臺灣博碩士論文加值系統

米糠是碾米過程中被低度利用的副產物，它的價錢並不高。米糠含有許多碳水化合物和其他營養素。移除米糠中的油脂可得到脫脂米糠（defatted rice bran，DRB），它可以被保存相當長的一段時間。在本篇研究中，使用脫脂米糠作為基質，接種一株真菌(Aureobasidium pullulans NCH-218)以生產半纖維素酶。Aureo. pullulans NCH-218產生的酵素(如聚甘露醣酶和聚木醣酶)可部分水解DRB，而產生DRB水解物。本篇研究探討在固態發酵和水解處理過程中，獲得高酵素活性和高寡醣含量的最適條件。DRB水解物隨後接種Lactobacillus kefiranofaciens BCRC 16059，以生產克弗蘭多醣，探討培養基的初始pH值，初始接種量和發酵時間的影響。此外，也測定了克弗蘭多醣的單醣組成和分子量。
兩階段的固態發酵-水解處理方面，脫脂米糠的固態發酵最佳條件：水分含量為50％(v/w)，Aureo. pullulans NCH-218接種量為2％(v/w)，在30℃下培養72小時。聚木醣酶活性最高為85.62 ± 1.84 U/g脫脂米糠，聚甘露聚醣酶的活性最高為30.47 ± 1.72 U/g脫脂米糠。在50℃下水解6小時後，葡萄糖濃度最高為20.51 ± 0.43 mg/mL，半乳糖濃度最高為2.93 ± 0.07 mg/mL，木糖濃度最高為0.97 ± 0.05 mg/mL，木三糖濃度最高為11.24±1.01 mg/mL。此DRB水解物可用於培養L. kefiranofaciens BCRC 16059及生產克弗蘭多醣。
接種2％(v/v) L. kefiranofaciens BCRC 16059到初始pH值為6.0的DRB水解物，在37℃下培養5天可得到最高濃度(0.58 ± 0.02 mg/mL)的克弗蘭多醣。使用DRB水解物培養一星期可得到分子量範圍為1.47∼1.93×106 Da的克弗蘭多醣。葡萄糖和半乳糖為組成克弗蘭多醣之單醣。L. kefiranofaciens BCRC 16059在25°C， 30℃ 和37℃生產的克弗蘭多醣的葡萄糖：半乳糖的重量比分別為1：0.55，1：0.59和1：0.78。結果顯示DRB可用於培養L. kefiranofaciens BCRC 16059以生產克弗蘭多醣。

Rice bran is an inexpensive, underutilized by-product from rice milling process. It contains a number of carbohydrates and other nutrients. After removing oil from rice bran, the product is called defatted rice bran (DRB). It can be preserved and can be stored for a long period of time. In this study, defatted rice bran was used as a substrate for producing hemicellulases by a fungal cell, Aureobasidium pullulans NCH-218 was chosen. DRB was partially hydrolyzed by the enzymes produced from Aureo. pulllulans NCH-218, such as mannanase and xylanase resulting in DRB hydrolyzates. The optimum conditions for the highest enzymatic acitivities and oligosaccharides contents obtained during solid-state fermentation and hydrolysis treatment were investigated. The DRB hydrolyzates were subsequently inoculated by Lactobacillus kefiranofaciens BCRC 16059 and cultivated. Effects of initial pH of culture medium, inoculums size and fermentation time were studied using DRB hydrolyzates as the useful nutrient source for kefiran production of L. kefiranofaciens BCRC 16059. Moreover, monosaccharide composition and molecular weight of kefiran were measured.

In a two-stage treatment comprising of solid-state fermentation and subsequent hydrolysis, the optimal solid-state fermentation conditions of defatted rice bran were 50% water content (v/w), 2% (v/w) of Aureo. pullulans NCH-218 inoculum and cultured at 30℃ for 72 hours. The highest xylanase acitivity was 85.62 ± 1.84 U/g of defatted rice bran and mannanase activity was 30.47 ± 1.72 U/g of defatted rice bran. After hydrolysis at 50℃ for 6 hours, the highest concentration of glucose was 20.51 ± 0.43 mg/mL, galactose was 2.93 ± 0.07 mg/mL, xylose was 0.97 ± 0.05 mg/mL and xylotriose was 11.24 ± 1.01 mg/mL. This DRB hydrolyzate can be used for cultivating L.kefiranofaciens BCRC 16059 and producing kefiran.

An initial pH 6.0 of DRB hydrolyzate incubated with 2% (v/v) inoculum of L. kefiranofaciens BCRC 16059 at 37℃ for 5 days showed the highest production of kefiran with the yield of 0.58 ± 0.02 mg/mL. The molecular weights of kefiran during one week of incubation time were in the range of 1.47 ~ 1.93 × 106 Da. The mass ratio of glucose: galactose which is composed in kefiran produced by L. kefiranofaciens BCRC 16059 at 25, 30 and 37℃ were 1:0.55, 1:0.59 and 1:0.78, respectively. The results showed that DRB supported well on bacterial growth as well as kefiran production by L.kefiranofaciens BCRC 16059.

摘要 i
Abstract ii
Table of contents iv
List of Tables x
List of Figures xii
Chapter 1 Introduction 1
Chapter 2 Literature Review 2
2.1 Rice 2
2.1.1 Introduction of rice 2
2.1.2 Biological characteristics of rice 2
2.1.3 Structure and composition of rice 2
2.1.4 The uses of rice 4
2.2 Rice bran 4
2.2.1 Introduction of rice bran 4
2.2.2 Composition of rice bran 6
2.2.2.1 Chemical composition of rice bran 10
2.2.2.1.1 Protein 10
2.2.2.1.2 Carbohydrates 10
2.2.2.1.3 Lipids 11
2.2.2.1.4 Crude fiber 12
2.2.2.1.5 Vitamin 12
2.2.2.1.6 Minerals 12
2.3 Defatted rice bran 13
2.4 Solid-state fermentation (SSF) 14
2.4.1 Introduction of solid-state fermentation 14
2.4.2 Factors affecting solid state fermentation 14
2.4.2.1 Carbon source 14
2.4.2.2 Moisture and water activity in SSF 16
2.4.2.3 pH 16
2.4.2.4 Temperature and heat transfer 17
2.4.2.5 Mass transfer 17
2.4.3 Applications of solid-state fermentation 18
2.4.3.1 Production of enzymes by SSF 18
2.4.3.2 Production of organic acids under SSF 18
2.4.3.3 Secondary metabolites production under SSF condition 18
2.5 Aureobasidium pullulans 18
2.5.1 Introduction of Aureobasidium pullulans 18
2.5.2 Products from Aureobasidium pullulans 19
2.5.2.1 Enzymes 19
2.5.2.2 Pullulan 22
2.5.2.3 Single cell protein (SCP) 22
2.5.2.4 Siderophore 22
2.6 Lactic acid bacteria (LAB) 23
2.6.1 Introduction of lactic acid bacteria 23
2.6.2 Classification of lactic acid bacteria 23
2.6.3 Industrial use of lactic acid bacteria 24
2.7 Exopolysaccharides 24
2.7.1 Introduction of exopolysaccharides 24
2.7.2 Classification of exopolysaccharides 26
2.7.3 Structure and pathway of exopolysaccharides biosynthesis 26
2.7.4 Exopolysaccharides production in lactic acid bacteria 29
2.8 Kefiran 30
2.8.1 Characteristics of kefiran 30
2.8.2 Properties of kefiran 32
2.8.3 Kefiran production 33
2.8.4 Factors affecting kefiran production in lactic acid bacteria 34
2.8.4.1 Culture medium composition 34
2.8.4.1.1 Carbon source 34
2.8.4.1.2 Nitrogen source 34
2.8.4.2 Culture condition 35
2.8.4.2.1 pH 35
2.8.4.2.2 Temperature 35
2.8.4.2.3 Aeration 35
2.8.4.2.4 Ethanol addition 36
Chapter 3 Objectives and Experimental Design 37
3.1 Objectives 37
3.2 Experimental Designs 38
Chapter 4 Materials and Methods 39
4.1 Materials 39
4.1.1 Raw materials 39
4.1.2 Microorganisms 39
4.1.3 Culture media 39
4.1.4 Chemicals 40
4.1.5 Instruments 42
4.1.6 Software 42
4.2 Experimental methods 43
4.2.1 Sample preparation 43
4.2.2 Preparation of stock culture and cell activation 43
4.2.2.1 Mold 43
4.2.2.1.1 Stock culture 43
4.2.2.1.2 Inoculum preparation 43
4.2.2.2 Lactic acid bacteria 44
4.2.2.2.1 Stock culture 44
4.2.2.2.2 Inoculum preparation 44
4.2.3 Raw material composition determination 44
4.2.3.1 Moisture content 44
4.2.3.2 Crude protein content 45
4.2.3.3 Crude lipid content 46
4.2.3.4 Dietary fiber content 47
4.2.3.5 Ash content 48
4.2.3.6 Starch content 48
4.2.4 Defatted rice bran (DRB) 49
4.2.5 Two-step treatment of solid-state fermentation and hydrolysis 49
4.2.5.1 Solid-state fermentation (SSF) 50
4.2.5.1.1 Initial water content 50
4.2.5.2 Hydrolysis 51
4.2.5.2.1 Hydrolysis temperature 51
4.2.5.2.2 Hydrolysis time 51
4.2.6 Lactic acid bacteria fermentation 51
4.2.6.1 Initial pH 52
4.2.6.2 Inoculum size 52
4.2.6.3 Comparison with MSR broth 52
4.2.7 Kefiran production 53
4.3 Analytical methods 53
4.3.1 Xylanase activity analysis 53
4.3.2 Mannanase activity analysis 54
4.3.3 Amylase activity analysis 54
4.3.4 Reducing sugar analysis 55
4.3.5 Peptide contents 55
4.3.6 Isolation and purification of the exopolysaccharide 55
4.3.7 Determination of titratable acidity 56
4.3.8 Analysis of carbohydrate composition in DRB hydrolyzate 57
4.3.9 Analysis of monosaccharide composition in kefiran 57
4.3.10 Measurement of molecular weight of purified kefiran 58
Chapter 5 Results and Discussion 59
5.1 Proximate composition of rice bran 59
5.2 Solid state fermentation of defatted rice bran 59
5.2.1 Selection of the microbial enzyme producer 59
5.2.2 Effects of initial water content 63
5.3 Enzymatic hydrolysis 66
5.3.1 Effect of hydrolysis temperature 70
5.3.2 Effect of hydrolysis time 74
5.3.3 Defatted rice bran hydrolyzate 78
5.4 The use of DRB hydrolyzate for kefiran production by L. kefiranofaciens BCRC 16059 78
5.4.1 Effect of initial pH 78
5.4.2 Effect of initial inoculum size 88
5.4.3 Effect of incubation time 93
5.4.4 Comparison with Lactobacilli MRS broth 97
5.5 Study on the characteristics of kefiran 100
5.5.1 Molecular weight of kefiran 100
5.5.2 Monosaccharide composition of kefiran 105
Chapter 6 Conclusions 108
6.1 Hydrolytic enzymes-producing fungal cells 108
6.2 Solid-state fermentation 108
6.3 Enzymatic hydrolysis 108
6.4 Kefiran production using DRB hydrolyzate 109
6.5 Characterization of kefiran 109
Chapter 7 Future Prospects 110
Chapter 8 References 111
Appendix 1 HPLC chromatograms showing molecular weights of polymer standards 121
Appendix 2 Standard curve plotting molecular weights of polymer standards 122

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