INTRODUCTION
Preface
Recent studies have focused on the use of bacteria in biotransformation to produce valuable secondary metabolites from medicinal plants, with ginseng being one of the most prominent examples Panax ginseng has a long history of use in traditional medicine across Asia, particularly in Korea, China, and Japan, where it is valued for its health benefits The primary active compounds in ginseng, known as ginsenosides, are responsible for its pharmacological effects, with over 150 derivatives identified in its roots, leaves, berries, and buds The pharmacological properties of ginsenosides are influenced by the specific types and locations of sugar attachments to the dammarane or oleanane aglycone molecules Notably, the roots of ginseng contain the main ginsenosides, including Rb1, Rb2, Rc, Rd, Re, and Rg1, which together represent 80-90% of the total ginsenoside content.
Alternative methods like acid hydrolysis, alkali separation, and microbial or enzyme decomposition are used to convert major ginsenosides into low-weight molecular forms such as Rg3, F2, Rh2, and compound K, facilitating easier metabolism and absorption by the human body Microbiological and enzyme hydrolysis techniques are favored over traditional chemical methods due to their operation under basic conditions and simpler, more efficient reaction steps However, research on enzyme-based production of low-weight molecular ginsenosides faces challenges, including low productivity, complexity of the enzyme methods, and food safety concerns.
Therefore, we conduct to research the subject: “Fermentation of ginseng extracts by probiotic bacteria and their antimicrobial and anti oxidant activity”
Objectives and requirements
• Determining the suitable concentration of ginseng for fermentation
• Establishing conditions that affect ginsenoside metabolism
• Researching and optimizing the conditions affect the ability of metabolize ginsenoside from bacteria NL812
• Determination the ginsenoside metabolism of β-glucosidase enzyme by thin- layer chromatography (TLC) analysis and high-pressure liquid chromatography (HPLC)
OVERVIEW
Introduction of ginseng
Ginseng (Panax ginseng) is a flowering plant belonging to the Araliaceae family, known for its roots that contain beneficial compounds like ginsenosides and gintonin This genus includes various types, such as Korean ginseng (P ginseng), South China ginseng (P notoginseng), and American ginseng (P quinquefolius).
Ginseng thrives in cooler climates, primarily found in the Korean Peninsula, Northeast China, and the Russian Far East, as well as in Canada and the United States However, certain species, such as South China ginseng, are native to warmer regions, specifically Southwest China and Vietnam.
Despite its long history in traditional medicine, modern clinical research has not established ginseng's medical effectiveness, and the US Food and Drug Administration (FDA) has not approved it as a prescription drug While ginseng is widely available as a dietary supplement, inconsistent manufacturing practices raise concerns about contamination with toxic metals or fillers Additionally, excessive use of ginseng may lead to adverse effects or negative interactions with prescription medications.
Modern medicine has validated the pharmacological benefits of ginseng, including its ability to boost energy, enhance memory, alleviate stress, and strengthen the immune system against inflammation Additionally, ginseng is known for its protective effects against cellular aging and its role in increasing the body's resistance Recent studies have also uncovered new properties of ginseng, such as its antioxidant and anti-cancer effects In Vietnam, various types of ginseng have been identified and utilized.
Ngoc Linh ginseng, also referred to as Vietnamese ginseng, is a highly valued herb found in the mountainous regions of Ngoc Linh, Quang Nam province Recognized as one of the top five most valuable ginseng varieties globally, it is renowned for its medicinal properties, including stopping bleeding, promoting wound healing, and serving as a tonic for rapid health restoration and fatigue relief Additionally, Ngoc Linh ginseng is celebrated for its anti-aging and anti-cancer benefits.
Bo Chinh ginseng, also known as Tho Hao ginseng (Abelmoschus sagittifolius), is a wild ginseng species primarily found in the southern central provinces and Tay Nguyen, including Phu Yen, Binh Dinh, and Gia Lai This ginseng is utilized for treating coughs, fevers, and general weakness, as well as for promoting health, regulating menstruation, and addressing conditions such as pneumonia, leukemia, pimples, and scabies.
Stone ginseng (Myxopyrum smilacifolium Bl), also known as Nhuong Le Kim Cang, is a member of the Oleaceae family recently found in the arid mountainous regions of Northwest provinces This herb is renowned for its health-restoring properties, enhancing vitality, and strengthening tendons, making it beneficial for treating kidney issues and body weakness Additionally, it serves as an excellent remedy for individuals with heart disease.
Forest areca ginseng (Curculigo orchioides Gaertn) is a natural ginseng variety found in the mountainous regions of Vietnam, including Dien Bien, Ha Giang, Cao Bang, and Bac Kan, as well as in countries like Thailand, Malaysia, India, and the Philippines In Oriental medicine, it is known as 'mao' and is valued for its medicinal properties, including its spicy taste This herb is used to treat various conditions such as diarrhea, rheumatism, impotence, and weakened sexual function in men.
Panax pseudoginseng, a rare medicinal plant, thrives in the mountainous regions of Vietnam, particularly in Ha Giang, Lao Cai, and Cao Bang, at altitudes of 1,500 meters or higher, where cold climates prevail The root of this ginseng species is rich in unique and beneficial pharmaceuticals, making it a valuable ingredient in traditional medicine Additionally, the flowers and buds of the plant are utilized for treating cardiovascular diseases and managing blood pressure.
Panax pseudoginseng is usually harvested after 5 to 7 years, people usually harvest in November every year after the tree has reached the age of 5-years or more
Analyzing the composition in the Panax pseudoginseng, the scientists discovered that there are many rare nutrients and pharmaceuticals, including amino
4 acids, sterols, sugars, Fe, Ca, especially 2 substances Saponin: Arasaponin A, Arasaponin B
Ginseng is known for its numerous benefits, including enhancing resistance, regulating the nervous system, and providing anti-aging properties Recent studies have revealed its potential anti-cancer effects, demonstrating its ability to inhibit metastasis, regenerate cancer cells, and promote cell differentiation.
Ginseng's chemical composition primarily includes ginsenosides, oligosaccharides, polysaccharides, amino acids, proteins, oils, and fatty acids, with ginsenosides being the key active ingredient These ginsenosides are categorized into two groups: Protopanaxadiol (PD), which includes Rb1, Rb2, Rc, and Rd, and Protopanaxatriol (PT), which consists of Re, Rf, Rg1, and Rg2 Notably, Rb1, Rd, and Re are the most abundant ginsenosides in ginseng, while Rg1, Rg3, Rh1, and Rh2 are present in smaller quantities but play a crucial role in the pharmacological properties of ginseng.
Ro: It has the effect of breaking down alcohol, preventing hepatitis and restoring bad breath
Rb1: It could inhibit the central nervous system so that pain is relieved
Rb2: Preventing diabetes restriction, liver sclerosis, and accelerate liver absorption Rc: Reduce the pain, speeds up protein absorption
Rg1, Rg2, Rg3: Anti-fatigue, reduce stress, restore memory, prevent cancer cells and protect the liver
Rh1, Rh2: Inhibits cancer cells, protects the liver, prevents platelet binding
Recent studies have highlighted the pharmacological potential of rare ginsenosides, including Panaxadiol Saponifier Rg3, Rh2, and CK, which exhibit properties such as anti-cancer, anti-inflammatory, and immune-boosting effects However, these rare ginsenosides are present in very low concentrations in ginseng, and some types are exclusively found in red ginseng (Park et al., 2014; Lee et al., 2016; Sun et al., 2017).
Over 150 ginsenoside derivatives have been identified from various parts of P ginseng, including roots, leaves, berries, and buds The pharmacological effects of these ginsenosides are influenced by the types of sugars attached to their aglycones, which can be either dammarane or oleanane The primary ginsenosides found in P ginseng roots—Rb1, Rb2, Rc, Rd, Re, and Rg1—constitute 80-90% of the total ginsenoside content However, their large molecular size, low solubility, and poor cell membrane permeability hinder their absorption in the human intestine In contrast, low-weight molecular ginsenosides such as Rg3, F2, and Rh2, which are hydrolyzed from the main ginsenosides, are more easily metabolized and absorbed by the body.
Small ginsenosides like Rg3 and Rh2 are highly soluble and easily absorbed through the human intestine Notably, Rg3, despite its low concentration of approximately 4.6 mg/g (about 1/6 of Rb1), exhibits significant pharmacological activities, including anti-cancer, anti-inflammatory, and immune-boosting effects (Park et al., 2014; Lee et al., 2016; Sun et al., 2017).
This study explores the metabolism and enhancement of saponins, highlighting research by health experts in Korea from 2012 that demonstrated the potential of ginsenoside in inhibiting cancer cell division, diminishing the vitality of abnormal cells, and preventing metastasis These findings suggest a promising new approach in modern medicine for cancer treatment.
Overview of fermented ginseng
The primary physical method involves heating, where high temperature and pressure disrupt the bonds in sugar However, this approach necessitates challenging reaction conditions, is difficult to control, generates numerous byproducts, and is unsuitable for industrial production.
Chemical methods include acid hydrolysis, alkali filtration, Smith decomposition
Acid hydrolysis mainly uses a solution of 50% acetic acid, 5% sulfuric acid to hydrolyze ginsenoside This method produces many byproducts, with low efficiency
The alkaline filtration method produces pure products, but the requirements on temperature, pressure, and pH are very strict
Smith’s decomposition method involves the oxidation of cis-glyoxal acidic acid to generate di-aldehydes, which are then reduced with sodium tetrahydroborate to yield diols While this approach eliminates the need for extreme temperature and pressure conditions, the resulting product lacks the original activity and is not suitable for the production of other ginsenosides.
Enzyme hydrolysis is an efficient method for producing ginsenoside by utilizing enzymes to extract sugars, resulting in minimal byproducts and simple reaction conditions This process is environmentally friendly, as the post-treatment does not lead to pollution Common industrial hydrolysis enzymes include amylase, peptidase, cellulase, pectinase, and glucose synthase.
The use of chemical methods to produce ginsenoside will produce many byproducts, react strongly, difficult to control hydrolysis, polluted environment, not suitable for industrial production (Liu et al., 2009)
The enzyme hydrolysis method offers several advantages for the industrial production of ginsenoside, but it also presents notable challenges The quality of the enzyme is crucial, necessitating purification from crude sources, which increases costs due to the large quantities required Additionally, biological enzymes lack stability under natural conditions, making them prone to isomerization and inactivation, complicating preservation efforts Some hydrolases require higher optimal reaction temperatures, resulting in increased energy consumption and higher industrial production costs.
Microbial fermentation utilizes enzymes from microbial cells to transform external substances into economically valuable products Specifically, the microbiological modification of ginsenosides involves their hydrolysis through enzymes generated by microbial metabolism This process primarily occurs through liquid fermentation, where ginsenosides are extracted from crude substances, and hydrolases are produced using microorganisms in a liquid medium.
Microbial fermentation is the primary method for producing ginsenosides in the industry, offering advantages such as the absence of the need to purify biological enzymes, stability of enzyme fluids, and ease of controlling reaction conditions However, this method has its drawbacks, as high concentrations of ginsenosides can inhibit normal microbial growth.
The inhibition of bacteria and enzyme production is particularly pronounced in liquid media, which impacts metabolism To enhance industrial production, it is essential to achieve high levels of ginsenoside through multiple batches and repeated processes.
2.2.2 Mechanism of the fermentation method
The fermentation process utilizes ginseng and other ingredients as nutrients, leveraging the physiological activities of microorganisms to alter the structure of ginseng and activate new biological activities This inverse fermentation reaction not only provides essential nutrients for microorganisms, promoting their growth, but also generates enzymes that transform the original ginsenoside structure into more active and valuable compounds.
Microorganisms produce enzymes that modify sugars at specific sites, including C-3, C-6, and C-20, facilitating the hydrolysis of glycoprotein bonds This process transforms primary ginsenosides into valuable variants, such as converting ginsenoside Rb1 into the more active ginsenoside Rg3 by removing two glucose molecules at the C-20 site using β-glucosidase.
Recent studies have highlighted the use of specific strains of microorganisms that produce high levels of β-glucosidase, which plays a crucial role in the metabolism of ginsenosides This microbial enzyme effectively cleaves 1-6 glucoside bonds in large molecular substances, such as Rb1, resulting in the formation of smaller compounds like Rg3 and CK.
In a study by Fu et al (2014), 58 strains of microorganisms were isolated from ginseng roots, leading to the selection of Penicillium simplicissimum GS33, which produces β-glucosidase on Esculin-R2A agar medium This strain effectively hydrolyzes ginsenosides Rb1, Rb2, Rc, and Rd into F2, Rg3, and C-K, while also transforming ginsenoside Rg1 into Rh1 and F1 Notably, ginseng products fermented by Penicillium simplicissimum GS33 demonstrated the ability to inhibit the growth of ES-2 cell lines, with an IC50 of 0.73 mg/ml.
Song et al (2015) investigated 147 strains of soil microorganisms that produce β-glucosidase, identifying strain K35 as effective in converting large ginsenosides into smaller, more valuable forms This strain is utilized for fermenting ginseng extract, with HPLC analysis revealing that the smaller compound was produced under optimal conditions over a 9-day period in Luria-Bertani agar medium at pH 7.
Renchinkhand et al (2011) identified and classified Paenibacillus sp MBT213, which exhibits active β-glucosidase activity, from raw milk They found that the optimal conditions for its metabolism are at 35 °C and pH 7 After 10 days, the strain completely converted ginsenoside Rb1 to Rd When applied to ferment 20% ginseng root, HPLC analysis after 14 days revealed a significant decrease in Rb1 concentration and a corresponding increase in Rd.
Yin Chengri et al (2017) demonstrated the transformation of ginsenoside Re to ginsenoside Rh1 using Sphingomonas 2-F2 The transformation mechanism involves the hydrolysis of ginsenoside Re, which leads to the removal of a sugar group and the formation of ginsenoside Rgl Subsequently, Rgl is catalyzed by specific enzymes produced by microorganisms, resulting in the removal of a β-glucosidase molecule and the production of a rare ginsenoside.
Enzyme β-glucosidase
β-glucosidase is a crucial enzyme found in the cellulase enzyme complex of bacteria, playing a significant role in cellulose degradation It works in conjunction with endo-cellulase, which cleaves the hydrophilic regions of cellulose, and exo-cellulase, which targets the hydrophobic regions, resulting in shorter polysaccharide molecules such as disaccharides, trisaccharides, and tetrasaccharides Ultimately, β-glucosidase hydrolyzes the 1-6 bonds of these polysaccharides, converting them into monosaccharides (Suto, 2014).
Fig 2.3 β-glucosidase in the hydrolysis
Cellulose-degradable β-glucosidase can be produced from diverse natural sources, including fungi and bacteria These microorganisms are crucial in various industries, such as textiles, paper production, and detergents.
1999), waste treatment (Ly Kim Bang et al., 1999), and production of microbiological organic fertilizers (Nguyen Lan Huong et al., 1999)
In fact, people receive enzymes β-glucosidase from microorganisms: fungi (Aspergillus niger, Aspergillus oryzae, Aspergillus candidus, ), Actinomycetes (Actinomyces griseus, Streptomyces reticuli, ), and bacteria (Acetobacter xylinum, Bacillus Subtilis, Bacillus pumilis, )
MATERIALS, CONTENT AND METHOD OF RESEARCH
Materials and equipments
Bacillus subtilis NL812 was received from the Department of Biochemistry,
Institute of Chemistry - Materials, Institute of Military Science and Technology - Ministry of Defense
Ginseng extract was received from the Department of Biochemistry, Institute of Chemistry - Materials, Institute of Military Science and Technology - Ministry of Defense
Standard sample Rg3, C-K was received from the Department of Biochemistry, Institute of Chemistry - Materials, Institute of Military Science and Technology - Ministry of Defense
Equipment: Incubator, sterilizers, shaker incubators, spectrometer
Instruments: cylinders, micropipettes, test tubes, Petri dishes, flasks and other necessary laboratory tools.
Chemicals
Chemicals used in the preparation of medium: glucose, CMC, agar, peptone, yeast extract, and some mineral salts such as MgSO4, KCl, CaCl2, K2HPO4
Chemicals used in analysis: 98% H2SO4, CHCl3, 96% alcohol.
Culture medium
PDA medium: Glucose 20 g/L, agar 20 g/L, potato extract liquid (boiling 200g potato in 1L distilled water for 30 minutes and subsequently filtered through a muslin cloth)
Potato dextrose medium: glucose 10 g/L, CMC 1 g/L, potato extract liquid
Table 3.1 List of medium for checking the optimum medium
All the medium was sterilized at 110 o C in 30 minutes and cooling down before used For PDA medium, the medium was poured into Petri dishes when it was 40-50 o C
Experimental method
A sterilized loop was used to pick one loop of Bacillus subtilis NL812, which was then distributed in a zig-zag pattern on the surface of a PDA plate, repeating this process three times The plates were incubated at 25-30°C for 24 hours to isolate a single colony This isolated colony was subsequently transferred to PDA slant agar and stored at 4°C for long-term preservation.
Fifty milliliters of potato dextrose medium were placed in a 250 ml flask and sterilized at 110°C for 30 minutes Once cooled, one loop of NL812 from slant agar was transferred to the medium The inoculated culture was then incubated at room temperature (25-30°C) in a shaker set to 120 rpm After 24 hours, the liquid culture was collected and stored at 4°C as a seeding culture.
3.4.2 Methods of optimizing culture conditions
Bacillus subtilis NL812 was inoculated on a 250ml flash which containing
50ml of sterilized MT3/MT4…./MT13 medium, at room temperature (25-30°C), in a shaker (120 rpm) After 48 hours, the liquid culture was collected for checking the minor saponin content
Bacillus subtilis NL812 was inoculated on a 250ml flash which containing
50ml of sterilized optimize medium under conditions of 15°C, 20°C, 25°C and 30°C, shake at 120 rpm After 48 hours, the liquid culture was collected for checking the minor saponin content
3.4.2.3 Effect of the initial pH value
Bacillus subtilis NL812 was inoculated on a 250ml flash which containing
The experiment utilized 50ml of sterilized optimized medium, maintaining an optimum temperature as specified in sections 4.2.1 and 4.2.2, with initial pH levels set at 5, 6, 7, and 8, while shaking at 120 rpm After a 48-hour incubation period, the liquid culture was collected for analysis of minor saponin content.
Bacillus subtilis NL812 was inoculated on a 250ml flash which containing
To determine the optimal carbon sources, 50ml of sterilized medium (as detailed in sections 4.2.1, 4.2.2, and 4.2.3) was prepared with an initial pH and temperature A 0.05% concentration of glucose, malt extract, or sucrose was added, and the mixture was shaken at 120 rpm After 48 hours, the liquid culture was collected for analysis of minor saponin content.
Bacillus subtilis NL812 was inoculated on a 250ml flash which containing
A 50ml sterilized optimize medium, with controlled temperature and initial pH, was prepared by adding 0.05% of yeast extract, peptone, NaNO3, and (NH4)2SO4 The mixture was shaken at 120 rpm to determine the optimal carbon sources After 48 hours, the liquid culture was collected for analysis of minor saponin content.
Bacillus subtilis NL812 was inoculated on a 250ml flash which containing
A 50ml sterilized optimize medium, with controlled parameters including temperature, initial pH, carbon sources, and nitrogen sources, was prepared as detailed in sections 4.2.1 to 4.2.5 The culture was agitated at 120 rpm, and samples were collected at 24, 48, 72, and 120 hours to assess the minor saponin content, aiming to determine the optimal inoculation period.
3.4.3 Analysis methods of saponin content
40 mg of ginsenoside Yonagenin was dissolved in 200ml ethyl acetate Dilute the solution of ginsenoside Yonagenin in concentrations of 20, 40, 60, 80, 100 àg/ml
After that, 1ml of each ginsenoside Yonagenin in concentration was mixed with
1 ml of solution A (consisting of 0.5 ml of vanillin and 99.5 ml of ethyl acetate) and 1 ml of solution B (consisting of 50 ml of concentrated sulfuric acid and 50 ml of ethyl acetate)
The mixed test tube was incubated in a water bath at 60 °C for 10 minutes to achieve a complete color reaction After allowing it to cool to room temperature for an additional 10 minutes, the test was conducted at a wavelength of 530 nm.
The total saponin concentration was determined against a calibration curve using yonagenin as the standard The standard curve equation takes the form: y = 0.0058x - 0.0815 R² = 0.9693
In which: y: OD index measured at a wavelength of 530nm x: amount of standard substance used in the reaction
R 2 is the coefficient of variance
Fig 3.1 Graph of standard Yonagenin
Determination of saponin content in samples:
The saponin content is measured using a spectroscopic method that relies on oxidation reactions between saponin triterpene and vanillin, as noted by Li et al (2010) In this process, sulfuric acid serves as the oxidizing agent, resulting in a characteristic purple color, which is colorimetrically analyzed at 530 nm (Hiai, Oura, & Hakajima, 1976).
A 1 ml sample was combined with 1 ml of solution A, which contains 0.5 ml of vanillin and 99.5 ml of ethyl acetate, and 1 ml of solution B, made up of 50 ml of concentrated sulfuric acid and 50 ml of ethyl acetate.
The test tube was incubated in a water bath at 60 °C for 10 minutes to ensure a complete color reaction After cooling to room temperature for an additional 10 minutes, the absorbance was measured at 530 nm The saponin content was calculated using the formula: \( y = 0.0058x - 0.0815 \), where \( y \) represents the optical density at 530 nm and \( x \) denotes the saponin content.
3.4.3.1 Saponin extraction after fermentation for TLC and HPLC
After choosing the optimum parameter (t o , pH, ….) for saponin fermentation by NL812, the liquid culture was obtained for saponin extraction by using n-Butanol saturated water with the ratio is 1:1 (V:V)
The n-Butanol fraction was collected and evaporated using a rotary evaporator under vacuum to achieve a moisture content of less than 8% at 45 °C, followed by dilution in 1 ml of methanol for subsequent TLC and HPLC analysis.
3.4.3.2 Determination of the ability to metabolize ginsenoside by thin-layer chromatography method (TLC)
TLC of methanol extract was performed on silica gel sheet 60 F254 with CHCl3-
CH3OH-H2O (65:35:10, v:v:v) Spots on TLC were detected by spraying 10% H2SO4 in ethanol and drying Dry in the dryer for 10 minutes (Intendra et al, 2014)
Antioxidant activity was determined using the free radical diphenylpicryhydrazyl (DPPH) This method is common used for determining the antioxidant activity of natural compounds in food and biology
The method utilizes diphenylpicryhydrazyl (DPPH), a stable free radical in ethanol, which exhibits a purple color absorbable at 517 nm When an antioxidant is introduced to the DPPH solution, it captures the free radicals, leading to a gradual loss of the purple hue The antioxidant activity is quantified by measuring the reduction in DPPH color before and after the addition of the antioxidant substrate.
100 àL of sample solution was added into 50 àL DPPH 0.2mM diluted in alcohol Mix gently to homogenize and allow to react for 30 minutes in the dark, at room temperature
Negative control: replace 100 àL of sample solution with distilled water
Positive control: replace 100 àL of sample solution with 1M ascorbic acid solution Measured at 517 nm
Antioxidant activity - Radical scaveging activity (RSA) is calculated as follows:
In which: Akc and Atn are photometric values at 517 nm of negative control and positive test/control samples
3.4.3.4 Determination of bacterial inhibitory activity
Three pathogenic microorganisms, including gram-negative strains Pseudomonas aeruginosa and Escherichia coli, as well as the gram-positive strain Staphylococcus aureus, were sourced from IMBT/VNU and cultured in a nutrient liquid medium containing 5% peptone and 3% meat extract for 24 hours In a 96-well microplate, 90 µL of the bacterial culture was diluted with an equal volume of distilled water and incubated with 20 µL of 50 mg/L ginseng extract and hydrolysates, or antibiotics as positive controls—gentamicin for P aeruginosa, ciprofloxacin for E coli, and penicillin for S aureus For the negative control, 90 µL of bacterial strains was combined with 120 µL of distilled water The absorbance of the mixtures was measured immediately and again after a 24-hour incubation at room temperature.
The antibacterial activity was evaluated using the agar plate diffusion method (Hadacek et al., 2000) Bacteria were activated from the original inoculum on solid LB medium, with one colony transferred to 5 ml of liquid LB medium and shaken overnight at 37 °C For the active test, 200 μL of bacterial suspension, approximately 4-5 × 10^8 CFU/ml, was inoculated onto a Petri dish containing concentrated LB medium, creating 5-6 wells, each about 6 mm in diameter and spaced 2-3 cm apart Subsequently, 50 μL of the test extract was added to the agar wells, and the dishes were kept at room temperature for 2 hours to allow diffusion into the bacterial culture medium Finally, the plates were incubated at 37 °C for 24 hours.
17 antibiotic solution (Ampicilin 0.1 mg / ml for E coli and P mirabillis; Kanamycin 5 mg / ml with S aureus and P vulgaris); The negative control was DMSO
The inhibitory activity was assessed by calculating the radius (R) of the microbial inhibition zone using the formula: R (mm) = D - d, where D represents the diameter of the sterile ring and d denotes the diameter of the agar bore This experiment was conducted three times to obtain average radius values.
3.4.3.5 Determination of the ability to metabolize ginsenoside by high pressure liquid chromatography (HPLC)
Model: Agilent 1260 Infinity LC, USA
Column: ZORBAX Eclipse XDB- C18 (4,6 x 250 mm; 5 μm)
Dissolve the sample in 1ml MeOH and conduct supersonic for 15 minutes
Sample is mixed with concentration 50mg / 1ml
The sample use centrifuge 4000 rpm for 10 minutes, (centrifuge 2 times)
Extract the fluid to run on HPLC
HPLC samples were prepared by diluting both standards and extracts (1:2 w/v ratio) in aqueous methanol 80% (v/v), vortexed and centrifuged for 5 min and kept at -
RESULTS AND DISCUSSION
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
Research on the morphological characteristics and 16S rRNA genetic sequence of strain NL812 (Nguyen Thi Huong Nhu-K61CNTP-VNUA) indicates that it is Bacillus subtilis This bacterium has been shown to be safe and holds promise for practical applications, confirming that strain NL812 meets safety standards for further research.
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
Fig 4.2 Saponin of ginseng medium
Strain NL812 demonstrates the capability to metabolize ginsenoside effectively The saponin content in the product increased steadily up to a 20% ginseng medium, after which it began to decline at a 25% concentration Notably, the saponin content at 20% CS concentration was significantly higher than at other concentrations, being 1.5 times greater than the second-highest medium (25% CS) and 9 times higher than the lowest medium (2% CS).
Optimizing culture conditions
This study investigates how various cultural conditions and nutritional mediums affect the metabolism of ginsenoside by different types of ginseng Key environmental factors examined include carbon and nitrogen sources, as well as culture conditions such as temperature, initial pH, seeding rate, and fermentation time, specifically focusing on their impact on the 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 25-45 o C and a fermentation time of about 24 to 72h
The temperature has a direct effect on the chemical metabolism during fermentation Surveyed temperatures during fermentation:
After inoculating 100 µL of the NL812 strain in ginseng media, the culture was incubated at temperatures of 15 °C, 20 °C, 25 °C, and 30 °C while shaking at 120 rpm for 48 hours Upon completion of the fermentation process, the saponin content in the resulting product was measured.
At a temperature of 20 °C, ginsenoside metabolism yielded the highest results, with saponin content exceeding that at 15 °C and 30 °C by more than two times However, while the saponin content at 25 °C was lower, it demonstrated greater antioxidant and antimicrobial resistance compared to the levels observed at 20 °C.
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 significantly influenced the ginsenoside metabolism of strain NL812 Saponin content increased gradually as the pH rose from 5 to 7, but sharply declined at pH 8 At pH 7, the saponin content was seven times higher than at other pH levels, which measured 1.2 and 1.38 These findings align with Se-Hwa Kim's research, which also indicated that optimal saponin production occurs at pH 7.
Experiments on antioxidant and antimicrobial resistance also showed that the first pH of 7 was the most active
Table 4.2 Effect of the initial pH value
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
Carbon is essential for the growth, development, and ginsenoside metabolism of strain NL812 The growth medium was prepared using glucose as the carbon source, which was then substituted with sucrose and malt extract Strain NL812 was cultivated at a temperature of 25 °C while shaking at 120 rpm for a duration of 48 hours.
Fig 4.5 Effect of carbon sources
Replacing glucose with malt extract in the product led to a decrease in saponin content In contrast, substituting the carbon source with sucrose resulted in an increase in saponin levels, with antioxidant capacity rising by 15% and antimicrobial activity increasing by 1.5 times.
The findings align with the research conducted by Wei-Nan Wang, which indicates that malt extract and glucose are capable of metabolizing ginsenoside only at Rb1 and Rd levels Furthermore, sucrose has the ability to convert ginsenoside into Rg3 and higher concentrations.
Table 4.3 Effect of carbon sources
Carbon sources Glu Malt Suc
The nitrogen source significantly impacts the metabolism of ginsenoside This study evaluated the effects of two inorganic nitrogen sources, ammonium sulfate and sodium sulfate, alongside two organic nitrogen sources, peptone and yeast extract, on the 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
25 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 (NH4)2SO4 NaNO3
This study's findings align with those of Wei-Nan Ưang, highlighting a nitrogen source that enhances ginsenoside metabolism Notably, ammonium sulfate emerges as the most effective option for converting compounds into C-K.
Time is one of the most important factors influencing the fermentation èficiency, growth and development ability of strain NL812
The experimental results indicated that the bacterial strain exhibited significant growth and development during the initial 48 hours, followed by a sharp decline in numbers in the subsequent days Notably, the saponin content reached its peak at the 48-hour mark.
A study conducted by Kim Hong-guk et al revealed that after 48 hours of fermentation, Korean red ginseng exhibited a significant increase in ginsenoside Rb1 by 180.94% and Rg3 by 235.85%, with the appearance of Rg1 post-fermentation These findings indicate that 48 hours is the optimal fermentation duration for ginseng using Bacillus subtilis bacteria.
Fig 4.7 Effect of fermented times
Analyze the transformation of ginseng by TLC and HPLC method
Raw Ginsenoside contains five substances identifiable through TLC, characterized by high molecular weight and significant content The primary substance matches the molecular weight of the standard Rg3, although its content is lower compared to the other substances present, as determined by sample area analysis.
Fig 4.8 TLC of ginseng extract before fermentation
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
Fig 4.11 Chromatogram of the Rg3 standard
Fig 4.12 Chromatogram of the TTB
Fig 4.13 The chromatogram of the TTB sample was fermented by NL812
After fermentation, substances are broken down into smaller compounds, notably Rg3 and CK Table 4.5 indicates that the CK content increased sixfold compared to its levels before fermentation, while the Rg3 content also saw a twofold increase.
CONCLUSIONS AND RECOMMENDATIONS
The best conditions for ginseng fermentation are 25 o C, pH 7 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
Research and evaluate the effects of substances and their applicability in practice
Optimize conditions to apply in industrial production and pharmaceutical
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1 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
2 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
3 Jieun J., Hye Ji J., Su Jin E., Nam S C., Na-Kyoung L & Hyun-Dong P (2017) Fermentation of red ginseng extract by the probiotic Lactobacillusplantarum KCCM 11613P: ginsenoside conversion and antioxidant effects Journal of Ginseng research 43: 20-26
4 Jitendra U., Min-Ji K., Young-Hoi K., Sung-Ryong K., Hee-Won P & Myung-Kon K
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A study by Lee et al (2016) published in the Journal of Immunology Research investigates the anti-inflammatory effects of ginsenoside Rg3 The research focuses on its impact through the NF-κB pathway in A549 cells and human asthmatic lung tissue, highlighting its potential therapeutic benefits for asthma-related inflammation.
7 Li Y., Ying Y & Ding W (2014) Dynamics of Panax ginseng rhizospheric soil microbial community and their metabolic function Evid Based Complement Alternat Med: 160373
8 Park E-H, Kim Y- J, Yamabe N, Park S-H, Kim H, Jang H-J & Kang K.S (2014) Stereospecific anticancer effects of ginsenoside Rg 3 epimers isolated from heat-processed American ginseng on human gastric cancer cell Journal of Ginseng Research 38(1): 22-27
9 Se-Hwa K., Jin-Woo M,, Lin-Hu Q,, Sungyoung L,, Dong-Uk Y Deok-Chun Y (2012) Enzymatic Transformation of Ginsenoside Rb1 by Lactobacillus pentosus Strain 6105 from Kimchi Journal of Ginseng research 36(3): 291-297.