CN114854628A - Lactobacillus fermentum TY-S07 and application thereof - Google Patents
Lactobacillus fermentum TY-S07 and application thereof Download PDFInfo
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- CN114854628A CN114854628A CN202210474550.1A CN202210474550A CN114854628A CN 114854628 A CN114854628 A CN 114854628A CN 202210474550 A CN202210474550 A CN 202210474550A CN 114854628 A CN114854628 A CN 114854628A
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- Prior art keywords
- lactobacillus fermentum
- eps
- lactobacillus
- dosage form
- peptone
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/143—Fermentum
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/225—Lactobacillus
Abstract
The invention belongs to the technical field of microorganisms and application thereof, and particularly relates to lactobacillus fermentum TY-S07(lactobacillus fermentum TY-S07) and application thereof, wherein the preservation number is CGMCC No.24628, and the lactobacillus fermentum has good gastrointestinal tract resistance; under certain fermentation conditions, the EPS yield reaches (469.0 +/-13.0) mg/L, and EPS monosaccharide mainly comprises glucose, galactose and fructose according to the molar ratio of 11.5:2.7: 1.0. The lactobacillus fermentum TY-S07 has high cholesterol removal capacity (17.9 +/-0.5)%, high oxidation resistance, and high DPPH free radical clearance rate (31.2 +/-0.6%).
Description
Technical Field
The invention belongs to the technical field of microorganisms and application thereof, and particularly relates to lactobacillus fermentum TY-S07(lactobacillus fermentum TY-S07) and application thereof.
Background
Lactobacillus (lactic acid bacteria) is a generic term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid, and mainly includes the genera lactobacillus, bifidobacterium, lactococcus lactis, streptococcus, and the like. Exopolysaccharides (EPS), one of the metabolites of lactobacillus, are water-soluble polysaccharides secreted outside the cell wall of some special microorganisms (including lactobacillus) during the growth and metabolism process, easily separated from the bacteria and secreted into the environment, belong to the secondary metabolites of microorganisms, and have important significance for the growth of microorganisms.
EPS is used as an important secondary metabolite of LAB (lactobacillus), can provide energy for organisms, participate in various life activities of the organisms, and has various biological functions. Therefore, the research on EPS is becoming the leading research field of related disciplines, and with the research, the biological functions of EPS, such as cholesterol reduction, antioxidation, antitumor, antivirus, immunoregulation and intestinal flora regulation, are being explored.
In view of the above-mentioned strong biological functions of EPS, EPS has become a research hotspot in the development and utilization of lactic acid bacteria resources. However, due to the low yield of certain EPS-producing microorganisms, many applications of EPS (e.g.cholesterol or antioxidant) are limited, making it essential to increase the EPS yield of the microorganisms.
Disclosure of Invention
The invention aims to provide Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07), the preservation number of which is CGMCC No. 24628. The strain is separated from naturally fermented yak acid milk of a Sichuan Hongyen herdsman, has good gastrointestinal tract resistance, higher EPS yield and higher cholesterol and DPPH free radical removal rate, and has the potential of reducing cholesterol and resisting oxidation.
Further, the lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) has a 16SrDNA sequence shown as SEQ ID No. 1.
The invention also aims to provide a fermentation composition for exopolysaccharide preparation, which comprises the lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07).
Further, the fermentation composition also comprises
Peptone and/or soy peptone. In general, when using Lactobacillus fermentum for the preparation of exopolysaccharides, MRS liquid medium containing 10mg/L peptone is used. In the invention, the peptone in the MRS liquid culture medium is replaced by the same quantity
Peptone or soyabean peptone, which obviously improves the EPS content. In actual production, the lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) is inoculated into a culture medium
The effect of increasing the EPS content can be achieved in a culture medium of peptone or soybean peptone.
Further, the preparation of the MRS liquid culture medium is as follows: adding distilled water and supplementing distilled water to 1000mL, adjusting pH to 6.3, stirring, heating, boiling for 2min, and sterilizing at 121 deg.C and 0.1Mpa for 30 min.
The invention also aims to provide a preparation method of the exopolysaccharide, which can improve the yield of the exopolysaccharide to (469.0 +/-13.0) mg/L at most. The preparation method comprises the following steps: prepared using any of the above described Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) or the fermented composition described above.
Further, the Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) is used for inoculation fermentation, and the inoculation amount is 2-4%.
Further, the Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) was inoculated into a culture medium containing
And (3) performing fermentation culture in MRS liquid medium (not containing peptone) of peptone and/or soybean peptone. Wherein, other components of the MRS liquid culture medium can be replaced by other equivalent substances.
Further, the method for preparing the exopolysaccharide comprises the following steps: lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) was inoculated at 3% inoculation rate
Culturing in MRS liquid culture medium with peptone and/or soybean peptone instead of peptone at 37 deg.C for 18 h; after the third generation, centrifuging at 4 ℃ and 10000r/min for 15min, removing precipitated thalli, and reserving strain fermentation supernatant; adding 95% ethanol with volume 3 times of the supernatant, standing at 4 deg.C for 24 hr, centrifuging for 15min, decanting the supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing at 4 deg.C for 24 hr in dialysis bag; to the liquid obtained from dialysis was added 25% volume fraction sevage reagent [ V (chloroform): V (n-butanol) ═ 4: 1]Oscillating in shaking table at room temperature for 30min to adsorb protein in organic phase, centrifuging at 8000r/min for 1min, and keeping water phase; repeating for 4-5 times until protein is completely removed to obtain EPS solution.
The invention also aims to provide the exopolysaccharide prepared by the preparation method of the exopolysaccharide. In certain embodiments, the exopolysaccharide of the present invention consists essentially of glucose, galactose, and fructose in a molar ratio of 11.5:2.7: 1.0.
The present invention also aims to provide a medicine or food having an antioxidant effect and a cholesterol-lowering effect. The medicine or food comprises an effective amount of the Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) or the extracellular polysaccharide and auxiliary materials; the auxiliary material is an edible carrier and/or diluent, or the auxiliary material is a medicinal carrier and/or diluent.
Further, the effective amount refers to an amount that produces exopolysaccharides or cholesterol-lowering or antioxidant effects.
Further, the edible or pharmaceutically acceptable carrier and/or diluent is suitable for a gastrointestinal administration form. The Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) has stronger gastrointestinal tract tolerance capability measurement, can survive in the gastric environment and keeps activity.
Further, the edible or pharmaceutically acceptable carrier and/or diluent is suitably used in any one of a solution form, a colloidal solution form, an emulsion form, a suspension form, a gas dispersion form, a fine particle dispersion form, or a solid dispersion form.
Further, Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) was prepared into probiotic beverage by the form of bacterial suspension; or prepared into probiotic tablets in the form of tablets for eating.
Further, the exopolysaccharide is prepared into candy, beverage or sugar tablet for eating.
Further, a fermentation broth of Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) was prepared in liquid or paste form for consumption.
Further, Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) or exopolysaccharide is combined with other probiotics or other medicines or foods beneficial to human body, and the respective effects are combined to act together.
The invention also provides application of any one of the Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) or any one of the fermented composition or any one of the extracellular polysaccharide in preparation of an antioxidant preparation.
Further, in the normal case, reactive oxygen species in the human body are byproducts of aerobic metabolism, and the production and elimination thereof maintain the oxidation-antioxidation balance, which plays an important role in regulating the conduction of signal pathways and cell proliferation. When the balance is disrupted, reactive oxygen species levels rise, leading to the production of free radicals, resulting in oxidative stress of the body, causing oxidative damage to cells and developing multisystemic diseases. The lactobacillus EPS has an antioxidant function in vivo or in vitro, and can participate in the scavenging of free radicals, thereby being used as a natural safe antioxidant. The removal rate of the lactobacillus or the extracellular polysaccharide prepared by the lactobacillus in the invention on DPPH free radicals is as high as (31.2 +/-0.6)%, and is obviously higher than that of the existing lactobacillus TY-G02, TY-G03 and TY-G04.
The invention also provides the application of any one of the Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) or any one of the fermented compositions or any one of the exopolysaccharides in preparing a preparation for reducing cholesterol.
Further, cholesterol, a derivative of cyclopentanoperhydrophenanthrene, is a major steroid compound in mammals and plays an important role in basic cell life activities. Nevertheless, when the content of cholesterol in a human body is too high, the cardiovascular health of the human body is damaged, so that the control of the level of cholesterol in the human body has important significance, and the EPS can inhibit the increase of blood pressure and reduce the total level of serum cholesterol; in addition, EPS can reduce the total cholesterol content in the experimental system by adsorption. The removal rate of cholesterol by the lactobacillus or the extracellular polysaccharide prepared by the lactobacillus in the invention can reach (17.8 +/-0.5)%, and is obviously higher than that of the existing lactobacillus TY-G02, TY-G03 and TY-G04 of the same species.
Further, in the above-mentioned application, the formulation includes a food or a medicine, and the dosage form includes any one of a solution dosage form, a colloidal solution dosage form, an emulsion dosage form, a suspension dosage form, a gas dispersion dosage form, a fine particle dispersion dosage form, or a solid dispersion dosage form.
Further, in the above application, other active substances with antioxidant effect or cholesterol-lowering effect can be added into the preparation for improving antioxidant effect or cholesterol-lowering effect.
The preservation information of the lactobacillus fermentum TY-S07(Lactobacillus fermentum LF15-3) in the invention is as follows: is preserved in China general microbiological culture Collection center (CGMCC), the preservation date is 2022 years, 4 months and 1 day, the preservation number is CGMCC No.24628, and the lactobacillus fermentum is classified and named as lactobacillus fermentum TY-S07(Lactobacillus 15-3).
In the present invention, the term "inoculum amount" refers to the ratio of the volume of the seed solution transferred to the volume of the culture solution after inoculation.
The invention has the beneficial effects that: the survival rate of the lactobacillus fermentum TY-S07 with the preservation number of CGMCC No.24628 in artificial gastric juice is as high as 58.46%, the survival rate in bile salt is 12.06%, and the lactobacillus fermentum has good gastrointestinal tract resistance; TY-S07 also has the highest EPS yield which is up to (318.0 +/-10.9) mg/L, and after the fermentation conditions are optimized, the EPS yield is up to (469.0 +/-13.0) mg/L, which is increased by 47.5% compared with the optimized conditions; TY-S07 also has higher cholesterol removal rate, which is up to (11.7 +/-0.2)%, and after the fermentation condition is optimized, the cholesterol removal rate is (17.8 +/-0.5)%, which is improved by 52.1% compared with that before optimization; TY-S07 also has higher DPPH free radical removal rate, and the highest rate is (31.2 +/-0.6)%.
Drawings
FIG. 1 shows the colony morphology of TY-S07.
FIG. 2 shows the results of TY-S07 gram-stain.
FIG. 3 shows the effect of TY-S07 colony stringiness.
FIG. 4 is a glucose standard curve.
FIG. 5 shows EPS contents in fermentation supernatants of 34 L.fermentum strains.
FIG. 6 shows the EPS content in TY-S07 fermentation supernatants after fermentation with different nitrogen sources.
FIG. 7 is a standard curve for cholesterol.
FIG. 8 shows cholesterol removal rates of different strains.
FIG. 9 shows DPPH radical removal rates of fermentation supernatants of different strains.
In fig. 2, the gram stain was actually purple, and black and white treatment was performed to meet the requirements of the application.
Detailed Description
The examples are given for the purpose of better illustration of the invention, but the invention is not limited to the examples. Therefore, those skilled in the art should make insubstantial modifications and adaptations to the embodiments of the present invention in light of the above teachings and remain within the scope of the invention.
In the examples of the present invention, comparative Lactobacillus fermentum TY-G02, Lactobacillus fermentum TY-G03, and Lactobacillus fermentum TY-G04 were obtained from Tianyou Dairy, Inc., Chongqing, and were publicly deposited. Wherein, the lactobacillus fermentum TY-G02 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCC No. 23754; the lactobacillus fermentum TY-G03 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms with the preservation number of CGMCCNo.23753; the lactobacillus fermentum TY-G04 is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No. 23752.
In the embodiment of the invention, the MRS broth culture medium is purchased from Beijing Luqiao technology GmbH, and the specific formula is as follows: 10.0g of peptone, 10.0g of beef powder, 5.0g of yeast powder, 20.0g of glucose, 5.0g of sodium acetate, 2.0g of ammonium citrate, 801.0g of tween, 2.0g of dipotassium phosphate, 0.1g of magnesium sulfate and 0.05g of manganese sulfate. The MRS liquid culture medium is prepared by weighing 55.2g MRS broth before use, culturing in 1L distilled water, adjusting pH to 6.3, stirring, heating, and sterilizing at 121 deg.C and 0.1Mpa for 15 min. The MRS solid culture medium is added with 2% (w/w) agar powder on the basis of the MRS liquid culture medium.
Example 1 separation, purification and characterization of TY-S07
In the embodiment of the invention, the experimental material source and the separation and purification are as follows: yak yogurt naturally fermented by herdsman in Qiang autonomous Zhou Hongyuan county of Akebia of Alba of Sichuan province. Taking naturally fermented yak yogurt in herdsman by using a small sterile spoon, putting the yak yogurt into a 15mL sterile spiral cover centrifuge tube containing a proper amount of sterile calcium carbonate and soluble starch (the mass ratio of the calcium carbonate to the soluble starch is 1: 1), uniformly stirring, screwing the spiral cover, putting the yak yogurt into a refrigerator, and conveying the yak yogurt back to a laboratory for immediately purifying and separating lactic acid bacteria.
(1) Separating and purifying
Under the aseptic condition, 1mL of sample is absorbed into 9mL of sterile physiological saline, and the sample is uniformly mixed by vortex to obtain 10 -1 The sample diluent is sequentially subjected to gradient dilution by 10 times to 10 -7 Choose 10 -5 、10 -6 、10 -7 mu.L of the diluted solution was uniformly spread on an MRS plate, and cultured in an inverted state at 37 ℃ for 48 hours. And after the culture is finished, observing the colony morphology on the MRS plate, selecting a typical colony of the lactobacillus, purifying the strain by plate streaking, and repeating the streaking operation until the purified strain is obtained.
The morphology of the purified colony is shown in figure 1, the bacterial strain forms a single colony in a solid culture medium after being purified, the colony is large, round, semitransparent, basically not convex, unsmooth in surface, irregular in edge and light yellow in color.
(2) Morphological structure observation
The purified strain was inoculated into 5mL of sterile MRS liquid medium and cultured at 37 ℃ for 18 h. Centrifuging 1mL of bacterial solution at 12000r/min for 1min, washing twice with sterile normal saline, adding equal volume of sterile normal saline to resuspend the thallus, taking a small amount of the thallus by using an inoculating loop, uniformly coating the thallus on a glass slide, fixing, performing gram staining, performing microscopic examination, and photographing. Cell morphology and gram staining results were observed and recorded. The gram-positive bacteria (G +) stained cells are bluish purple, and the gram-negative bacteria (G-) stained cells are red.
The gram staining result is shown in FIG. 2, the cells are purple under the microscope, are gram-positive bacteria (G +), are rod-shaped, and have the same characteristics with lactobacillus and uniform morphological structure, which indicates that the strain is pure.
(3) PCR amplification of 16S rDNA sequences
PCR amplification was performed using a 25. mu.L reaction: mu.L of template, 1. mu.L of upstream primer (10. mu.M in concentration and SEQ ID No.2 in sequence), 1. mu.L of downstream primer (10. mu.M in concentration and SEQ ID No.3 in sequence), and 2 XTaqPCRMasterMix12.5. mu.L, and the final volume was 25. mu.L with sterile ultrapure water. PCR amplification conditions: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 1min for 35 cycles; terminal extension at 72 ℃ for 10 min. After the sequence amplification, the PCR amplification product qualified by detection is sequenced by the company of Weitusheng bioengineering (Shanghai) and the obtained sequence is shown as SEQIDNo.1 and is identified as lactobacillus fermentum.
Example 2 TY-S07 determination of gastrointestinal tolerability
In the embodiment of the invention, gastrointestinal tract tolerance of the lactobacillus fermentum TY-S07 (the yak milk isolated from natural fermentation of Sichuan Hongyuan herdsman and preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24628) isolated in the example 1 is measured.
(1) TY-S07 determination of resistance to pH3.0 Artificial gastric juice
Probiotics entering the human body exert corresponding functional activity, so that the probiotics have good gastrointestinal tract resistance. Before entering the human intestinal tract to exert the functions of the probiotics, the probiotics firstly pass through the stomach (the pH of gastric juice is about 3.0, and the retention time is 1-3h), and the strongly acidic gastric environment is not beneficial to the survival of the probiotics, so that the probiotics serving as the probiotics are required to exert the corresponding functional activity and have the primary condition of being capable of tolerating the gastric juice in the human body.
TY-S07 is inoculated in MRS liquid culture medium according to the inoculation amount of 3 percent, cultured for 18 hours at 37 ℃ and activated for three generations; centrifuging 2mL of the suspension at 8000r/min for 10min, collecting thallus precipitate, washing with sterile PBS solution for 2 times, and suspending in equal volume of sterile physiological saline to obtain a thallus suspension; mixing the prepared bacterial suspension with artificial gastric juice (0.2% of NaCl and 0.35% of pepsin in a volume ratio of 1: 10000, adjusting the pH value to 3.0 by using 1mol/L of HCl, filtering and sterilizing for later use) according to a ratio of 1: 9(v/v), mixing uniformly, placing in a constant temperature oscillator at 37 ℃ for 3h at 100r/min, and measuring the viable count of 0h and 3h respectively by adopting a pour plate method. The survival rate of the strain tolerant to pH3.0 artificial gastric juice was calculated according to the formula (1):
the experimental result shows that the survival rate of TY-S07 cultured in artificial gastric juice with the pH value of 3.0 for 3h is 58.46%, and the gastric juice tolerance is better.
(2) TY-S07 determination of 0.3% bile salt tolerance
After entering the intestinal tract, the probiotics survived by gastric juice treatment can be inhibited and poisoned by bile salts in the small intestine, so that the tolerance of the strain to the bile salts is also one of important indexes for screening the probiotics, and the mass concentration of the bile salts in a human body fluctuates within the range of 0.03-0.3%.
TY-S07 is inoculated in MRS liquid culture medium according to the inoculation amount of 3 percent, cultured for 18 hours at 37 ℃ and activated for three generations; centrifuging 2mL at 8000r/min for 10min to collect thallus precipitate, washing with sterile PBS solution for 2 times, suspending in sterile physiological saline containing 0.0% and 0.3% of ox-gall salt, mixing, placing in a constant temperature oscillator at 37 deg.C, centrifuging at 100r/min, culturing for 2h, and measuring viable count in 0.0% and 0.3% of ox-gall salt culture solution by pour plate method. The survival rate of the strain in bile salts was calculated according to the formula (2):
the experimental result shows that the growth efficiency of TY-S07 in 0.3% bile salt is 12.06%, and the bile salt has better tolerance.
Example 3 EPS production test
In the embodiment of the invention, EPS yield test is carried out on the lactobacillus fermentum TY-S07 (isolated from natural fermented yak yogurt of Sichuan Hongyuan herdsman, preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24628) isolated in the example 1.
(1) Bacterial colony wire drawing performance test
EPS is a capsule secreted by microorganisms during the growth metabolism to the extracellular mucus or attached to the cell surface. Therefore, colonies of the high EPS producing strain should have good stringing property, which is also a common method for screening high EPS producing lactic acid bacteria according to phenotype.
In the embodiment of the invention, the colony wire drawing performance test comprises the following steps: inoculating the strain into MRS liquid culture medium according to the inoculation amount of 3%, culturing at 37 ℃ for 18h, after activating for three generations, using an inoculating loop to dip bacterial liquid, streaking on MRS solid culture medium, and culturing at 37 ℃ for 48 h; the single colonies obtained by streaking were picked with a toothpick, and the lengths of the streaks were compared.
The 118L lactobacillus fermentum in the probiotic reservoir of Tianyou dairy gmbh, Chongqing, was tested for stringiness according to the method described above, wherein TY-S07 had the longest stringiness, and the stringiness is shown in FIG. 3.
(2) Determination of EPS content
Drawing a glucose standard curve
Taking 0.2mg/mL glucose as a standard solution, taking 0.0mL, 0.1mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL of the standard solution, and supplementing water to 2.0 mL; transferring 200 μ L of glucose solution with different concentrations into a clean test tube, adding 100 μ L of 5% phenol solution, shaking, and slowly adding 500 μ L of concentrated H 2 SO 4 Preventing the liquid drops from splashing; standing in dark place for 30min, placing the reaction solution in a 96-well plate, and determining the concentration of glucose as abscissa and OD 490nm The values are plotted on the ordinate and the standard curve is plotted, which is shown in FIG. 4.
② measuring the EPS content in the supernatant fluid of the bacterial strain fermentation
In the embodiment of the invention, the content of EPS in the strain fermentation supernatant is measured by adopting a phenol-sulfuric acid method, and the method specifically comprises the following steps: transferring the EPS200 μ L into a clean test tubeAdding 100 μ L of 5% phenol solution, shaking, and slowly adding 500 μ L concentrated H 2 SO 4 Preventing the liquid drops from splashing; standing in dark place for 30min, measuring OD 490nm And calculating the EPS content according to the standard curve.
In the embodiment of the invention, the EPS content in the fermentation supernatant of 34 strains of lactobacillus fermentum is measured in total, and the experimental result is shown in FIG. 5, the EPS content in the fermentation supernatant of the TY-S07 strain is higher and reaches (318.0 +/-10.9) mg/L, which shows that the TY-S07 strain has the capability of high-yield EPS.
Example 4 fermentation condition optimization
In the embodiment of the invention, experiments for obtaining different EPS yields under different fermentation conditions are carried out on the fermented lactobacillus TY-S07 (the yak yogurt which is separated from natural fermentation of Sichuan Hongyuan herdsman and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No. 24628).
(1) Determination of EPS content under different nitrogen source conditions
The EPS production capacity of the same lactic acid bacteria strain under different fermentation conditions is different, and the peptone is formed by decomposing protein by acid, alkali or protease, is rich in organic nitrogen compounds and can provide a nitrogen source for the growth of microorganisms.
Replacing the peptone in the MRS liquid culture medium with tryptone in the same amount (10g/L) respectively,
Peptone, casein peptone, bacterial peptone and soy peptone; inoculating the strain into MRS liquid culture medium replacing peptone according to the inoculation amount of 3%, culturing at 37 ℃ for 18h, and activating for three generations; centrifuging at 4 deg.C and 10000r/min for 15min, removing precipitate thallus, and retaining strain fermentation supernatant; adding 95% ethanol with volume 3 times of the supernatant, standing at 4 deg.C for 24 hr, centrifuging for 15min, decanting the supernatant, dissolving the precipitate with appropriate amount of distilled water, and dialyzing at 4 deg.C for 24 hr in dialysis bag; adding sevage reagent with volume fraction of 25% (V (chloroform):V (n-butanol) ═ 4: 1) to the liquid obtained by dialysis]Oscillating in shaking table at room temperature for 30min to adsorb protein in organic phase,centrifuging at 8000r/min for 1min, and keeping water phase; repeating for 4-5 times until protein is completely removed to obtain EPS solution.
The method for measuring the EPS content in the strain fermentation supernatant by adopting a phenol-sulfuric acid method comprises the following steps: transferring 200 μ L of EPS into a clean test tube, adding 100 μ L of 5% phenol solution, shaking, and slowly adding 500 μ L of concentrated H 2 SO 4 Preventing the liquid drops from splashing; standing in dark place for 30min, measuring OD 490nm And calculating the EPS content according to the standard curve.
TY-S07 EPS yield under different nitrogen source conditions is shown in FIG. 6, in the original MRS liquid culture medium (not replacing peptone), TY-S07 EPS yield is (318.0 + -10.9) mg/L; replacing peptone in MRS liquid medium with equal amount
After peptone, the EPS yield is (469.0 +/-13.0) mg/L, which is improved by 47.5%; after the peptone in the MRS liquid medium is replaced by the same amount of soybean peptone, the EPS yield is (444.7 +/-1.6) mg/L, and is improved by 39.8%.
(2) Viable count under different nitrogen source conditions
In the embodiment of the invention, viable bacteria count is carried out for exploring the reason of the EPS production capacity improvement after nitrogen source replacement. The method comprises the following specific steps: replacing the peptone in the MRS liquid culture medium with tryptone in the same amount (10g/L) respectively,
Peptone, casein peptone, bacterial peptone and soy peptone; inoculating the strain into MRS liquid culture medium substituted with peptone according to the inoculation amount of 3%, culturing at 37 deg.C for 18h, activating for three generations, and counting viable bacteria by pouring method.
The viable count results of different nitrogen sources are shown in Table 1, and the viable count of TY-S07 is one order of magnitude under different nitrogen sources, which indicates that the growth capacity is unchanged, but the EPS production capacity is improved.
TABLE 1 live TY-S07 count under different nitrogen sources
Example 5EPS monosaccharide composition analysis
In the present invention, the composition of monosaccharides of EPS produced by lactobacillus fermentum TY-S07 (yak yogurt isolated from nature fermentation of herdsman in red origin, chuanxiong, and deposited in the general microbiological culture collection center of the china committee for culture collection of microorganisms, with the collection number of CGMCC No.24628) isolated in example 1 was analyzed.
The same amount (10g/L) of peptone in MRS liquid medium is replaced by
Peptone, EPS solution was prepared by the method in example 4, and EPS powder was obtained after freeze-drying; weighing 10mg of EPS powder, adding 2mL of 2M trifluoroacetic acid, sealing the bottle, and hydrolyzing in an oven at 120 ℃ for 2 h; after cooling, trifluoroacetic acid is removed by evaporation with a rotary evaporator, a small amount of methanol is added and evaporated to dryness to remove residual trifluoroacetic acid, and the process is repeated for 3 times; finally, a small amount of water is added for dissolving to obtain a monosaccharide sample for complete acid hydrolysis, the monosaccharide composition is measured by an ion chromatograph, and the ion chromatographic analysis conditions are shown in the following table 2.
TABLE 2 ion chromatography conditions
In the embodiment of the invention, the monosaccharide composition of the EPS generated by TY-S07 after the optimization of the culture medium is analyzed, wherein the EPS mainly comprises glucose, galactose and fructose according to the molar ratio of 11.5:2.7: 1.0.
Example 6 determination of Cholesterol lowering ability
In the embodiment of the invention, the lactobacillus fermentum TY-S07 (the yak yogurt which is separated from natural fermentation of Sichuan monarch herdsman and is preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24628) separated in the example 1 is subjected to cholesterol reduction capability measurement and is compared with the lactobacilli TY-G02, TY-G03 and TY-G04.
In the embodiment of the invention, the MRS-CHOL culture medium is configured as follows: dissolving cholesterol in anhydrous ethanol to obtain 10.0mg/mL cholesterol solution, sterilizing with 0.45 μm microporous membrane, and adding into sterile MRS liquid culture medium at a ratio of 1% (v/v) to obtain MRS-CHOL culture medium containing 0.1mg/mL cholesterol solution.
(1) Drawing a cholesterol standard curve
Taking 5 test tubes, numbering according to 1-5, adding MRS-CHOL culture medium 0.0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL, 0.5mL respectively in sequence, and supplementing the test tubes to 0.5mL with glacial acetic acid; adding 0.2mL (1mg/mL, absolute ethyl alcohol is dissolved) of the o-phthalaldehyde working solution into each test tube, and shaking and uniformly mixing; standing for 10min, respectively adding 4.0mL of mixed acid (glacial acetic acid and concentrated sulfuric acid are mixed according to a volume ratio of 1: 1), and uniformly mixing; standing at room temperature for 10min, placing the reaction solution in a 96-well plate, and determining the concentration of cholesterol as abscissa and OD 550nm The values are plotted on the ordinate of a standard curve, and the standard curve of cholesterol is shown in FIG. 7.
(2) Determination of Cholesterol removal Rate
Culturing the strain at 37 ℃ for 18h, activating the third generation, adding the strain into 5mL of MRS-CHOL culture medium according to the inoculation amount of 3%, and culturing at 37 ℃ for 24 h; simultaneously, rapidly determining a sample for 0h according to an o-phthalaldehyde colorimetric method, centrifuging a culture medium of the just-inoculated bacteria for 10min at 9000r/min, taking 0.5mL of supernatant, adding 0.2mL of o-phthalaldehyde working solution, standing for 10min after fully shaking, adding 4.0mL of mixed acid, standing for 10min at room temperature, placing the reaction solution into a 96-well plate, and determining the OD of the reaction solution 550nm The value is obtained. After the strain is cultured for 24h, determining the OD of the fermentation liquor according to the o-phthalaldehyde method 550nm The value is obtained. And finally, determining the cholesterol content in the fermentation liquor according to a fitting equation of a cholesterol standard curve, and calculating the removal rate of the cholesterol according to a formula (3):
wherein: a is the cholesterol content in the supernatant after the fermentation of the strain; b is the cholesterol content of the supernatant before fermentation of each test strain.
In the embodiment of the invention, the cholesterol removal rates of TY-S07, TY-G02, TY-G03 and TY-G04 lactobacilli before and after optimization of a culture medium are determined, and the experimental result is shown in FIG. 8, the cholesterol removal rate of the unoptimized TY-S07 is (11.7 +/-0.2)%, which is obviously higher than that of TY-G02, TY-G03 and TY-G04 lactobacilli, which indicates that TY-S07 has higher cholesterol removal rate; the cholesterol removal rate of the optimized TY-S07 is (17.8 +/-0.5)%, which is improved by 52.1% compared with that before optimization, and the improvement is remarkable.
Example 7 antioxidant capacity assay
In the embodiment of the invention, the antioxidant capacity of the lactobacillus fermentum TY-S07 (isolated from natural fermented yak yogurt of Sichuan Hongyuan herdsman, preserved in China general microbiological culture Collection center with the preservation number of CGMCC No.24628) isolated in the example 1 is measured and compared with the lactobacilli TY-G02, TY-G03 and TY-G04.
Inoculating the strain in MRS liquid culture medium according to 3% of inoculum size, culturing at 37 deg.C for 18h, activating for three generations, centrifuging at 4 deg.C and 10000r/min for 15min, removing precipitated thallus, and collecting strain fermentation supernatant; adding 1ml of 0.2mmol/LDPPH absolute ethyl alcohol solution, fully and uniformly mixing, and reacting for 30min in the dark at room temperature; then centrifuging at 6000r/min for 10min, and taking supernatant for determination.
OD was determined by the same procedure as above, except that the same volume of MRS liquid medium was used instead of the sample solution 517nm Value, OD in the formula (4) 517nm (blank). And the volume of the mixed solution of physiological saline and absolute ethyl alcohol is equal to the volume of the mixed solution, and the mixed solution is used as a blank for zero adjustment. The DPPH radical clearance was calculated according to equation (4):
in the embodiment of the invention, the DPPH free radical clearance rates of TY-S07, TY-G02, TY-G03 and TY-G04 lactobacilli before and after culture medium optimization are determined, and the experimental result is shown in FIG. 9, the DPPH free radical clearance rate of unaptimized TY-S07 is (31.2 +/-0.6)%, the TY-G02, TY-G03 and TY-G04 lactobacilli are obviously high, which indicates that TY-S07 has higher DPPH free radical clearance rate; the DPPH free radical removal rate of the TY-S07 after optimization is (28.0 +/-0.3)%, although the DPPH free radical removal rate is not as high as that before optimization, the DPPH free radical removal rate and the DPPH free radical removal rate are not obviously different and are also obviously higher than TY-G02, TY-G03 and TY-G04 lactobacilli.
All the above examples are combined to test that the lactobacillus fermentum TY-S07 of the present embodiment has good gastrointestinal tract resistance. The bacterial colony has stronger wiredrawing property and higher EPS output; the strain has high cholesterol and DPPH free radical removal rate, and has the potential of reducing cholesterol and resisting oxidation.
Finally, the above embodiments are only intended to illustrate the technical solution of the present invention and not to limit the same, and although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that modifications or equivalent substitutions may be made to the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention, which shall be covered by the claims of the present invention.
Claims (10)
1. Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) with preservation number of CGMCC No. 24628.
2. Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) according to claim 1, having the 16SrDNA sequence as shown in SEQ ID No. 1.
3. A fermented composition for exopolysaccharide production, characterized in that it comprises lactobacillus fermentum TY-S07(lactobacillus fermentum TY-S07) according to claim 1 or 2.
4. The fermentation composition of claim 3, further comprising
Peptone and/or soy peptone.
5. A method for preparing exopolysaccharide, which is characterized by comprising the following steps: prepared using lactobacillus fermentum TY-S07(lactobacillus fermentum TY-S07) according to claim 1 or 2 or a fermented composition according to claim 3 or 4.
6. The method according to claim 5, wherein the fermentation is carried out by inoculation with Lactobacillus fermentum TY-S07(Lactobacillus fermentum TY-S07) according to claim 1 in an amount of 2-4%.
7. Exopolysaccharide prepared by the method of preparation according to claim 5 or 6.
8. A medicament or food product comprising lactobacillus fermentum TY-S07(lactobacillus fermentum TY-S07) according to claim 1 or 2 or an exopolysaccharide according to claim 7, and adjuvants; the auxiliary material is an edible carrier and/or diluent, or the auxiliary material is a medicinal carrier and/or diluent.
9. The medicament or food product according to claim 8, wherein the edible or pharmaceutically acceptable carrier and/or diluent is suitable for use in any one of a solution dosage form, a colloidal solution dosage form, an emulsion dosage form, a suspension dosage form, a gas dispersion dosage form, a particulate dispersion dosage form, or a solid dispersion dosage form.
10. Use of lactobacillus fermentum TY-S07(lactobacillus fermentum TY-S07) according to claim 1 or 2, or of the fermented composition according to claim 3 or 4, or of the exopolysaccharide according to claim 7, as or for the preparation of a formulation for anti-oxidant formulation or for reducing cholesterol.
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