 30 ml of water. The mixture was kept in the shaking incubator at hydrolysis temperature which is 55?. Various of enzyme concentration was added. The enzymatic hydrolysis were performed at pH 5.5, 200 rpm and 24 hours at shaking incubator. The procedure was repeated for various of hydrolysis temperature, which is 50?, 55?, and 60? (modified from ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1590/S1516-89132012000200002”, “ISBN” : “1516-8913 UL – http://www.scielo.br/scielo.php?script=sci_arttext&pid=S1516-89132012000200002&nrm=iso”, “ISSN” : “15168913”, “abstract” : “The enzymatic hydrolysis of food waste by commercially available enzymes and the subsequent ethanol fermentation of the hydrolysates by Saccharomyces cerecisiae H058 were studied in this work. The optimum batch enzymatic conditions were found to be saccharification , solid-liquid ratio of 1: 0.75 (w/w). Fed batch hydrolysis process was started with a solid-liquid ratio of 1: 1 (w/w), with solid fpH of 4.5, temperature of 55u2103, glucoamylase concentration of 120 u/g, u03b1-amylase concentration of 10 u/good waste added at time lapse of 2 h to get a final solid-liquid ratio of 1: 0.5 (w/w). After 4 h of reaction, the reducing sugar concentration reached 194.43 g/L with a enzymatic digestibility of 93.12%. Further fermentation of the batch and fed batch enzymatic hydrolysates, which contained reducing sugar concentration of 131.41 and 194.43 g/L respectively, was performed using Saccharomyces cerevisiae H058, 62.93 and 90.72 g/L ethanol was obtained within 48 h.”, “author” : { “dropping-particle” : “”, “family” : “Yan”, “given” : “Shoubao”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yao”, “given” : “Jianming”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yao”, “given” : “Liming”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhi”, “given” : “Zhijun”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Chen”, “given” : “Xiangsong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Wu”, “given” : “Jingyong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Brazilian Archives of Biology and Technology”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2012” }, “title” : “Fed batch enzymatic saccharification of food waste improves the sugar concentration in the hydrolysates and eventually the ethanol fermentation by saccharomyces cerevisiae H058”, “type” : “article-journal” }, “uris” : “http://www.mendeley.com/documents/?uuid=5abef9f5-d40f-32e0-aa71-cce9c93a4e38” } , “mendeley” : { “formattedCitation” : “(Yan et al., 2012)”, “manualFormatting” : “Yan et al., (2012)”, “plainTextFormattedCitation” : “(Yan et al., 2012)”, “previouslyFormattedCitation” : “(Yan et al., 2012)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Yan et al., (2012)). After that, the hydrolysate of soya bean waste were centrifuged at 9000 rpm for 10 minutes. The supernatant was kept in frozen (-35? ) for further analysis.

3.6 Total carbohydratePhenol sulphuric acid solutions allow measuring the concentration of total carbohydrate obtained as a result of enzymatic hydrolysis of soya bean waste. It is the most common assay for determination of total carbohydrate presented in the soya bean waste. An amount of 0.2 ml of sample solution was pipetted into a tube and add with distilled water until 1.0 ml. After that, 1.0 ml of the phenol solution was added to the solution. 5.0 ml of concentrated sulphuric acid was added rapidly into the mixture. After allowing the test tubes to stand for 10 min, they were vortexed for 30 s and placed for 20 min in a water bath at room temperature for color development. For the blank sample, 1.0 ml of distilled water was mixed with 1.0 ml of phenol solutions and 5.0 ml of sulphuric acid solution was added ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “abstract” : “Carbohydrates are an essential source of energy for the body to perform its normal functions. Having a diet that does not contain carbohydrate can lead to muscle breakdown, ketosis and dehydration. This can be prevented by taking 50 to 100 grams of carbohydrate per day. In the present work, quantitative estimation of total carbohydrate present in different dry fruits was done using phenol sulfuric acid method. This is a simple and rapid colorimetric method to determine total carbohydrate in a sample. Concentrated sulfuric acid breaks down all the polysaccharides, oligosaccharides and disaccharides to monosaccharide. Therefore this method determines total sugar present in a sample. In our study, the percentage of total carbohydrate present in cashew came out to be 30.0%, in peanut 24.0%, in almond 18.5%, in coconut 16.5% and in walnut 10.5%.Therefore, from cashew and peanut we get more carbohydrate as compared to almond, coconut and walnut to maintain the energy level of our body.”, “author” : { “dropping-particle” : “”, “family” : “Shri”, “given” : “Swami”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Saraswati”, “given” : “Swaroopanand”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bhilai”, “given” : “Mahavidyalya Hudco”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Agrawal”, “given” : “Neeru”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Minj”, “given” : “Divya K”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rani”, “given” : “Khushboo”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “IOSR Journal of Environmental Science Toxicology and Food Technology”, “id” : “ITEM-1”, “issue” : “6”, “issued” : { “date-parts” : “0” }, “page” : “2319-2402”, “title” : “Estimation of Total Carbohydrate Present In Dry Fruits”, “type” : “article-journal” }, “uris” : “http://www.mendeley.com/documents/?uuid=8c239334-3c9d-387a-ad0a-a8ec03028772” } , “mendeley” : { “formattedCitation” : “(Shri et al., n.d.)”, “plainTextFormattedCitation” : “(Shri et al., n.d.)”, “previouslyFormattedCitation” : “(Shri et al., n.d.)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Shri et al., n.d.). Afterwards, the colour intensity was measured with the spectrophotometer at wavelength 490 nm.
3.6.1 Preparation of arabinose standard curveDifferent concentration of arabinose solution is prepared. 0.2ml of the arabinose solution is being pipetted to different test tube respectively. Each test tube will fill with distilled water up to 1 ml of solution. 1.0 ml of the phenol solution was added to the solution. 5.0 ml of concentrated sulphuric acid was added rapidly into the mixture. After allowing the test tubes to stand for 10 min, they were vortexed for 30 s and placed for 20 min in a water bath at room temperature for color development. The colour intensity was measured with the spectrophotometer at wavelength 490 nm.

3.7 Total reducing sugar
Dinitrosalicylic acid solutions allow measuring the concentration of total reducing sugars obtained as a result of enzymatic hydrolysis of soya bean waste. It is the most common assay for determination of accumulation of reducing sugars. An amount of 0.2 ml of sample solution was pipetted into a tube. Afterwards, DNS reagent was added to have the total volume of the sample 2 ml. For the blank sample, 0.2 ml of water was measured and 2 ml of DNS reagent was added. All the tubes were placed in boiling water for exactly 10 minutes, transferred to ice to rapidly cool down and then brought to room temperature by placing them in the water bath at 25?. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.biombioe.2011.09.013”, “ISBN” : “3317780266”, “ISSN” : “09619534”, “abstract” : “3,5-Dinitrosalicylic acid (DNS) reagent is widely used in the estimation of reducing sugars. The reagent shows a differential behaviour towards mono- and di-saccharides. This phenomenon has been misinterpreted in the literature. Contrary to the facts, it has been reported that the DNS test is less sensitive for the estimation of cellobiose than it is for the estimation of glucose. This communication clarifies the concept. In addition, the study also compares the reaction of different mono- and di-saccharides and discusses the difference in their reactivity. u00a9 2011 Elsevier Ltd.”, “author” : { “dropping-particle” : “”, “family” : “Saqib”, “given” : “Abdul Aala Najmus”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Whitney”, “given” : “Philip John”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Biomass and Bioenergy”, “id” : “ITEM-1”, “issue” : “11”, “issued” : { “date-parts” : “2011” }, “page” : “4748-4750”, “publisher” : “Elsevier Ltd”, “title” : “Differential behaviour of the dinitrosalicylic acid (DNS) reagent towards mono- and di-saccharide sugars”, “type” : “article-journal”, “volume” : “35” }, “uris” : “http://www.mendeley.com/documents/?uuid=18a80029-429c-4870-931c-7bd64d583d77” } , “mendeley” : { “formattedCitation” : “(Saqib & Whitney, 2011)”, “plainTextFormattedCitation” : “(Saqib & Whitney, 2011)”, “previouslyFormattedCitation” : “(Saqib & Whitney, 2011)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Saqib ; Whitney, 2011). The initial bright yellow colour of the mixture of DNS reagent and sample turned into brown colour. The intensity of brown colour indicated the level of total reducing sugars in the sample. Afterwards, the colour intensity was measured with the spectrophotometer at wavelength 540 nm. (Miller 1959.).
3.7.1 Preparation of Dinitrosalicylic acid reagentDinitrosalicylic acid reagent is prepared using Coughlan and Monoley method. 1.5 g of the dinitrosalicyclic acid (DNS) and 75 g of sodium potassium tartrate was added to 20 ml of 0.5 N of sodium hydroxide and was gently heated to dissolve the reagent. The volume is then made up to 250 ml with distilled water.

3.7.2 Preparation of arabinose standard curveDifferent concentration of arabinose solution is prepared. 0.2ml of glucose solution is being pipetted to different test tube respectively. 2 ml of DNS reagent was added. All the tubes were placed in boiling water for exactly 10 minutes, transferred to ice to rapidly cool down and then brought to room temperature by placing them in the water bath at 25?. The colour intensity was measured with the spectrophotometer at wavelength 540 nm.

3.8 Experimental design and analysisIn this research study, Response Surface Methodology (RSM) was introduced by applying central composite design (CCD). Basically, two process parameters were focused to study the effect on the production of total carbohydrate and total reducing sugar. The results were analyzed by the software and expected to accomplish with an optimization value. An analysis of variance which known as (ANOVA) will use to identify the significance of the variables by computing the F value at p-value less than 0.5 while the fitness to the response was assessed using coefficient R2. A 3-D model was generated using the RSM software.

3.8.1 Central Composite Design (CCF)In this research study, total of two independent variable and two dependent variable were being determined. The two independent variables for this study are the hydrolysis temperature and concentration of enzyme used. The hydrolysis temperature to be studied were selected between 50°C – 60°C and the enzyme concentrations to be studied were 0.7% – 2.0%. With the aid of central composite design, a total of 13 runs were sequenced and constructed.
The tables below show the detailed element of independent and dependent variable, the factors with levels in the central composite design and the specific process parameters for each sequenced run. After that, statistical analysis, namely (Analysis of Variance) ANOVA was carried out to determine the optimization value by using Response Surface Methodology.

Table SEQ Table * ARABIC 5: Detailed elements of independent variables and dependent variablesType of variable Element Unit
Independent Hydrolysis temperature °C
Independent Concentration of enzyme %
Dependent Total carbohydrate mg/L
Dependent Total reducing sugar mg/L
Table SEQ Table * ARABIC 6: Specific factor with two levels in central composite designLevel -1.41 -1 0 +1 +1.41
Concentration of enzyme (%) 0.4 0.7 1.35 2.0 2.3
Hydrolysis temperature (°C) 48 50 55 60 62
Table SEQ Table * ARABIC 7: Specific process parameters for each sequenced runRun Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis Temperature (°C)
1 0.7(-1) 50(-1)
2 2(1) 50(-1)
3 0.7(-1) 60(1)
4 2(1) 60(1)
5 0.4(-1.41) 55(0)
6 2.3(+1.41) 55(0)
7 1.35(0) 48(-1.41)
8 1.35(0) 62(+1.41)
9 1.35(0) 55(0)
10 1.35(0) 55(0)
11 1.35(0) 55(0)
12 1.35(0) 55(0)
13 1.35(0) 55(0)
3.9 Analysis of monosaccharide and oligosaccharideAccording to Pascal, 2013, 1ml of sample solution was transferred into the High Performance Liquid Chromatography injection column. The conditions used for the analysis are:
Injection volume: 20 ?lMobile phase: distilled water
Flow rate: 0.3 ml/min
Column temperature: 55 ?Detector: Refractive index
Run time: 40 minutes
3.10 Fourier transform infrared spectroscopy (FT-IR) analysis
Fourier transform infrared spectrophotometry was used to examine the functional group of soya bean waste. The spectra were measured with a resolution over the range of 650 cm-1 to 4000 cm-1 using Fourier transform infrared spectrometer.

CHAPTER 4RESULTS AND DISCUSSIONS4.1 Proximate analysisProximate analysis is a quantitative analysis of the different macronutrients in a material that was developed in 1860 by Henneberg and Stohmann in Germany. This technique is used to estimate the amount of moisture content, ash, fat and oils, protein and carbohydrate that presented in the material. The result of the proximate analysis of soya bean waste is shown in the table below with 75.93 ± 0.01 % of moisture content, 0.59 ± 0.06 % of ash, 0.98 ± 0.06 % of fats and oils, 6.24 ± 0.13 % of protein and 16.26 ± 0.26 % of carbohydrate.

Table SEQ Table * ARABIC 8: Proximate analysis of soya bean wastePhysiochemical properties Concentration (%)
Moisture content 75.93 ± 0.01 %
Ash 0.59 ± 0.06 %
Fats and Oils 0.98 ± 0.06%
Protein 6.24 ± 0.13 %
Carbohydrate 16.26 ± 0.26 %
The moisture content of the soya bean waste in wet weight is 75.93 ± 0.01 %. This result reveals that the short shelf life of this material while fresh and long storage results in spoilage due its susceptibility of microbial infections. The presence of free moisture is directly related to water activity. The higher the water activity, the more susceptible the food will be interactions with microbes and its environment. 
The ash content of the soya bean waste is 0.59 ± 0.06 %. The ash content represents the mineral content of the okara. After the water is being removed from the soya bean waste by heating, the remaining inorganic residues are the ash, which represents the total amount of mineral presented in the material. The soya bean waste has similar ash content as reported in 0.4 % (Tseng et al., 2013) and 0.7 % ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1155/2013/423590”, “abstract” : “The production of soybean products has been increasing throughout the world, and there has been a corresponding increase in the quantity of soybean curd residue (SCR) being thrown out. The dumping of SCR has become a problem to be solved due to its contamination to the environment. SCR is rich in fiber, fat, protein, vitamins, and trace elements. It has potential for value-added processing and utilization; options that simultaneously hold the promise of increased economic benefit as well as decreased pollution potential for the environment. The objective of this study is to fully investigate, review, and summarize the existing literature in order to develop a comprehensive knowledge base for the composition and reuse of SCR. It is evident from the literature survey that SCR shows good potential as a functional food material. However, there are several drawbacks to the use of SCR and corresponding solutions presented in this paper.”, “author” : { “dropping-particle” : “”, “family” : “Li”, “given” : “Shuhong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhu”, “given” : “Dan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Kejuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Yingnan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lei”, “given” : “Zhongfang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhang”, “given” : “Zhenya”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bermejo”, “given” : “M D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rajabathar”, “given” : “J”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “ISRN Industrial Engineering”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2013” }, “publisher” : “Hindawi Publishing Corporation”, “title” : “Soybean Curd Residue: Composition, Utilization, and Related Limiting Factors”, “type” : “article-journal”, “volume” : “8” }, “uris” : “http://www.mendeley.com/documents/?uuid=5c761e1d-5a21-3cda-acba-4fc3eeeeaec7” } , “mendeley” : { “formattedCitation” : “(S. Li et al., 2013)”, “plainTextFormattedCitation” : “(S. Li et al., 2013)”, “previouslyFormattedCitation” : “(S. Li et al., 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(S. Li et al., 2013).
The protein content of the soya bean waste is 6.24 ± 0.13 %. The okara is considered a moderate source of protein. Soya bean waste contains about 4 – 7% proteins with good nutritional quality and superior protein efficiency ratio (PER), suggesting a potential source of low-cost vegetable protein for human consumption. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.4028/www.scientific.net/AMR.726-731.2908”, “ISSN” : “1662-8985”, “author” : { “dropping-particle” : “”, “family” : “Tseng”, “given” : “Yu Chang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Tseng”, “given” : “Hou Chia”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Weng”, “given” : “Yih Ming”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Advanced Materials Research”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2013” }, “page” : “2908-2916”, “title” : “Hydrolyzed Okara in Low-Fat Pork Burgers under Pilot Scale”, “type” : “article-journal”, “volume” : “726-731” }, “uris” : “http://www.mendeley.com/documents/?uuid=b6f7a0f3-c814-43da-b45e-41c57bf1efa7” } , “mendeley” : { “formattedCitation” : “(Tseng, Tseng, & Weng, 2013)”, “manualFormatting” : “(Tseng et al., 2013)”, “plainTextFormattedCitation” : “(Tseng, Tseng, & Weng, 2013)”, “previouslyFormattedCitation” : “(Tseng, Tseng, & Weng, 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Tseng et al., 2013).
The fat and oil content of the soya bean waste is 0.98 ± 0.06 %. Fat and oil content is an excellent source of energy and increase the transportation of fat soluble vitamins, insulate and protect internal tissues and even contribute to essential cell processes. The soya bean waste has similar crude lipid content as soybean curd residue (1.4%) ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1155/2013/423590”, “abstract” : “The production of soybean products has been increasing throughout the world, and there has been a corresponding increase in the quantity of soybean curd residue (SCR) being thrown out. The dumping of SCR has become a problem to be solved due to its contamination to the environment. SCR is rich in fiber, fat, protein, vitamins, and trace elements. It has potential for value-added processing and utilization; options that simultaneously hold the promise of increased economic benefit as well as decreased pollution potential for the environment. The objective of this study is to fully investigate, review, and summarize the existing literature in order to develop a comprehensive knowledge base for the composition and reuse of SCR. It is evident from the literature survey that SCR shows good potential as a functional food material. However, there are several drawbacks to the use of SCR and corresponding solutions presented in this paper.”, “author” : { “dropping-particle” : “”, “family” : “Li”, “given” : “Shuhong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhu”, “given” : “Dan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Kejuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Yingnan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lei”, “given” : “Zhongfang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhang”, “given” : “Zhenya”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bermejo”, “given” : “M D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rajabathar”, “given” : “J”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “ISRN Industrial Engineering”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2013” }, “publisher” : “Hindawi Publishing Corporation”, “title” : “Soybean Curd Residue: Composition, Utilization, and Related Limiting Factors”, “type” : “article-journal”, “volume” : “8” }, “uris” : “http://www.mendeley.com/documents/?uuid=5c761e1d-5a21-3cda-acba-4fc3eeeeaec7” } , “mendeley” : { “formattedCitation” : “(S. Li et al., 2013)”, “plainTextFormattedCitation” : “(S. Li et al., 2013)”, “previouslyFormattedCitation” : “(S. Li et al., 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(S. Li et al., 2013).

The analysis shown that the carbohydrate content in soya bean waste is 16.26 ± 0.26 %. The carbohydrate consist of complex carbohydrate, sugar and starch. Carbohydrates can act as energy storage molecules in our health. It is a good dietary fiber, which cannot be digested in the small intestine, but can be fermented by microbes in the large intestine. It is reported that dietary fiber in soya bean waste can reduce blood fat and blood pressure, lower the level of cholesterol in the blood, protect against coronary heart disease, and prevent the occurrence of constipation and colon cancer ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1155/2013/423590”, “abstract” : “The production of soybean products has been increasing throughout the world, and there has been a corresponding increase in the quantity of soybean curd residue (SCR) being thrown out. The dumping of SCR has become a problem to be solved due to its contamination to the environment. SCR is rich in fiber, fat, protein, vitamins, and trace elements. It has potential for value-added processing and utilization; options that simultaneously hold the promise of increased economic benefit as well as decreased pollution potential for the environment. The objective of this study is to fully investigate, review, and summarize the existing literature in order to develop a comprehensive knowledge base for the composition and reuse of SCR. It is evident from the literature survey that SCR shows good potential as a functional food material. However, there are several drawbacks to the use of SCR and corresponding solutions presented in this paper.”, “author” : { “dropping-particle” : “”, “family” : “Li”, “given” : “Shuhong”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhu”, “given” : “Dan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Li”, “given” : “Kejuan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Yang”, “given” : “Yingnan”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Lei”, “given” : “Zhongfang”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhang”, “given” : “Zhenya”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bermejo”, “given” : “M D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rajabathar”, “given” : “J”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “ISRN Industrial Engineering”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2013” }, “publisher” : “Hindawi Publishing Corporation”, “title” : “Soybean Curd Residue: Composition, Utilization, and Related Limiting Factors”, “type” : “article-journal”, “volume” : “8” }, “uris” : “http://www.mendeley.com/documents/?uuid=5c761e1d-5a21-3cda-acba-4fc3eeeeaec7” } , “mendeley” : { “formattedCitation” : “(S. Li et al., 2013)”, “plainTextFormattedCitation” : “(S. Li et al., 2013)”, “previouslyFormattedCitation” : “(S. Li et al., 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(S. Li et al., 2013).

4.2 Statistical analysis of total carbohydrate and total reducing sugar by enzymatic hydrolysisWith the Design Expert software, statistical analysis, namely (Analysis of Variance), ANOVA was carried out by using Response Surface Methodology (RSM) for the response of total carbohydrate and total reducing sugar by enzymatic hydrolysis that affected by temperature and concentration of enzyme. The effect of these two factors was evaluated and an empirical model was formed based on the relationship. The design chooses is a central composite design with 2 levels of all two independent variables (temperature and concentration of enzyme) and two dependent variable (total carbohydrate and total reducing sugar).

4.2.1 Model fitting of total carbohydrate
By employing a regression analysis for the experimental data, the predicted response Y for the yield of total carbohydrate could be obtained using the following second-order polynomial equation:
Y = 1948.19 + 172.70X1 +76.79X2 – 74.75 X1X2 – 166.26X21 – 197.97 X22 ,
Where Y represents the predicted yields of the total carbohydrate, X1 and X2 represent the independent variables of the enzyme concentration and hydrolysis temperature, respectively.

The results of ANOVA were represented in the table. The model showed high significant with a relatively high model F-value of 23.34. The lack of fit-F value of 1.30 showed that the lack of Fit is not significant relative to the pure error. This shows the model is a good fit with experimental data of total carbohydrate produced. In terms of adequate precision which measure the signal of noise, a ratio that is greater than 4 is always desirable and the ration of 12.78 shows the model is an adequate signal. So, this model can be used to investigate the design space. Relatively small of coefficient of variation (CV) which is 4.60% recommended good reproducibility of the investigation system. Furthermore, the high R2 value of 0.9434 indicates the model has less variation around the mean. The model of total carbohydrate being produced is extremely fit and able to use in the future for optimization of the model.
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Total carbohydrate produced (mg/L)
1 0.7 50 1345.00
2 2 50 1793.00
3 0.7 60 1611.00
4 2 60 1760.00
5 0.4 55 1268.00
6 2.3 55 1837.00
7 1.35 48 1382.00
8 1.35 62 1650.00
9 1.35 55 2009.00
10 1.35 55 2008.60
11 1.35 55 1839.20
12 1.35 55 1978.40
13 1.35 55 1902.00
Table SEQ Table * ARABIC 9: Analysis of variance (ANOVA) for optimization of total carbohydrate yield by the hydrolysis of okaraSource Sum of squares DF Mean square F value P value Model 733400 5 146700 23.34 0.0003 Significant
A 246700 1 246700 39.26 0.0004 Significant
B 46705.46 1 46705.46 7.43 0.0295 Significant
AB 22350.25 1 22350.25 3.56 0.1013 Not significant
A2 213000 1 213000 33.89 0.0006 Significant
B2 263400 1 263400 41.91 0.0003 Significant
Residual 43995.75 7 6285.11 Lack of fit 21726.36 3 7242.12 1.30 0.3895 Not significant
Pure of error 22269.39 4 5567.35 Cor total 777400 12 R2 0.9434 Adjusted R2 0.9030 Predicted R2 0.7559 Adequate decision 12.7829
Figure SEQ Figure * ARABIC 12: Response surface plot of temperature-enzyme concentration for the total carbohydrate yield4.2.2 Model fitting of total reducing sugar
By employing a regression analysis for the experimental data, the predicted response Y for the yield of total reducing sugar could be obtained using the following second-order polynomial equation:
Y = 674.25 + 52.03X1 + 64.65X2 – 39.37X1X2 + 13.03X21 + 110.93X22 ,
where Y represents the predicted yields of the total reducing sugar , X1 and X2 represent the independent variables of the enzyme concentration and hydrolysis temperature, respectively.

The results of ANOVA were represented in the table. The model showed high significant with a relatively high model F-value of 29.94. The lack of fit-F value of 3.94 showed that the lack of Fit is not significant relative to the pure error. This shows the model is a good fit with experimental data of total reducing sugar produced. In terms of adequate precision which measure the signal of noise, a ratio that is greater than 4 is always desirable and the ration of 16.8789 shows the model is an adequate signal. So, this model can be used to investigate the design space. Relatively small of coefficient of variation (CV) which is 4.14% recommended good reproducibility of the investigation system. Furthermore, the high R2 value of 0.9553 indicates the model has less variation around the mean. This model of total reducing sugar being produced is extremely fit and able to use in the future for optimization of the model.
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Total reducing sugar produced (mg/ml)
1 0.7 50 662.50
2 2.0 50 845.00
3 0.7 60 880.00
4 2.0 60 1105.00
5 0.4 55 602.50
6 2.3 55 755.00
7 1.35 48 752.50
8 1.35 62 950.00
9 1.35 55 660.00
10 1.35 55 709.10
11 1.35 55 665.00
12 1.35 55 660.00
13 1.35 55 675.00
Table SEQ Table * ARABIC 10: Analysis of variance (ANOVA) for optimization of total reducing sugar yield by the hydrolysis of okaraSource Sum of squares DF Mean square F value P value Model 144400 5 28882.83 29.94 0.0001 Significant
A 22392.01 1 22392.01 23.21 0.0019 Significant
B 33098.99 1 33098.99 34.31 0.0006 Significant
AB 6201.56 1 6201.56 6.43 0.0389 Significant
A2 1307.48 1 1307.48 1.36 0.2825 Not significant
B2 82700.40 1 82700.40 85.74 ? 0.0001 Significant
Residual 6752.20 7 964.60 Lack of fit 5046.36 3 1682.12 3.94 0.1091 Not significant
Pure of error 1705.85 4 426.46 Cor total 151200 12 R2 0.9553 Adjusted R2 0.9234 Predicted R2 0.7455 Adequate decision 16.8789
Figure SEQ Figure * ARABIC 13: Response surface plot of temperature-enzyme concentration for the total reducing sugar yield4.2.3 Relationship of temperature towards total carbohydrate and total reducing yield.Based on the both ANOVA, the hydrolysis temperature is significant to the total carbohydrate and total reducing sugar yield. Increasing the hydrolysis temperature can increase the yield of total carbohydrate and total reducing sugar yield. Total carbohydrate is interrelated with total reducing sugar as carbohydrate consists of complex carbohydrate, sugar and starch. At higher hydrolysis temperature, more energy has been provided for liquefaction to produce a higher amount of reducing sugars. This allows more collisions of substrate molecules per time. According to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “author” : { “dropping-particle” : “”, “family” : “Garba”, “given” : “Mohammed U”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mohammed”, “given” : “Abubakar”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Etim”, “given” : “Effiong D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “id” : “ITEM-1”, “issue” : “11”, “issued” : { “date-parts” : “2012” }, “page” : “65-70”, “title” : “A Kinetic Study of the Enzymatic Hydrolysis of Cassava Starch”, “type” : “article-journal”, “volume” : “1” }, “uris” : “http://www.mendeley.com/documents/?uuid=287315ea-bce5-4740-baf2-c9432712c705” } , “mendeley” : { “formattedCitation” : “(Garba, Mohammed, & Etim, 2012)”, “manualFormatting” : “Garba, Mohammed, & Etim (2012)”, “plainTextFormattedCitation” : “(Garba, Mohammed, & Etim, 2012)”, “previouslyFormattedCitation” : “(Garba, Mohammed, & Etim, 2012)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Garba, Mohammed, ; Etim (2012), increment of sugar yield occurs with increasing the temperature. Although that temperature can accelerate the production of total carbohydrate and total reducing sugar yield, prolong heating at high temperature would lead to the formation of sugar degraded products and resulted in a decline in reducing sugars and total carbohydrate yield.
4.2.4 Relationship of enzyme concentration towards total carbohydrate and total reducing yield.Based on the both ANOVA, the enzyme concentration is very significant to the total carbohydrate and slightly significant to total reducing sugar yield. The production of total carbohydrate and total reducing sugar is higher when the enzyme concentration increase. This is because more collisions occur between enzyme molecules and substrate molecules. Although the high enzyme concentration favored on the production of reducing sugar, the yield increment might not be sufficient for reducing sugar production at large scale. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “abstract” : “u2014 Glucose syrup is a concentrated aqueous solution of various sugars like glucose, maltose and other nutritive saccharides obtained from the hydrolysis of edible starch. Cassava is the preferred starch source since it is a high source of carbohydrate while being organic and free of pesticides. The purpose of this paper is to review the information available on the enzymatic hydrolysis of cassava and investigate the best possible conditions and source of enzyme that would give a better yield of glucose syrup. The hydrolysis of cassava using different sources of amylase enzymes like rice, maize, sorghum and wheat have been explored and optimum conditions for germination and subsequent hydrolysis have been analysed. The effect of temperature, pH, germinating period, malting period etc. has been studied and different methods for obtaining the best possible yield has also been reviewed”, “author” : { “dropping-particle” : “”, “family” : “Ramachandran”, “given” : “Veena”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Pujari”, “given” : “Nisha”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Matey”, “given” : “Tanmay”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kulkarni”, “given” : “Sunil”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Rice”, “given” : “A”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Journal of Science,Engineering and Technology Research”, “id” : “ITEM-1”, “issue” : “10”, “issued” : { “date-parts” : “2013” }, “page” : “1937-1942”, “title” : “Enzymatic Hydrolysis for Glucose-A Review”, “type” : “article-journal”, “volume” : “2” }, “uris” : “http://www.mendeley.com/documents/?uuid=28cd68f2-6ca7-4310-a5f7-c581ccd23701” } , “mendeley” : { “formattedCitation” : “(Ramachandran, Pujari, Matey, Kulkarni, & Rice, 2013)”, “manualFormatting” : “Ramachandran et al., (2013)”, “plainTextFormattedCitation” : “(Ramachandran, Pujari, Matey, Kulkarni, & Rice, 2013)”, “previouslyFormattedCitation” : “(Ramachandran, Pujari, Matey, Kulkarni, & Rice, 2013)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Ramachandran et al., (2013) reported that high enzyme dose showed highest increment of sugar yield as temperature rises.
4.2.5 Optimization of operating conditions on enzymatic hydrolysis on producing total carbohydrate and total reducing sugar.To get the best condition of production of sugar from okara by enzymatic hydrolysis, factors optimization will be the important element to maximize the production of sugar. The production of sugar is based on the temperature during hydrolysis and the enzyme concentration used. The objective of this study is to determine the optimum operating conditions on enzymatic hydrolysis.

Optimization of the operating conditions of enzymatic hydrolysis was carried out by maximizing the response using the Design Expert Software. As a result, by ANOVA, the best operating parameters were shown in table below. A little adjustment has been made in the optimized hydrolysis parameters of concentration of enzyme and temperature. After running the experiments at these optimal conditions (enzyme concentration: 1.3 % and 2 %, and hydrolysis temperature: 59? ), the experimental response of total carbohydrate and total reducing sugar was in agreement with the predicted values with small differences. The table below shows the optimal conditions of the hydrolysis parameters to yield the optimum carbohydrate and reducing sugar.

Table SEQ Table * ARABIC 11: Optimization conditions and predicted response values of hydrolysate okaraEnzyme concentration (%) Temperature (c) Total carbohydrate
(mg/L) Total reducing sugar (mg/L)
Predicted 1.362 59.141 1877.92 804.24
Experiment 1 1.35 59.0 1871.89 798.75
Difference (%) 0.32 0.68
Predicted 1.916 59.395 1829.73 841.84
Experiment 2 2.0 59.0 1830.78 836.89
Difference (%) 0.06 0.59

Figure SEQ Figure * ARABIC 14: Contour surface plot (experiment 1) with total carbohydrate yield
Figure SEQ Figure * ARABIC 15: Contour surface plot (experiment 2) with total carbohydrate yield
Figure SEQ Figure * ARABIC 16: Contour surface plot (experiment 1) with total reducing sugar yield
Figure SEQ Figure * ARABIC 17: Contour surface plot (experiment 2) with total reducing sugar yield4.3 Analysis of monosaccharide and oligosaccharideAccording to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1016/j.jchromb.2014.10.039”, “ISBN” : “5713165000”, “ISSN” : “1873376X”, “PMID” : “25462107”, “abstract” : “A High Performance Liquid Chromatography (HPLC) method was developed and validated to quantify sucrose (non-reducing sugar), glucose, and fructose (reducing sugars) in raw tubers of Solanum tuberosum Group Phureja. Chromatographic analysis was performed using an AMINEX HPX 87H column, at 18. u00b0C, linked to a refraction index detector, at 35. u00b0C. The eluent was 10. mM sulfuric acid. The conditions established for the method provided an optimum separation of sugars, citric acid, and malic acid, with resolution values higher or equal to one. Among the four sugar extraction methods tested, the double 50% (v/v) aqueous methanol extraction gave the highest level of analytes. Recovery of this extraction method ranged between 94.14 and 99.77%. The HPLC method was validated for repeatability, reproducibility, linearity, and limits of detection, and quantification. Relative standard deviation was found to be lower than five, when testing repeatability and reproducibility, which is suitable considering a range of acceptability from 5.3 to 7.3. Additionally, the regression analyses supported the method linearity in a range of quantification from 3 to 100. mg/L with regression coefficients values greater than 0.998 for the three analytes. Limits of detection were 3.0. mg/L for the three sugars and limits of quantification were 2.0. mg/L for sucrose and 3.0. mg/L for glucose and fructose. Four Colombian commercial cultivars (Criolla Guaneu00f1a, Criolla Paisa, Criolla Galeras, and Criolla Colombia) and five landrace accessions from the Colombian Core Collection of Group Phureja were grown in the district of Usme (Bogotu00e1) fields to analyze their sugar contents. Sucrose, glucose, and fructose contents were found ranging from 0.93 to 3.11. g/100. g tuber dried weight (DW), from 0.25 to 4.53. g/100. g tuber DW, and from 0.10 to 1.49. g/100. g tuber DW, respectively. Therefore, a high range in the variability of sugar contents was found among genotypes. However, the variability was low among technical replicates of the same genotype, revealing an accurate quantification of sugars in Group Phureja. This method can be used to assess the amount of reducing and non-reducing sugars accumulation in potato germplasm.”, “author” : { “dropping-particle” : “”, “family” : “Duarte-Delgado”, “given” : “Diana”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Narvu00e1ez-Cuenca”, “given” : “Carlos Eduardo”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Restrepo-Su00e1nchez”, “given” : “Luz Patricia”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kushalappa”, “given” : “Ajjamada”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mosquera-Vu00e1squez”, “given” : “Teresa”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2015” }, “page” : “18-23”, “publisher” : “Elsevier B.V.”, “title” : “Development and validation of a liquid chromatographic method to quantify sucrose, glucose, and fructose in tubers of Solanum tuberosum Group Phureja”, “type” : “article-journal”, “volume” : “975” }, “uris” : “http://www.mendeley.com/documents/?uuid=8e55f3cd-1f3a-4aa6-8967-975cb9371cc1” } , “mendeley” : { “formattedCitation” : “(Duarte-Delgado, Narvu00e1ez-Cuenca, Restrepo-Su00e1nchez, Kushalappa, & Mosquera-Vu00e1squez, 2015)”, “manualFormatting” : “Duarte-Delgado et al., (2015)”, “plainTextFormattedCitation” : “(Duarte-Delgado, Narvu00e1ez-Cuenca, Restrepo-Su00e1nchez, Kushalappa, & Mosquera-Vu00e1squez, 2015)”, “previouslyFormattedCitation” : “(Duarte-Delgado, Narvu00e1ez-Cuenca, Restrepo-Su00e1nchez, Kushalappa, & Mosquera-Vu00e1squez, 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Duarte-Delgado et al., (2015), chromatographic methods are the most powerful analytical techniques for the identification and quantification of monosaccharide and oligosaccharides in foods. High Performance Liquid Chromatography (HPLC) is currently the most common chromatographic method for analyzing these compounds as it is capable of rapid, specific, sensitive, and precise measurements.

Figure SEQ Figure * ARABIC 18: Production of sucrose in 50?, 55? and 60?
Figure SEQ Figure * ARABIC 19: Production of arabinose in 50?,55? and 60?From the analysis of High Performance Liquid Chromatography, the oligosaccharide contain in okara is sucrose whereas the monosaccharide containing in okara is arabinose. From figure 19 and 20, it is clearly shown that the production of sucrose and arabinose increase when the enzyme concentration increase. According to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1002/star.200900253”, “ISSN” : “00389056”, “author” : { “dropping-particle” : “”, “family” : “Mangat”, “given” : “Manraj”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kalra”, “given” : “Krishan Lal”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Kocher”, “given” : “Gurvinder Singh”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Phutela”, “given” : “Rajpal”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Sharma”, “given” : “Savita”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Starch/Staerke”, “id” : “ITEM-1”, “issue” : “12”, “issued” : { “date-parts” : “2010” }, “page” : “647-651”, “title” : “Comparative ethanol production for two corn varieties by commercial enzymes”, “type” : “article-journal”, “volume” : “62” }, “uris” : “http://www.mendeley.com/documents/?uuid=693ccf35-13af-4558-8424-c0f3268bffe4” } , “mendeley” : { “formattedCitation” : “(Mangat, Kalra, Kocher, Phutela, & Sharma, 2010)”, “manualFormatting” : “Mangat et al., (2010)”, “plainTextFormattedCitation” : “(Mangat, Kalra, Kocher, Phutela, & Sharma, 2010)”, “previouslyFormattedCitation” : “(Mangat, Kalra, Kocher, Phutela, & Sharma, 2010)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Mangat et al., (2010), the speed of reaction is directly proportional to the concentration of enzyme. According to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “author” : { “dropping-particle” : “”, “family” : “Garba”, “given” : “Mohammed U”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mohammed”, “given” : “Abubakar”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Etim”, “given” : “Effiong D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “id” : “ITEM-1”, “issue” : “11”, “issued” : { “date-parts” : “2012” }, “page” : “65-70”, “title” : “A Kinetic Study of the Enzymatic Hydrolysis of Cassava Starch”, “type” : “article-journal”, “volume” : “1” }, “uris” : “http://www.mendeley.com/documents/?uuid=287315ea-bce5-4740-baf2-c9432712c705” } , “mendeley” : { “formattedCitation” : “(Garba et al., 2012)”, “manualFormatting” : “Garba et al., (2012)”, “plainTextFormattedCitation” : “(Garba et al., 2012)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Garba et al., (2012), authors such as Ayenor et al. (2002) also found that increase in the concentration of enzyme enhances the level of sugar concentration in the cassava flour system .The highest sucrose production is on 55? with 2.3 % enzyme concentration which is 202.45 mg/L whereas the lowest sucrose production is on 50? with 2.0 % of enzyme concentration which is 136.24 mg/L. The highest arabinose production is on 60? , 2.0 % of enzyme concentration which is 1489.66 mg/L whereas the lowest sucrose production is on 50?, 0.7% enzyme concentration which is 693.65 mg/L.

Figure SEQ Figure * ARABIC 20: Production of sucrose and arabinose in optimization point (59?)From figure 21, it had showed the production of sucrose and arabinose of the optimum conditions chosen from Response Surface Methodology. For experiment 1, the production of sucrose and arabinose is 176.02 mg/L and 1243.13 mg/L respectively. For experiment 2, the production of sucrose and arabinose is 140.82 mg/L and 1291.91 mg/L.

By comparing the result from the total reducing sugar based on DNS method and HPLC method, it can be confirmed that HPLC is more accurate and precise than DNS method. HPLC have a better absorptivity onto either in the mobile phase and stationary phase. HPLC method can separate pure compound from the substances while the DNS method is analytical. ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “ISSN” : “1406894X”, “author” : { “dropping-particle” : “”, “family” : “Tihomirova”, “given” : “K.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Dalecka”, “given” : “B.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mezule”, “given” : “L.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Agronomy Research”, “id” : “ITEM-1”, “issue” : “5”, “issued” : { “date-parts” : “2016” }, “page” : “1713-1719”, “title” : “Application of conventional HPLC RI technique for sugar analysis in hydrolysed hay”, “type” : “article-journal”, “volume” : “14” }, “uris” : “http://www.mendeley.com/documents/?uuid=6d023871-f84b-437d-86d2-80b5008ef4d2” } , “mendeley” : { “formattedCitation” : “(Tihomirova, Dalecka, & Mezule, 2016)”, “manualFormatting” : “Tihomirova et al., (2016)”, “plainTextFormattedCitation” : “(Tihomirova, Dalecka, & Mezule, 2016)”, “previouslyFormattedCitation” : “(Tihomirova, Dalecka, & Mezule, 2016)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Tihomirova et al., (2016) proved that the sugar concentration from HPLC data are more than two times higher than the DNS data.ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1080/10942912.2013.837064”, “ISSN” : “15322386”, “abstract” : “ABSTRACTFructose, glucose, and sucrose in sugarcane molasses are determined simultaneously by high performance liquid chromatography (HPLC) using maltose as internal standard (IS) with refractive index detector (RID). The mixture of the diluted samples and IS was purified with Sep-Pak C18 solid-phase extraction (SPE) and filtered through a 0.22-?m membrane before injection. The results showed that the linear ranges for fructose, glucose and sucrose were 3.30-16.48 g/L, 1.80-9.02 g/L and 5.94-29.70 g/L with the squared correlation coefficients (R2) being 0.9986, 0.9987 and 0.9955, respectively. The method is simple, quantified, and time-saving for determination of sugars in sugarcane molasses. ABSTRACTFructose, glucose, and sucrose in sugarcane molasses are determined simultaneously by high performance liquid chromatography (HPLC) using maltose as internal standard (IS) with refractive index detector (RID). The mixture of the diluted samples and IS was purified with Sep-Pak C18 solid-phase extraction (SPE) and filtered through a 0.22-?m membrane before injection. The results showed that the linear ranges for fructose, glucose and sucrose were 3.30-16.48 g/L, 1.80-9.02 g/L and 5.94-29.70 g/L with the squared correlation coefficients (R2) being 0.9986, 0.9987 and 0.9955, respectively. The method is simple, quantified, and time-saving for determination of sugars in sugarcane molasses.”, “author” : { “dropping-particle” : “”, “family” : “Xu”, “given” : “Wanxia”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Liang”, “given” : “Lei”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Zhu”, “given” : “Mingjun”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “International Journal of Food Properties”, “id” : “ITEM-1”, “issue” : “3”, “issued” : { “date-parts” : “2015” }, “page” : “547-557”, “publisher” : “Taylor & Francis”, “title” : “Determination of sugars in molasses by HPLC following solid-phase extraction”, “type” : “article-journal”, “volume” : “18” }, “uris” : “http://www.mendeley.com/documents/?uuid=98626e87-4701-4c3e-99f1-539ab2e22bea” } , “mendeley” : { “formattedCitation” : “(Xu, Liang, & Zhu, 2015)”, “manualFormatting” : ” According to Xu et al., (2015)”, “plainTextFormattedCitation” : “(Xu, Liang, & Zhu, 2015)”, “previouslyFormattedCitation” : “(Xu, Liang, & Zhu, 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” } According to Xu et al., (2015) , authors such as Nejib et al., (2011) and Sims (1995) have reported that HPLC method as an established and preferred method for the determination of the individual sugars in carbohydrate mixtures, for its accuracy and simplicity.

4.4 Determination of functional group of hydrolyzate of soya bean waste by Fourier transform infrared spectroscopyFourier-transform infrared spectroscopy is a routine analytical technique. It can used to determine chemical compositions of food being quick and environmental friendly. Absorption bands in the infrared region are characteristic linkages and functional groups of a molecule. These absorption bands characterize a molecule state, including stretching or bending, so that the whole spectrum is a fingerprint of a specific compound or product ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.15835/nbha4319879”, “ISSN” : “18424309”, “abstract” : “Individual sugars were analyzed by high performance liquid chromatography (HPLC) in samples of apple juices obtained from the fruits of ‘Jonathan’, ‘Starkrimson’ and ‘Golden Delicious’ cultivars. Samples were harvested from the inside and the periphery of the crown, at different periods during fruits growth, from 7 to 144 days after full bloom (DAFB). Values from 0.42 to 14.33%, 0.29 to 4.06% and 0 to 4.28% were determined for fructose, glucose and sucrose, respectively. The values of fructose and glucose have increased significantly (p < 0.05), starting with the seventh DAFB, regardless of the studied cultivar, while sucrose increased slowly at the beginning and then faster starting 65 DAFB. Fourier transform mid-infrared (FT-MIR) analysis confirmed the differences between juice samples, the region 900-1500 cm-1 being the most specific to sugars signals. FT-MIR coupled to partial least squares (PLS) calibration models for predicting individual sugars of apple juices were developed. The optimal regions and pre-treatments of the spectra were 900-1500 cm-1 and Savitzky Golay first derivative (d1) for fructose, 900-1200 cm-1 and d2 for glucose and 900-1200 cm-1 and standard normal variate for sucrose. In cross-validation, the PLS calibration models showed very good performance for fructose (Rcval2=0.95; standard error of cross-validation (SECV) =0.907) and acceptable for glucose (Rcval2=0.85; SECV=0.424), while for sucrose showed only satisfactory performance (Rcval2=0.75; SECV=0.561). For practical relevance, the FT-MIR predicted values were compared against the HPLC determined reference values in external validations tests. The best results were achieved for fructose (p2=0.94; RPD=4.9), while glucose (p2=0.84; RPD=2.61) and sucrose (p2=0.7; RPD=2.08) models reached satisfactory values.”, “author” : { “dropping-particle” : “”, “family” : “Mureu015fan”, “given” : “Andruu0163a E.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Muste”, “given” : “Sevastiu0163a”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Vlaic”, “given” : “Romina A.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bobiu015f”, “given” : “Otilia”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mureu015fan”, “given” : “Crina”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Socaciu”, “given” : “Carmen”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mureu015fan”, “given” : “Vlad”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Notulae Botanicae Horti Agrobotanici Cluj-Napoca”, “id” : “ITEM-1”, “issue” : “1”, “issued” : { “date-parts” : “2015” }, “page” : “222-228”, “title” : “HPLC determination and FT-MIR prediction of sugars from juices of different apple cultivars during fruit development”, “type” : “article-journal”, “volume” : “43” }, “uris” : “http://www.mendeley.com/documents/?uuid=8213a805-34cd-4895-951b-b7e67662bb33” } , “mendeley” : { “formattedCitation” : “(Mureu015fan et al., 2015)”, “plainTextFormattedCitation” : “(Mureu015fan et al., 2015)”, “previouslyFormattedCitation” : “(Mureu015fan et al., 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Mure?an et al., 2015).

32880307810500
Figure SEQ Figure * ARABIC 21: Fourier-transform infrared spectroscopyFrom the graph above, it is noticed that the spectra showed strong and broad absorption at peak 3380cm-1. According to ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1063/1.4973109”, “ISBN” : “9780735414693”, “ISSN” : “15517616”, “author” : { “dropping-particle” : “”, “family” : “Patty”, “given” : “Diana Julaidy”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Loupatty”, “given” : “Grace”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Sopalauw”, “given” : “Fitria”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “AIP Conference Proceedings”, “id” : “ITEM-1”, “issued” : { “date-parts” : “2017” }, “page” : “1-7”, “title” : “Interpretation FTIR spectrum of seawater and sediment in the Ambon Bay (TAD)”, “type” : “article-journal”, “volume” : “1801” }, “uris” : “http://www.mendeley.com/documents/?uuid=37e0107b-43d3-4c8f-810f-e8ddf7cd4856” } , “mendeley” : { “formattedCitation” : “(Patty, Loupatty, & Sopalauw, 2017)”, “manualFormatting” : “Patty et al., (2017)”, “plainTextFormattedCitation” : “(Patty, Loupatty, & Sopalauw, 2017)”, “previouslyFormattedCitation” : “(Patty, Loupatty, & Sopalauw, 2017)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }Patty et al., (2017), for regions 3300 – 3450 cm-1, the functional group is alcohols, hydrogen bonded O-H and the peak intensity is strong and broad. A very broad peak in the region between 3100 and 3600 cm-1 indicates the presence of exchangeable protons, typically from alcohol, amine, amide or carboxylic acid group. Spectra showed strong absorption bands, characteristic to water molecules between 2800 – 3700 cm-1, indicating an apparent similarity to all solutions ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.15835/nbha4319879”, “ISSN” : “18424309”, “abstract” : “Individual sugars were analyzed by high performance liquid chromatography (HPLC) in samples of apple juices obtained from the fruits of ‘Jonathan’, ‘Starkrimson’ and ‘Golden Delicious’ cultivars. Samples were harvested from the inside and the periphery of the crown, at different periods during fruits growth, from 7 to 144 days after full bloom (DAFB). Values from 0.42 to 14.33%, 0.29 to 4.06% and 0 to 4.28% were determined for fructose, glucose and sucrose, respectively. The values of fructose and glucose have increased significantly (p < 0.05), starting with the seventh DAFB, regardless of the studied cultivar, while sucrose increased slowly at the beginning and then faster starting 65 DAFB. Fourier transform mid-infrared (FT-MIR) analysis confirmed the differences between juice samples, the region 900-1500 cm-1 being the most specific to sugars signals. FT-MIR coupled to partial least squares (PLS) calibration models for predicting individual sugars of apple juices were developed. The optimal regions and pre-treatments of the spectra were 900-1500 cm-1 and Savitzky Golay first derivative (d1) for fructose, 900-1200 cm-1 and d2 for glucose and 900-1200 cm-1 and standard normal variate for sucrose. In cross-validation, the PLS calibration models showed very good performance for fructose (Rcval2=0.95; standard error of cross-validation (SECV) =0.907) and acceptable for glucose (Rcval2=0.85; SECV=0.424), while for sucrose showed only satisfactory performance (Rcval2=0.75; SECV=0.561). For practical relevance, the FT-MIR predicted values were compared against the HPLC determined reference values in external validations tests. The best results were achieved for fructose (p2=0.94; RPD=4.9), while glucose (p2=0.84; RPD=2.61) and sucrose (p2=0.7; RPD=2.08) models reached satisfactory values.”, “author” : { “dropping-particle” : “”, “family” : “Mureu015fan”, “given” : “Andruu0163a E.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Muste”, “given” : “Sevastiu0163a”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Vlaic”, “given” : “Romina A.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Bobiu015f”, “given” : “Otilia”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mureu015fan”, “given” : “Crina”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Socaciu”, “given” : “Carmen”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Mureu015fan”, “given” : “Vlad”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Notulae Botanicae Horti Agrobotanici Cluj-Napoca”, “id” : “ITEM-1”, “issue” : “1”, “issued” : { “date-parts” : “2015” }, “page” : “222-228”, “title” : “HPLC determination and FT-MIR prediction of sugars from juices of different apple cultivars during fruit development”, “type” : “article-journal”, “volume” : “43” }, “uris” : “http://www.mendeley.com/documents/?uuid=8213a805-34cd-4895-951b-b7e67662bb33” } , “mendeley” : { “formattedCitation” : “(Mureu015fan et al., 2015)”, “plainTextFormattedCitation” : “(Mureu015fan et al., 2015)”, “previouslyFormattedCitation” : “(Mureu015fan et al., 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Mure?an et al., 2015).
Based on the graph, the small peak on range 1048 to 1216.50cm-1 can be determined as presence of functional group of sucrose, which is ether (C-O-C). According to Mure?an et al., (2015), characteristic absorption bands of sucrose are on peak 995, 1055, 1113 and 1138cm-1. According to Coates (2006), the peak on (1150 – 1050cm-1) represents the ether, C-O-C stretch. The particular frequency ranges from (1320–1000 cm-1) is C-O stretching, which is the presence of alcohols, carboxylic acids, esters, ethers ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “ISSN” : “09763333”, “abstract” : “Fourier Transform Infrared FT-IR Spectoroscopic Analysis of Spirulina”, “author” : { “dropping-particle” : “”, “family” : “Venkatesan”, “given” : “S”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Pugazhendy”, “given” : “K”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Sangeetha”, “given” : “D”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Vasantharaja”, “given” : “C”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Prabakaran”, “given” : “S”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Meenambal”, “given” : “M”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” } , “container-title” : “Journal of Medicinal Plants Studies”, “id” : “ITEM-1”, “issue” : “4”, “issued” : { “date-parts” : “2015” }, “page” : “30-32”, “title” : “Fourier Transform Infrared ( FT-IR ) Spectoroscopic Analysis of Spirulina”, “type” : “article-journal”, “volume” : “3” }, “uris” : “http://www.mendeley.com/documents/?uuid=12c1681d-8627-4e37-b9d0-055cb9406b17” } , “mendeley” : { “formattedCitation” : “(Venkatesan et al., 2015)”, “plainTextFormattedCitation” : “(Venkatesan et al., 2015)”, “previouslyFormattedCitation” : “(Venkatesan et al., 2015)” }, “properties” : { “noteIndex” : 0 }, “schema” : “https://github.com/citation-style-language/schema/raw/master/csl-citation.json” }(Venkatesan et al., 2015). For the control which is without enzyme, there is no peak in this range which proved that the enzyme did hydrolyze the sucrose from the soya bean waste sample.

Based on the graph, the spectra showed the absorption band at 1641cm-1. According to the infrared spectrum table, the peak at range (1740-1660cm-1 ) show the presence of a simple carbonyl compound such as a carboxylic acid or aldehyde ADDIN CSL_CITATION { “citationItems” : { “id” : “ITEM-1”, “itemData” : { “DOI” : “10.1002/9780470027318.a5606”, “ISBN” : “9780470027318”, “ISSN” : “00219606”, “PMID” : “4931998”, “abstract” : “The vibrational spectrum of a molecule is considered to be a unique physical property and is characteristic of the molecule. As such, the infrared spectrum can be used as a fingerprint for identification by the comparison of the spectrum from an u2018u2018unknownu2019u2019 with previously recorded reference spectra. This is the basis of computer-based spectral searching. 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The azomethines 3a-e were prepared from the corresponding aryl aldehydes and the acid hydrazide 2. Treatment of compound 2 with formic acid gave the N-formyl acid hydrazide 4, which upon refluxing with phosphorous pentoxide or phosphorous pentasulphide in xylene yielded the corresponding 2-(3-chloro-1-benzothien-2-yl)-1,3,4-oxadiazole (5) and 2-(3-chloro-1-benzo-thien-2-yl)-1,3,4-thiadiazole (6). Reaction of 1-thiosemicarbazide 7 with NaOH leads to ring closure giving 5-(3-chloro-1-benzothien-2-yl)-4Hu2013triazole-3-thiol (8) which is converted into a number of derivatives 9-12 Reaction of 2 with phenyl isothiocyanate and NaOH afforded 5-(3-chloro-1-benzothien-2-yl)-4-(phenyl)-4H-1,2,4-triazole-3-thiol (14).”, “author” : { “dropping-particle” : “”, “family” : “Sharba”, “given” : “A. Hussain K.”, “non-dropping-particle” : “”, “parse-names” : false, “suffix” : “” }, { “dropping-particle” : “”, “family” : “Al-Bayati”, “given” : “R. 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So, it can deduce that the hydrolyzate of soya bean waste contains the functional group of aldehyde which is the presence of arabinose.
CHAPTER 5CONCLUSION AND RECOMMENDATION5.1 ConclusionThe objective of this project is to investigate the effect of temperature and enzyme concentration towards the enzymatic hydrolysis of soya bean waste. The food material used in this project is soya bean waste, a food waste from soya bean and the enzyme used is enzyme fungal alpha amylase. The production of sugar can be said is directly proportional to the enzyme concentration. When the enzyme concentration is high, it will cleaves more glycosidic bonds in the substrate. At high temperature, more energy has been provided for enzyme to produce a higher amount sugars.

In this project, the second objective also had been achieved. From High Performance Liquid Chromatography, the monosaccharide and oligosaccharide of soya bean waste is arabinose (1243.13 mg/L and 1291.91 mg/L) and sucrose (176.20 mg/L and 149.82 mg/L) respectively. This also been confirmed by the Fourier Transform Infrared Spectroscopy, where an ester group that represents the functional group of sucrose and aldehyde group that represents the functional group of arabinose had been confirmed.

All of the objectives in this project had been achieved. This had encouraged further study on the potential of enzyme amylase on other types of food material. In overall, enzyme amylase able to hydrolyze the sucrose (oligosaccharide) and arabinose (monosaccharide) in high amount of yield.

5.2 Future recommendationsFor future studies, several recommendations and ideas are being proposed to continue this research:
Altering of hydrolysis parameter including the concentration of substrate, hydrolysis time and pH can be investigated to check on the significance of these independent variables over a wide range.

Comparison between several types of enzyme such as enzyme amyloglucosidase and enzyme beta-amylase for enzymatic hydrolysis.

Comparison study of total carbohydrate and total reducing sugar using different techniques such as subcritical water treatment and acid hydrolysis.

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CHAPTER 7APPENDIXAppendix A: Proximate analysis of soya bean waste sample
Table A1: Moisture content of soya bean waste sample
Sample 1 Sample 2
Sample before drying (g) 5.0097 g 5.0095 g
Sample after drying (g) 1.2065 g 1.2055 g
Moisture content (%) 75.92 % 75.94 %
Average moisture content (%) 75.93 ± 0.01 %
Table A2: Ash content of soya bean waste sample
Sample 1 Sample 2
Sample weight (g) 2.0399 2.0126
Sample weight loss during ashing (g) 2.0264 2.0020
Ash content (%) 0.6618 0.5267
Average ash content (%) 0.59 ± 0.06 %
Table A3: Crude protein content of soya bean waste sample
Sample 1 Sample 2
Crude protein content (%) 6.1134 % 6.3761 %
Average crude protein content (%) 6.24 ± 0.13 %
Table A4: Crude lipid content of soya bean waste sample
Sample 1 Sample 2
Weight of thimble (g) 44.8154 44.8262
Sample after drying + thimble (g) 45.2286 45.2902
Crude lipid content (%) 0.9222 % 1.0351 %
Average crude lipid content (%) 0.98 ± 0.06 %
Table A5: Carbohydrate content of soya bean waste sample
Sample 1 Sample 2
Carbohydrate content (%) 100 – 75.92 – 0.53 – 6.11 – 0.92 = 16.52 100 – 75.94 – 0.65 – 6.37 – 1.04 = 16.00
Average carbohydrate content (%) 16.26 ± 0.26 %
Appendix B: Standard curve with various concentration of glucose solution

Figure SEQ Figure * ARABIC 23: Calibration curve for determination of total carbohydrate by phenol sulphuric acid
Figure SEQ Figure * ARABIC 24: Calibration curve for determination of total reducing sugar by DNS methodAppendix C: Standard curve with various concentration of arabinose solution

Figure SEQ Figure * ARABIC 25: Calibration curve for determination of total carbohydrate by phenol sulphuric acid
Figure SEQ Figure * ARABIC 26: Calibration curve for determination of total carbohydrate by DNS methodAppendix D: Total carbohydrate and reducing sugar produced (Design Expert)
Appendix D1: Total carbohydrate of soya bean waste sample (based on glucose standard curve)
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Absorbance 1 Absorbance 2 Average absorbance Total carbohydrate produced (mg/L)
1 0.7 50 0.687 0.658 0.6725 336.25
2 2 50 0.916 0.877 0.8965 493.25
3 0.7 60 0.788 0.823 0.8055 402.75
4 2 60 0.867 0.893 0.8800 440.00
5 0.4 55 0.637 0.631 0.6340 317.00
6 2.3 55 0.854 0.983 0.9185 459.25
7 1.35 48 0.699 0.683 0.6910 345.50
8 1.35 62 0.799 0.851 0.8250 412.50
9 1.35 55 0.998 1.011 1.0045 502.25
10 1.35 55 0.986 1.023 1.0043 502.15
11 1.35 55 0.956 0.982 0.9691 484.55
12 1.35 55 1.006 0.972 0.9892 494.60
13 1.35 55 0.996 0.906 0.9510 475.50
Appendix D2: Total carbohydrate of soya bean waste sample (based on arabinose standard curve)
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Absorbance 1 Absorbance 2 Average absorbance Total carbohydrate produced (mg/L)
1 0.7 50 0.687 0.658 0.6725 1345.00
2 2 50 0.916 0.877 0.8965 1793.00
3 0.7 60 0.788 0.823 0.8055 1611.00
4 2 60 0.867 0.893 0.8800 1760.00
5 0.4 55 0.637 0.631 0.6340 1268.00
6 2.3 55 0.854 0.983 0.9185 1837.00
7 1.35 48 0.699 0.683 0.6910 1382.00
8 1.35 62 0.799 0.851 0.8250 1650.00
9 1.35 55 0.998 1.011 1.0045 2009.00
10 1.35 55 0.986 1.023 1.0043 2008.60
11 1.35 55 0.956 0.982 0.9691 1839.20
12 1.35 55 1.006 0.972 0.9892 1978.40
13 1.35 55 0.996 0.906 0.9510 1902.00
Appendix D3: Total reducing sugar of soya bean waste sample (based on glucose standard curve)
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Absorbance 1 Absorbance 2 Average absorbance Total reducing sugar produced (mg/L)
1 0.7 50 0.133 0.132 0.1325 120.45
2 2.0 50 0.168 0.150 0.1590 144.55
3 0.7 60 0.181 0.171 0.1760 160.00
4 2.0 60 0.191 0.171 0.1810 164.55
5 0.4 55 0.131 0.110 0.1205 109.55
6 2.3 55 0.154 0.148 0.1510 137.27
7 1.35 48 0.150 0.151 0.1505 136.82
8 1.35 62 0.209 0.175 0.1900 172.73
9 1.35 55 0.139 0.125 0.1320 120.00
10 1.35 55 0.156 0.128 0.1418 128.91
11 1.35 55 0.138 0.128 0.1330 120.91
12 1.35 55 0.139 0.125 0.1320 120.00
13 1.35 55 0.144 0.131 0.1350 122.73
Appendix D4: Total reducing sugar of soya bean waste sample (based on arabinose standard curve)
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Absorbance 1 Absorbance 2 Average absorbance Total reducing sugar produced (mg/L)
1 0.7 50 0.133 0.132 0.1325 662.50
2 2.0 50 0.168 0.170 0.1690 845.00
3 0.7 60 0.181 0.171 0.1760 880.00
4 2.0 60 0.191 0.171 0.1810 1105.00
5 0.4 55 0.131 0.110 0.1205 602.50
6 2.3 55 0.154 0.148 0.1510 755.00
7 1.35 48 0.150 0.151 0.1505 752.50
8 1.35 62 0.209 0.175 0.1900 950.00
9 1.35 55 0.139 0.125 0.1320 660.00
10 1.35 55 0.156 0.128 0.1418 709.00
11 1.35 55 0.138 0.128 0.1330 665.00
12 1.35 55 0.139 0.125 0.1320 660.00
13 1.35 55 0.144 0.131 0.1350 675.00
Appendix F: Standard curve of various sucrose concentration (HPLC)

Appendix G: Standard curve of various arabinose concentration (HPLC)

Appendix H: Production of sugar (HPLC) in hydrolysate soya bean waste sample
Appendix H1: Production of sucrose in soya bean waste sample
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Peak
area 1 Peak area 2 Average peak area Concentration of sucrose produced (mg/L)
1 0.7 50 143582 116511 130047 144.89
2 2.0 50 128363 116188 122276 136.24
3 0.7 60 125131 124968 125050 139.33
4 2.0 60 179865 160373 170119 189.54
5 0.4 55 163316 160428 161872 180.35
6 2.3 55 189502 173914 181708 202.45
7 1.35 48 118202 127987 123095 137.15
8 1.35 62 123724 110490 117107 130.48
9 1.35 55 172006 173167 172587 192.29
Appendix H2: Production of arabinose in soya bean waste sample
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Peak
area 1 Peak area 2 Average peak area Concentration of arabinose produced (mg/L)
1 0.7 50 199511 678622 439067 693.65
2 2.0 50 586034 576713 581374 918.47
3 0.7 60 470730 469248 469989 742.50
4 2.0 60 945978 939873 942926 1489.66
5 0.4 55 831538 725610 778574 1230.01
6 2.3 55 765716 818687 792202 1251.54
7 1.35 48 525361 543436 534399 844.26
8 1.35 62 587158 599234 593196 937.15
9 1.35 55 833305 917556 875431 1383.03
10 Control 50 199511 – 199511 315.19
11 Control 55 – – – –
12 Control 60 78146 – 78146 123.46
Appendix H3: Production of fructose in soya bean waste sample
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Peak
area 1 Peak area 2 Average peak area Concentration of fructose produced (mg/L)
1 Control 50 – – – –
2 Control 55 49116 – 49116 59.13
3 Control 60 76325 – 76325 91.89
Appendix H4: Production of sucrose in optimization sample
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Peak
area 1 Peak area 2 Average peak area Concentration of sucrose produced (mg/L)
1 1.35 59 152229 164064 158147 176.20
2 2.0 59 117250 135538 126394 140.82

Appendix H5: Production of arabinose in optimization sample
Run Factor 1
Concentration of enzyme (%) Factor 2
Hydrolysis temperature (°C) Peak
area 1 Peak area 2 Average peak area Concentration of arabinose
produced (mg/L)
1 1.35 59 850321 723434 786878 1243.13
2 2.0 59 893027 742477 817752 1291.91
Appendix I: Functional group on hydrolyzate soya bean waste by using FTIR
Sample Wavelength (cm-1) Wavelength range (cm-1) Functional group
Control 3380.53 Highest sugar yield based on RSM 3382.23 3300-3450 O-H
Experiment 1 3381.38 Experiment 2 3381.81 Sample Wavelength (cm-1) Wavelength range (cm-1) Functional group
Control – Highest sugar yield based on RSM 1047.55 1050-1250 C-O-C
Experiment 1 1048.07 Experiment 2 1216.50 Sample Wavelength (cm-1) Wavelength range (cm-1) Functional group
Control 1641.46 Highest reducing sugar yield based on RSM 1641.19 1740-1660 C=O-H
Experiment 1 1640.96 Experiment 2 1640.93 3516979131%T
0%T
45805975081710cm-100cm-1544243943434001641.46
1641.46
433753818405232138.96
2138.96

FTIR data from control sample (without enzyme)
548938043901461641.22
1641.22
687802714712461047.55
1047.55
151535444344003381.32
3381.32
430823018112152136.09
2136.09
45363435160939cm-100cm-15275487923%T
0%T

FTIR data from highest sugar yield in RSM
547760844401151640.53
01640.53
150612243989383380.73
3380.73
687558515034851048.07
1048.07
433705018312422136.02
2136.02
45720005152292cm-100cm-16154670338%T
0%T

FTIR data from experiment 1
605091513561161375.32
1375.32
653473619194581216.29
1216.29
150607444163273382.50
3382.50
548044144693261640.80
1640.80
431687719694772136.43
2136.43
45008805143158cm-100cm-17913170338%T
0%T

FTIR data from experiment 2