Fermentation with pure and mixed cultures of Lactobacillus plantarum, Lactobacillus buchneri, Saccharomyces cerevisiae and Gluconacetobacter enhances the phytochemical content and biological activities of Momordica charantia

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羅傑(Rajan Jaiswal) 查詢紙本館藏

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化學工程與材料工程學系

論文名稱

(Fermentation with pure and mixed cultures of Lactobacillus plantarum, Lactobacillus buchneri, Saccharomyces cerevisiae and Gluconacetobacter enhances the phytochemical content and biological activities of Momordica charantia)

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摘要(中)

糖尿病、痛風等慢性疾病的患病率急劇上升，造成人們的過早死亡和巨大的經濟損失。據報導，用於治療此類疾病的化學合成現代藥物具有不良副作用，促使人們大力關注開發富含天然植物化學物質並引起輕微或無副作用的新型功能性食品和飲料。自古以來，使用微生物發酵技術對水果和蔬菜進行改良以增強營養功能、風味和保質期就很受歡迎。混合培養發酵有利於實現更高的生長速率、產品產量、更好的原料運用、有效的多步轉化和減少污染的機會。本次研究旨在利用已知乳酸菌、醋酸菌和酵母菌在混合培養中共存並共生，期能優化研究結果。
Momordica charantia (MC) 也稱為苦瓜，在世界不同地區廣泛種植，用作蔬菜和傳統藥物。苦瓜中不同植物化學物質的存在和治療應用推動了對其生物活性研究的巨大研究。然而，關於微生物發酵在利用苦瓜開發不同功能食品和飲料方面的應用研究有限。因此，在本研究中，我們嘗試使用混合培養發酵開發使用 MC 培養液和豆漿的非乳製飲料。使用植物乳桿菌和布氏乳桿菌的 MC 提取液混合培養厭氧發酵，在 30°C條件下添加 10% 豆漿，將 α- 澱粉酶抑制活性提高到 73.15%，將苦瓜苷含量提高到 14.2 ppm。發酵產生71.9%的DPPH自由基清除活性。在不同的培養溫度中，發現 30°C對 α-澱粉酶抑制、苦瓜苷含量和 DPPH 自由基清除活性為最佳。
在發酵的第 16 天，含有 10% 豆漿的 Gluconacteobacter 純培養物在 125 rpm 下產生 40.33% 的黃嘌呤氧化酶抑制 (XOI) 活性和 1215.34 ppb 的苦瓜苷含量。該結果表明，與未發酵的 MC 粉末相比，XOI 活性增加了 29.61%，Charantin 含量增加了 251.89 ppb。葡萄糖醋桿菌和釀酒酵母與 10% 豆漿的共培養在發酵的第 14 天產生 45.65% 的 XOI 活性。用豆漿多菌發酵葡萄糖醋桿菌、釀酒酵母和植物乳桿菌，發酵第4天XOI活性提高到47.38%。此外，進行了兩階段發酵，其中釀酒酵母、植物乳桿菌和布氏乳桿菌與 10% 豆漿在第一階段厭氧培養前 12 小時，然後添加葡糖醋桿菌，在第二階段進行好氧發酵階段。兩階段發酵在發酵第4天產生最高的XOI活性，達到63.57%，與純培養相比，XOI活性提高了23.24%，發酵時間縮短了12天。
本研究使用新的發酵策略成功地證明了苦瓜的生物活性和植物化學含量可以通過微生物發酵得到增強。結果表明，30°C的兩階段發酵對XOI活性的提升最為顯著，而植物乳桿菌和布氏乳桿菌的厭氧混合培養對促進α-澱粉酶抑制活性和提高苦瓜苷含量為最佳。培養溫度、加入接種物的比例和時間、添加豆漿均會影響微生物發酵的結果，因此需要進一步優化工藝的經濟可行性。

摘要(英)

The prevalence of lifestyle diseases including diabetes mellitus and gout has increased tremendously causing large number of untimely deaths and huge financial losses. Chemically synthesized modern medicines used for treatment of such ailments are reported to have adverse side effects driving huge attention to development of novel functional foods and beverages which are rich in natural phytochemicals and cause mild or null side effects. Modification of fruit and vegetables using microbial fermentation technologies to enhance nutritional functionality, flavor and shelf life has been popular since time immemorial. Mixed culture fermentation has been beneficial in achieving higher growth rate, product yield, better utilization of substrates, effective multistep transformation and reduced chances of contamination. Lactic acid bacteria, acetic acid bacteria and yeast have been known to co-exist and function symbiotically in mixed culture fermentation.
Momordica charantia [MC) also known as bitter melon or balsam pear is cultivated widely in different regions of the world for its consumption as vegetable and traditional medicines. The presence of different phytochemicals in M. charantia with therapeutic application has driven tremendous research on the study of the biological activities. However, there have been limited studies on the application of microbial fermentation for development of different functional food and beverages using M. charantia. Therefore, in this study we have attempted to develop non-dairy beverages using MC broth and soymilk using mixed culture fermentation. Mixed culture anaerobic fermentation of MC extract broth using L. plantarum and L. buchneri with the addition of 10% soymilk at 30oC increased the α- amylase inhibition activity to 73.15% and charantin content to 14.2 ppm. The fermentation resulted 71.9% DPPH radical scavenging activity. Of the different culture temperatures, 30 oC was found to be optimum for α-amylase inhibition, charantin content and DPPH radical scavenging activity.
Pure cultures of Gluconacteobacter fermented MC media broth with 10% soymilk at 125 rpm demonstrated 40.33% xanthine oxidase inhibition (XOI) activity and 1215.34 ppb charantin content on the 16th day of fermentation. These results showed XOI activity increment of 29.61% and charantin content increment of 251.89 ppb as compared to unfermented MC powder. Co-cultures of Gluconacetobacter and Saccharomyces cerevisiae fermented MC media broth with 10% soymilk produced 45.65 % XOI activity on 14th day of the fermentation. Multi-strain fermentation using Gluconacetobacter, Saccharomyces cerevisiae and L. plantarum fermented MC media broth with soymilk resulted XOI activity to 47.38% on the 4th day of fermentation. Furthermore, two stage fermentation was carried out in which Saccharomyces cerevisiae, L. plantarum and L. buchneri were cultured anaerobically in MC media broth with 10% soymilk for first 12 hours in the first stage followed by the addition of Gluconacetobacter and the fermentation carried out aerobically in the second stage. Two stage fermentation produced the highest XOI activity of 63.57% on the 4th day of the fermentation thereby showing XOI activity increment of 23.24% and shortening the fermentation time by 12 days as compared to pure culture.
This study used novel fermentation strategies to successfully demonstrate that the bioactivities and phytochemical content of M. charantia can be enhanced through microbial fermentation. The results indicated that the two stage fermentation at 30 oC was optimum for XOI activity while anaerobic mixed culture of L. plantarum and L. buchneri was optimum for promoting α- amylase inhibition activity as well as enhancing charantin content. Culture temperature, ratio and time of addition of the inoculum, addition of soymilk affected the outcomes of the microbial fermentation and hence required optimization for economic viability of the process.

關鍵字(中)

★ 也稱為苦瓜

關鍵字(英)

★ Momordica charantia
★ Mixed culture
★ charantin
★ coumarin
★ bioactivities

論文目次

Table of Contents
摘要 i
Abstract iii
Acknowledgement v
List of figures ix
List of tables xi
Abbreviations xii
Chapter 1. Background and research motivation 1
Chapter 2. Literature review 5
2.1 Momordica charantia 5
2.1.1 General introduction and geographical distribution 5
2.1.2 Traditional use of M. charantia 6
2.1.3 Nutritional content 7
2.1.4 Phytochemicals 8
2.1.5 Bioactivity of the phytochemicals in M. charantia 14
2.2 Type II Diabetes mellitus (T2DM) 17
2.2.1 Treatments of T2DM 18
2.2. 2 Charantin 21
2.3 Gout 23
2.3.1 Treatment of gout 24
2.3.2 Xanthine oxidase and XOI mechanism 25
2.3.3 Coumarin 26
2.4 Microbial fermentation to enhance phytochemicals and bioactivities 27
2.5 Basic introduction of the bacterial strain used in this study 28
2.5.1 Lactic acid bacteria and its role in biotransformation 28
2.5.2 Acetic acid bacteria 29
2.5.3 Saccharomyces cerevisiae 30
2.6 Soymilk 31
2.7 Fermentation strategies and selection of microorganisms for mixed culture fermentation 32
Chapter 3. Materials and methods 37
3.1 Momordica charantia collection and processing 43
3.2 Bacterial strains and maintenance 43
3.3 Inoculum preparation 48
3.4 Soymilk preparation 51
3.5 Antidiabetic bioactivity improvement fermentation 51
3.6 Anti-gout improvement fermentation 52
3.7 pH analysis 54
3.8 Analysis of bacterial growth 54
3.9 Reducing sugar concentration analysis 54
3.10 Ethanol and acetic acid concentration analysis 56
3.11 Lactic acid concentration analysis 57
3.12 Charatin concentration analysis 58
3.13 Coumarin concentration analysis 59
3.14 Antioxidant activity evaluation 60
3.14.1 Total phenolic content (TPC) 60
3.14.2 DPPH radical scavenging activity 61
3.15 Determination of α-amylase inhibition activity 62
3.16 Determination of xanthine oxidase inhibition (XOI) activity 62
3.17 Ultrasound assisted aqueous extraction 63
3.18 Statistical analysis 63
Chapter 4. Results and discussion 64
4.1 Growth curves of the bacterial strains used in this study 64
4.2 Ultrasound assisted aqueous extraction of MC 67
4.3 Effect of mixed culture of L. plantarum and L. buchneri on MC extract broth 68
4.4 Mixed culture fermentation of non-extracted MC broth 72
4.5 Addition of 10 % soymilk in mixed culture fermentation 74
4.6 Effect of temperature on mixed culture fermentation of MC extracted broth with 10% soymilk 78
4.6.1 Growth and production of lactic acid 78
4.6.2 Antioxidant properties 81
4.6.3 Antidiabetic activity 83
4.7 Effect of mixed culture fermentation on soymilk only 85
4.8 Isolation and identification of Gluconacteobacter sp. Sc-01 87
4.9 Effect of Pure cultures of Gluconacetobacter fermentation of MC media broth with 10% soymilk on xanthine oxidase inhibition activity 89
4.10 Effect of temperature on Gluconacetobacter fermentation of MC media broth with soymilk 94
4.11 Co-culture of Gluconacetobacter and Saccharomyces cerevisiae for fermentation of MC media broth supplemented with 10% soymilk 97
4.12 Multistrain culture involoving Gluconacteobacter, Saccharomyces cerevisiae and Lactobacillus plantarum for fermentation of MC broth media with 10% soymilk 100
4.13 Two stage using multistrain culture of Gluconacteobacter, Saccharomyces cerevisiae and Lactobacillus plantarum for fermentation of MC broth media with 10% soymilk 102
4.14 Multistrain involving culture of Gluconacteobacter, Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus buchneri for fermentation of MC broth media with 10% soymilk 104
4.15 Optimization of xanthine oxidase inhibition activity and coumarin content using two stage multistrain cultures of Gluconacteobacter, Saccharomyces cerevisiae, Lactobacillus plantarum and Lactobacillus buchneri for fermentation of MC broth media with 10% soymilk 106
Chapter 5. Conclusion 108
Chapter 6. Future works 111
References 112
Appendix 132

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指導教授

徐敬衡(Chin-Hang Shu)

審核日期

2022-9-8

推文