Fermentation of ginseng extracts by probiotic bacteria and their antimicrobial and anti oxidant activity (khóa luận tốt nghiệp)

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: FERMENTATION OF GINSENG EXTRACTS BY PROBIOTIC BACTERIA AND THEIR ANTIMICROBIAL AND ANTI-OXIDANT ACTIVITY HANOI, 2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: FERMENTATION OF GINSENG EXTRACTS BY PROBIOTIC BACTERIA AND THEIR ANTIMICROBIAL AND ANTI-OXIDANT ACTIVITY Student Supervisor : : Student code Class Location : : : Le Minh Vy Dr Bui Thi Thu Huong Dr Le Thi Hoang Yen Dr Vu Duy Nhan 610691 K61CNSHE Institute of Microbiology and Biotechnology, VNUA HANOI, 2021 DECLARATION I hereby declare that this paper was my own work All results and data in this title were absolutely honest and have not been submitted before to any institution for assessment purposes All sources used in this paper were cited in references Student Le Minh Vy i ACKNOWLEDGEMENTS I would like to express my respect and deep gratitude to Dr Bui Thi Thu Huong, Dr Le Thi Hoang Yen and Dr Vu Duy Nhan, for giving me the opportunity to carry out this work, and their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis I would like to express sincere gratitude to all the staff of the Institute of Microbiology and Biotechnology who were enthusiastic to help me through the project Finally, I would like to thank my family, those with us and those sadly departed You provided fantastic support and inspiration throughout the duration of this undergraduate thesis, and without you all, this would never have been possible Student Le Minh Vy ii CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLE v LIST OF FIGURE vi PART I: INTRODUCTION 1.1 Preface 1.2 Objectives and requirements PART II: OVERVIEW 2.1 Introduction of ginseng 2.1.1 Ginseng 2.1.2 Ingredients and uses 2.2 Overview of fermented ginseng 2.2.1 Methods of ginsenoside metabolism 2.2.2 Mechanism of the fermentation method 2.3 Enzyme β-glucosidase PART III: MATERIALS, CONTENT AND METHOD OF RESEARCH 11 3.1 Materials and equipments 11 3.1.1 Material 11 3.1.2 Experimental equipment and tools 11 3.2 Chemicals 11 3.3 Culture medium 11 3.4 Experimental method 12 3.4.1 Culture methods 12 3.4.2 Methods of optimizing culture conditions 12 3.4.3 Analysis methods of saponin content 13 iii PART IV: RESULTS AND DISCUSSION 19 4.1 Check for the survival of bacteria 19 4.2 Evaluation of ginsenoside metabolism of strain NL812 in ginseng medium 19 4.3 Optimizing culture conditions 20 4.3.1 Effect of temperature 21 4.3.2 Effect of the initial pH value 22 4.3.3 Effect of carbon sources 23 4.3.4 Effect of nitrogen sources 24 4.3.5 Effect of fermented times 25 4.4 Analyze the transformation of ginseng by TLC and HPLC method 26 4.4.1 TLC method 26 4.4.2 HPLC method 27 PART V CONCLUSIONS AND RECOMMENDATIONS 31 REFERENCES 32 iv LIST OF TABLE Table 4.1 Effect of temperature 21 Table 4.2 Effect of the initial pH value 22 Table 4.3 Effect of carbon sources .24 Table 4.4 Effect of nitrogen sources 25 Table 4.5 Quantitative C-K 29 Table 4.6 Quantitative Rg3 29 v LIST OF FIGURE Fig 2.1 Structure of saponins Fig 2.2 Mechanism of β - glucosidase 10 Fig 2.3 β-glucosidase in the hydrolysis 10 Fig 3.1 Graph of standard Yonagenin 14 Fig 4.1 Colony of bacterial NL812 .19 Fig 4.2 Saponin of ginseng medium .20 Fig 4.3 Effect of temperature 21 Fig 4.4 Effect of the initial pH value 22 Fig 4.5 Effect of carbon sources 23 Fig 4.6 Effect of nitrogen sources 24 Fig 4.7 Effect of fermented times 26 Fig 4.8 TLC of ginseng extract before fermentation 26 Fig 4.9 TLC of ginseng extract after fementation 27 Fig 4.10 Chromatogram of the C-K standard .27 Fig 4.11 Chromatogram of the Rg3 standard .28 Fig 4.12 Chromatogram of the TTB .28 Fig 4.13 The chromatogram of the TTB sample was fermented by NL812 29 vi PART I: INTRODUCTION 1.1 Preface In recent years, the application of bacteria in biotransformation to produce pharmacologically valuable secondary ingredients is being studied extensively in medicinal plants One of the most valuable and researched medicinal plants is ginseng Panax ginseng has been used as a folk medicine for healing and maintaining health for thousand years in Asian countries such as Korea, China and Japan Ginseng saponins, known as ginsenosides, have been considered as the main ingredients of ginseng's pharmacological effects Nowadays, more than 150 ginsenoside derivatives have been found from the roots, leaves, berries, and buds of ginseng The pharmacological effects of ginsenosides depend on the types or location of sugars attach to dammarane or oleanane with aglycones molecule The main ginsenosides in the roots of ginseng are ginsenosides Rb1, Rb2, Rc, Rd, Re and Rg1, which account for 80-90% of the total number of ginsenosides (Jitendra et al., 2016) Other methods, such as acid hydrolysis, separate alkali , and microbial and enzyme decomposition to produce low-weight molecular ginsenosides Rg3, F2, Rh2 and compound K from major ginsenosides, so that the human body metabolize and absorb easier Microbiological or enzyme hydrolysis methods are more prevalent than conventional chemical methods because the reaction occurs in basic conditions, reaction steps are simple and intensive However, some research in produce lowweight molecular ginsenoside by enzyme have been limited by low productivity, the complexity of enzyme method, or food safety problem Therefore, we conduct to research the subject: “Fermentation of ginseng extracts by probiotic bacteria and their antimicrobial and anti oxidant activity” 1.2 Objectives and requirements 1.2.1 Objectives • Determining the suitable concentration of ginseng for fermentation • Establishing conditions that affect ginsenoside metabolism 1.2.2 Requirements • Researching and optimizing the conditions affect the ability of metabolize ginsenoside from bacteria NL812 • Determination the ginsenoside metabolism of β-glucosidase enzyme by thinlayer chromatography (TLC) analysis and high-pressure liquid chromatography (HPLC) PART II: OVERVIEW 2.1 Introduction of ginseng 2.1.1 Ginseng Ginseng (Panax ginseng) is a species of flowering plant in the Araliaceae family Ginseng is the root of plants in the genus Panax, such as Korean ginseng (P ginseng), South China ginseng (P notoginseng), and American ginseng (P quinquefolius), typically characterized by the presence of ginsenosides and gintonin Ginseng is found in cooler climates – Korean Peninsula, Northeast China, and Russian Far East, Canada and the United States, although some species grow in warm regions – South China ginseng being native to Southwest China and Vietnam Although ginseng has been used in traditional medicine over centuries, modern clinical research is inconclusive about its medical effectiveness There is no substantial evidence that ginseng is effective for treating any medical condition, and its use has not been approved by the US Food and Drug Administration (FDA) as a prescription drug Although ginseng is commonly sold as a dietary supplement, inconsistent manufacturing practices for supplements have led to analyses showing that ginseng products may be contaminated with toxic metals or unrelated filler compounds, and its excessive use may have adverse effects or untoward interactions with prescription drugs Nowadays, modern medicine has many studies that confirm pharmacological effects of ginseng as boost energy, improve memory, reduce stress, effects on the immune system against inflammation, protect cells against aging, increase the body's resistance Moreover, many new effects of ginseng were discovered, such as antioxidants, anti-cancer Meanwhile, in Vietnam, people discovered many kinds of ginseng: Ngoc Linh ginseng is also known as Vietnamese ginseng, grows in the mountainous districts of Ngoc Linh, Quang Nam province As one of the most valuable ginseng in the world It is known to many people for its use in stopping bleeding, healing wounds, tonic to help restore health quickly, dispel fatigue Especially Ngoc Linh ginseng also has anti-aging and anti-cancer effects PART IV: RESULTS AND DISCUSSION 4.1 Check for the survival of bacteria Fig 4.1 Colony of bacterial NL812 After 2-4 days of culture on agar plates of normal agar medium, colonies are round, slightly small, ivory, size 0.5-2.5 mm Based on previous research on morphological characteristics and genetic sequence encoding for rARN 16S of strain NL812 (Nguyen Thi Huong NhuK61CNTP-VNUA), we can roughly conclude that strain NL812 is Bacillus subtilis Research has demonstrated that this bacterium is safe and has the potential for practical applications Therefore, strain NL812 meets the safety standards to be able to proceed to the continous research 4.2 Evaluation of ginsenoside metabolism of strain NL812 in ginseng medium Performed fermentation experiments on the ability to metabolize ginsenoside in medium containing ginseng in the ratio at 25 °C, shaking at 120 rpm, for 48 hours 19 Fig 4.2 Saponin of ginseng medium From the examine results, strain NL812 has the ability to metabolize ginsenoside The saponin content in the product increased gradually to the 20% ginseng medium and began to decrease when it reached the 25% concentration of ginseng The saponin content at 20% CS concentration was superior to other concentrations Moreover, it was 1.5 times higher than the second-highest medium (25% CS) and times compared with the lowest medium (2% CS) 4.3 Optimizing culture conditions Under different culture conditions and nutritional mediums, different types of ginseng will have different abilities to metabolize ginsenoside In this study, some environmental components: carbon source, nitrogen source and some culture conditions, such as: temperature, initial pH, seeding rate, influence of fermentation time on ginsenoside metabolism of strain NL812 In a study by Kim Kwang Soo et al and patented, which showed that ginseng fermentation is best at a 5-20% concentration of ginseng, a temperature of 25-45oC and a fermentation time of about 24 to 72h 20 4.3.1 Effect of temperature The temperature has a direct effect on the chemical metabolism during fermentation Surveyed temperatures during fermentation: Fig 4.3 Effect of temperature After inoculation of 100 µL strain of NL812 in the ginseng media was cultured under the conditions of 15 °C, 20 °C, 25 °C and 30 °C, shaking at 120 rpm in 48 hours Finish the fermentation process, determine the saponin content in the product With four examine temperatures, at a temperature of 20 oC, it shows that ginsenoside metabolism has the highest result The saponin content at 20 °C was superior to that at 15 °C and 30 °C, which was more than times However, research experiments on antioxidant and antimicrobial resistance showed that saponin content at 25 °C was low but its antioxidant and anti-microbial resistance was higher than it was at 20 °C Table 4.1 Effect of temperature Temperature Saponin (µg/ml) RSA(%) Bacteria inhibitory E.coli (mm) S.aure 15 123.62 32.05 4.5 21 20 246.46 74.13 6.5 25 219.39 81.9 8.55 6.5 30 105.43 63.46 7.5 4.3.2 Effect of the initial pH value Test of pH survey was performed with basic environment with initial pH values of 5,6,7,8: Fig 4.4 Effect of the initial pH value The initial pH value of the fermentation medium changed was affected the ability to metabolize ginsenoside of strain NL812 The saponin content increased gradually when the first pH increased from to 7, then decreased sharply at pH The saponin content obtained at the first pH was times compared with the remaining pH values, which were 1.2 and 1.38 The results of examine was similar to the research results of Se-Hwa Kim’s study, Saponin content generated the most at pH Experiments on antioxidant and antimicrobial resistance also showed that the first pH of was the most active Table 4.2 Effect of the initial pH value Initial pH value Saponin (µg/ml) 232.5 246.21 282.66 218.79 RSA(%) 47.86 51.5 73.48 34.94 E.coli 1.5 4.5 5.5 1.5 S.aure 1.5 Bacteria inhibitory (mm) 22 Thus, the initial pH of the medium is either acidic or alkaline, in addition, the average first pH is the best for ginsenoside development and metabolism of strain NL812 4.3.3 Effect of carbon sources Carbon is an important source of nutrients for the growth and development as well as ginsenoside metabolism of strain NL812 The prepared medium with glucose carbon source and replaced with sucrose, malt extract Strain NL812 was raised in a temperature of 25 ° C, shaking at 120 rpm in 48 hours Fig 4.5 Effect of carbon sources The saponin content in the product decreased when replacing the source of carbon glucose with malt extract But when replacing the carbon source was sucrose, the results showed that the saponin content in the product increased, especially the antioxidant capacity increased by 15% and anti-microbial increased by 1.5 times This result was consistent with previous research of Wei-Nan Wang In WeiNan Wang's study, malt extract and glucose can only metabolize ginsenoside at Rb1 and Rd In addition, sucrose can convert ginsenoside to Rg3 and higher levels 23 Table 4.3 Effect of carbon sources Carbon sources Glu Malt Suc Saponin (µg/ml) 235.08 169.92 252.93 RSA(%) 50.89 61.91 64.85 E.coli 4.5 4.5 6.5 S.aure 1.5 Bacteria inhibitory (mm) 4.3.4 Effect of nitrogen sources Nitrogen source is also one of the main factors influencing the ability to metabolize ginsenoside In this study, two sources of inorganic nitrogen, which are ammonium sulfate and sodium sulfate, and two sources of organic nitrogen, which are peptone and yeast extract, were used to evaluate the effects of these sources on ginsenoside metabolism of strain NL812 Fig 4.6 Effect of nitrogen sources The above results indicated that when using inorganic nitrogen sources, the ginsenoside conversion capacity of strain NL812 was higher than that of organic 24 nitrogen sources In which, nitrogen ammonium sulfate for conversion yield and saponin content was the highest In addition, nitrogen ammonium sulfate also showed superior resistance to microorganisms and especially oxidation resistance, which had 1.5 times higher than other nitrogen sources Table 4.4 Effect of nitrogen sources Nitrogen sources Yeast Peptone (NH ) SO NaNO Saponin (µg/ml) 174.82 244.48 332.92 273.78 RSA(%) 34.84 35.15 54.79 33.05 E.coli 4.5 5.5 5.5 S.aure 3.5 2.5 4.5 Bacteria inhibitory (mm) The results of this study are similar to that of Wei-Nan Ưang’s study, a nitrogen source capable of promoting ginsenoside metabolism However, only ammonium sulfate is most suitable for converting compounds into C-K 4.3.5 Effect of fermented times Time is one of the most important factors influencing the fermentation èficiency, growth and development ability of strain NL812 From the experimental results, the first 48 hours showed growth and development of the bacterial strain, but then the bacteria started to decline drastically over the next days In addition, the saponin content at 48 hours showed the highest result A study on Korean red ginseng fermentation by Kim Hong-guk et al., After 48 hours of fermentation, the product ginsenoside Rb1 increased 180.94%, Rg3 increased 235.85% and appeared Rg1 after fermentation The results of this examine showed that 48 hours is the appropriate time to ferment ginseng with bacillus subtilis bacteria 25 Fig 4.7 Effect of fermented times 4.4 Analyze the transformation of ginseng by TLC and HPLC method 4.4.1 TLC method Raw Ginsenoside has substances expressed on TLC, substances with high molecular weight below and large content The top substance has the same molecular weight as the standard Rg3 The content of Rg3 is smaller than that of the remaining substances (based on sample area) Fig 4.8 TLC of ginseng extract before fermentation 26 After strain NL812 converts ginsenoside, substances with large molecular weight below have been converted to higher compounds such as Rg3, CK, Fig 4.9 TLC of ginseng extract after fementation 4.4.2 HPLC method Fig 4.10 Chromatogram of the C-K standard 27 Fig 4.11 Chromatogram of the Rg3 standard Fig 4.12 Chromatogram of the TTB 28 Fig 4.13 The chromatogram of the TTB sample was fermented by NL812 Table 4.5 Quantitative C-K Time Pic area (mAU.s) Concentration (μg/ml) Content (%) TTB (30.1 μg/l) 70.128 5.526 3.165 0.011 Sample 3.10 (15000μg/l) 70.359 32.745 10.301 0.0687 Table 4.6 Quantitative Rg3 STT Time Pic area (mAU.s) Concentration (μg/ml) Content (%) TTB (30.1 μg/l) 64.536 261.671 193.694 0.644 Sample 3.10 (1500μg/l) 64.454 21.970 15.808 1.0539 29 After fermentation, substances are cut into smaller compounds, for example Rg3 and CK According to table 4.5, CK content increased times compared to before fermentation In addition, the Rg3 content also increased times 30 PART V CONCLUSIONS AND RECOMMENDATIONS Conclusions The best conditions for ginseng fermentation are 25oC, pH and 48 hours The carbon source suitable for ginseng fermentation reaction is succrose The nitrogen source suitable for ginseng fermentation reaction is amoni sulfate Through TLC and HPLC, substances in ginseng have been converted into smaller and more valuable compounds, such as Rg3, CK Recommendation Research and evaluate the effects of substances and their applicability in practice Optimize conditions to apply in industrial production and pharmaceutical 31 REFERENCES Vietnamese documents Nguyễn Thượng Dong, Trần Công Luận & Nguyễn Thị Thu Hương (2007) Sâm Việt Nam số thuốc họ Nhân sâm Nhà xuất Khoa học Kĩ thuật: 70-78 Foreign documents Fu Le, T.H Van, Lee S Y., Kim T R., Kim J Y., Kwon S W., Nguyen N K & Nguyen M.D (2014) “Processed Vietnamese ginseng: Preliminary results in chemistry and biological activity”, Journal of Ginseng Research 38(2): 154-159 Hong-guk K., Young-ho C., Geun-seop K., Ha-young K &Byeong-soo K (2018) Effect of Korean red ginseng marc fermented by Bacillus subtilis on swine immunity Korean Journal of Livestock Hygiene 41(3): 141-147 Jieun J., Hye Ji J., Su 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