LU502581B1 - Lactobacillus fermentum TY-S07 and application thereof - Google Patents
Lactobacillus fermentum TY-S07 and application thereof Download PDFInfo
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- LU502581B1 LU502581B1 LU502581A LU502581A LU502581B1 LU 502581 B1 LU502581 B1 LU 502581B1 LU 502581 A LU502581 A LU 502581A LU 502581 A LU502581 A LU 502581A LU 502581 B1 LU502581 B1 LU 502581B1
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- lactobacillus fermentum
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- fermentation
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Abstract
The present invention belongs to the technical field of microorganisms and applications thereof, and specifically relates to a Lactobacillus fermentum TY-S07 and an application thereof, its preservation number is CGMCC No. 24628, and it has the good gastrointestinal tract resistance; and under a certain fermentation condition, the exopolysaccharides (EPS) yield thereof is up to (469.0±13.0) mg/L, and the EPS monosaccharide is mainly composed of a glucose, a galactose and a fructose in a molar ratio of 11.5:2.7:1.0. The Lactobacillus fermentum TY-S07 has the relatively high cholesterol removal capacity of (17.9±0.5)%, and also has the relatively high antioxidant capacity, and its 1,1-diphenyl-2-picryl-hydrazyl (DPPH) free radical scavenging rate reaches 31.2±0.6%.
Description
Lactobacillus fermentum TY-S07 and application thereof
LUSO2BRhos58 1
The present invention belongs to the technical field of microorganisms and applications thereof, and specifically relates to a Lactobacillus fermentum TY-S07 and an application thereof.
Lactobacillus (lactic acid bacteria) is a general term for a class of bacteria that may utilize fermentable carbohydrates to produce a large amount of a lactic acid, mainly including bacterial genuses of lactic acid bacillus, bifidobacterium, lactococcus lactis and streptococcus and the like.
As one of lactobacillus metabolites, an exopolysaccharides (EPS) is a water-soluble polysaccharide that is secreted outside cell walls by some special microorganisms (including the lactobacillus) in the growth and metabolism processes, is easily separated from the bacteria, and secreted into the environment. It belongs to a secondary metabolite of the microorganisms, and is of great significance to the growth of the microorganisms.
EPS, as an important secondary metabolite of the lactobacillus (LAB), may not only provide energy for a body, and participate in various life activities of the body, but also have a variety of biological functions. Therefore, researches related to EPS gradually become the frontier research field of related disciplines. With the continuous deepening of the researches, the biological functions of EPS such as cholesterol lowering, anti-oxidation, anti-tumor, anti-virus, immune regulation and intestinal flora regulation are continuously explored.
In view of the above powerful biological functions of EPS, EPS already becomes a research hotspot in the development and utilization of lactic acid bacteria resources. However, due to the lower yield of some EPS-generating microorganisms, many applications of EPS (such as the cholesterol lowering or the anti-oxidation) are limited, so that the improvement of the EPS yield of the microorganisms is crucial.
A purpose of the present invention is to provide a Lactobacillus fermentum TY-S07, and its preservation number is CGMCC No.24628. The strain is separated from natural fermented yak yoghurt in Hongyuan herdsmen's house in Sichuan, it has the good gastrointestinal resistance, has the higher EPS yield and higher cholesterol and DPPH free radical removal rate, and has the potential of cholesterol lowering and anti-oxidation.
Further, a 16SrDNA sequence of the Lactobacillus fermentum TY-S07 is shown in SEQ ID
No.l.
A purpose of the present invention is to further provide a fermentation composition for preparation of EPS, and it contains the above Lactobacillus fermentum TY-S07. "°PÉ0S02581
Further, the fermentation composition further includes a proteose peptone and/or a soy peptone. Generally, while the Lactobacillus fermentum is used to prepare EPS, an MRS liquid medium is used, and the MRS liquid medium contains 10 mg/L of a peptone. In the present invention, the peptone in the MRS liquid medium is replaced with the same amount of the proteose peptone or the soy peptone, the content of EPS is significantly increased. In the actual production, as long as the Lactobacillus fermentum TY-S07 is inoculated into a medium containing the proteose or the soy peptone, the effect of increasing the content of EPS may be achieved.
Further, the preparation of the MRS liquid medium is as follows: 10.0 g of the peptone, 10.0 g of a beef extract, 5.0 g of a yeast extract, 20.0 g of glucose, 5.0 g of a sodium acetate, 2.0 g of a citric acid diamine, 1.0 g of Tween (Tween 80), 0.4 g of a dipotassium hydrogen phosphate, 0.58 g of a magnesium sulfate, 0.29 g of a manganese sulfate, 20.0 g of a calcium carbonate, and 15.0 gof an agar are added in sequence with distilled water and the distilled water is supplemented to 1000 mL, a pH value is adjusted to 6.3, it is heated after being stirred, boiled for 2 min, and sterilized at 121°C and 0.1 Mpa for 30 min.
A purpose of the present invention is to further provide a preparation method for EPS, and the method may increase the yield of EPS to (469.0+13.0) mg/L at the highest. The preparation method thereof includes: using any one of the above Lactobacillus fermentum TY-S07 or the above fermentation composition to prepare.
Further, any one of the above Lactobacillus fermentum TY-S07 is used for inoculation and fermentation, and the inoculation amount thereof is 2%-4%.
Further, the Lactobacillus fermentum TY-S07 is inoculated in the MRS liquid medium (without containing the peptone) containing the proteose peptone and/or the soy peptone for fermentation culture. Herein, other components of the MRS liquid medium may be replaced with other equivalent substances.
Further, the method for preparing EPS is as follows: by 3% of the inoculation amount, the
Lactobacillus fermentum TY-S07 is inoculated in the MRS liquid medium in which the peptone is replaced with the proteose peptone and/or the soy peptone, and cultured at 37°C for 18 h; after three generations of activation, it is centrifuged at 4°C and 10000 r/min for 15 min, to remove precipitated bacteria, and a strain fermentation supernatant is retained; 3-fold volume of 95% ethanol is added to the supernatant, it is standing at 4°C for 24 h, and then centrifuged for 15 min, the supernatant is poured out, and after a precipitate is dissolved with an appropriate amount of the distilled water, it is placed in a dialysis bag and dialyzed at 4°C for 24 h; 25% of a sevage reagent [V(chloroform):V(n-butyl)=4:1] in volume fraction is added to liquid obtained by the dialysis, it is placed on a shaker at a room temperature and shaken for 30 min, so that a protein = PVÉ0S02581 adsorbed in an organic phase, and then it is centrifuged at 8000 r/min for 1 min, an aqueous phase is retained; and it is repeated for 4-5 times, until the protein is completely removed, to obtain EPS solution.
A purpose of the present invention is to further provide EPS prepared by any one of the preparation methods for EPS. In some specific embodiments, EPS in the present invention is mainly composed of glucose, galactose, and fructose in a molar ratio of 11.5:2.7:1.0.
A purpose of the present invention is to further provide a drug or food, and the drug or food has the antioxidant effect and cholesterol lowering effect. The drug or food includes an effective amount of any one of the above Lactobacillus fermentum TY-S07 or an effective amount of the above EPS and an auxiliary material; and the auxiliary material is an edible carrier and/or diluent, or the auxiliary material is a pharmaceutically acceptable carrier and/or diluent.
Further, the effective amount refers to an amount that may produce EPS or cholesterol lowering or antioxidant effect.
Further, the edible or pharmaceutically acceptable carrier and/or diluent is suitable for a gastrointestinal administration dosage form. The Lactobacillus fermentum TY-S07 has the stronger gastrointestinal tract endurance capacity, and may survive and maintain activity in a gastric environment.
Further, the edible or pharmaceutically acceptable carrier and/or diluent is suitable for 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 particle dispersion dosage form or a solid dispersion dosage form.
Further, the Lactobacillus fermentum TY-S07 is prepared into a probiotic beverage in the form of bacterial suspension; or prepared into a probiotic tablet and the like in the form of tablets for consumption.
Further, EPS is prepared into a candy, a beverage or a sugar tablet and other forms for consumption.
Further, fermentation broth of the Lactobacillus fermentum TY-S07 is prepared into a liquid or paste form for consumption.
Further, the Lactobacillus fermentum TY-S07 or EPS is used in combination with other probiotics or other beneficial drugs or foods for a human body, and the respective effects work together.
A purpose of the present invention is to further provide an application of any one of the above Lactobacillus fermentum TY-S07 or the above fermentation composition or the above EPS as or in preparing a preparation having the antioxidant effect.
Further, under a normal condition, an active oxygen substance in the human body is a PVÉ0S02581 by-product of aerobic metabolism, and its production and elimination maintains an oxidation-antioxidation balance, this plays an important role in regulating the conduction of signaling pathways and cell proliferation. While the balance is disrupted, the level of the active oxygen substance is increased, resulting in the generation of a free radical, and the oxidative stress in the body, the cell oxidative damage is caused and developed into a multi-system disease.
Lactobacillus EPS has the antioxidant function both in vivo and in vitro, and may participate in free radical scavenging, thus it is used as a natural safe antioxidant. The lactobacillus in the present invention or EPS prepared by the lactobacillus in the present invention has a high removal rate of a DPPH free radical, it is as high as (31.2+0.6)%, and is significantly higher than that of existing lactobacillus TY-G02, TY-G03, and TY-G04.
A purpose of the present invention is to further provide an application of any one of the above Lactobacillus fermentum TY-S07 or the above fermentation composition or the above EPS as or in preparing a preparation for lowering a cholesterol.
Further, the cholesterol, a cyclopentane polyhydrophenanthrene derivative, is a main steroid compound in mammals, and plays an important role in basic cellular life activities. However, while the cholesterol content in the human body is too high, the cardiovascular health of the human body may be endangered, so it is of great significance to control the cholesterol level in the human body. EPS may inhibit the increase of a blood pressure and reduce the total level of a serum cholesterol; and in addition, EPS may reduce the total cholesterol content in an experimental system by adsorption. The lactobacillus in the present invention or EPS prepared by the lactobacillus in the present invention has a high removal rate of the cholesterol, and the highest removal rate may reach (17.8+0.5)%, and is significantly higher than that of the same species of the existing lactobacillus TY-G02, TY-G03 and TY-G04.
Further, in the above application, the preparation includes a food or a drug, and the dosage form includes any one of the solution dosage form, the colloidal solution dosage form, the emulsion dosage form, the suspension dosage form, the gas dispersion dosage form, the particle dispersion dosage form or the solid dispersion dosage form.
Further, in the above application, other active substances with the anti-oxidative effect or cholesterol lowering effect may also be added in the preparation to work together to improve the anti-oxidative effect or cholesterol lowering effect.
Preservation information of the Lactobacillus fermentum TY-S07 (Lactobacillus fermentum
LF15-3) in the present invention is as follows: it is preserved in China General Microbiological
Culture Collection Center (CGMCC), the preservation date is April 11, 2022, the preservation number is CGMCC No. 24628, and the classification is named as Lactobacillus fermentum
TY-S07 (Lactobacillus fermentum LF15-3). "°PÉ0S02581
In the present invention, a term "inoculation amount" refers to a ratio of the volume of inoculated seed solution and the volume of culture solution after inoculation. 5 The beneficial effects of the present invention are as follows: the survival rate of the
Lactobacillus fermentum TY-S07 with the preservation number CGMCC No.24628 in the present invention is up to 58.46% in artificial gastric juice, the survival rate in bile salt is 12.06%, and it has the good gastrointestinal tract resistance; TY-S07 also has the highest EPS yield, it is up to (318.0+10.9) mg/L, and after a fermentation condition is optimized, its EPS yield reaches (469.0+13.0) mg/L, it is increased by 47.5% compared to that before the optimization; TY-S07 also has the higher cholesterol removal rate, it is up to (11.7+0.2)%, and after the fermentation condition is optimized, the cholesterol removal rate is (17.8+£0.5)%, it is increased by 52.1% compared to that before the optimization; and TY-S07 also has the higher DPPH free radical removal rate, and the highest removal rate may reach (31.2+0.6)%.
Fig. 1 is a TY-S07 colony morphology.
Fig. 2 is a TY-S07 Gram staining result.
Fig. 3 is a TY-S07 colony drawing effect.
Fig. 4 is a glucose standard curve.
Fig. 5 is an EPS content in a fermentation supernatant of 34 strains of lactobacillus fermentum.
Fig. 6 is an EPS content in a TY-S07 fermentation supernatant after fermentation of different nitrogen sources.
Fig. 7 is a cholesterol standard curve.
Fig. 8 is cholesterol removal rates of different strains.
Fig. 9 is DPPH free radical removal rates of fermentation supernatants of the different strains.
Herein, in Fig. 2, the Gram staining result is actually purple, in order to meet the application text requirements, black and white processing is performed.
Embodiments listed are intended to describe the present invention better, but the content of the present invention is not limited to the embodiments listed. Therefore, those skilled in the art make non-essential improvements and adjustments to implementation schemes according to the above content of the present invention, which still belong to a scope of protection of the present invention. 5920602581
In the embodiment of the present invention, Lactobacillus fermentum TY-G02,
Lactobacillus fermentum TY-G03 and Lactobacillus fermentum TY-G04 as contrasts are all from
Chongqing Tianyou Dairy Co., Ltd, and all perform public preservation. Herein, the
Lactobacillus fermentum TY-GO2 is preserved in CGMCC, and the preservation number is
CGMCC No. 23754; the Lactobacillus fermentum TY-G03 is preserved in CGMCC, and the preservation number is CGMCC No. 23753; the Lactobacillus fermentum TY-G04 is preserved in CGMCC, and the preservation number is CGMCC No. 23752.
In the embodiment of the present invention, an MRS broth culture medium is purchased from Beijing Land Bridge Technology Co., Ltd., and a specific formula is: 10.0 g of a peptone, 10.0 g of beef powder, 5.0 g of yeast powder, 20.0 g of glucose, 5.0 g of a sodium acetate, 2.0 g of an amine citrate, 1.0 g of Tween 80, 2.0 g of a dipotassium hydrogen phosphate, 0.1 g of a magnesium sulfate, and 0.05 g of a manganese sulfate. The MRS liquid medium is prepared as follows: 55.2 g of the MRS broth culture medium is weighed before use, and cultured in 1 L of distilled water, a pH value is adjusted to 6.3, it is heated after being stirred, and sterilized at 121°C and 0.1 Mpa for 15 min. An MRS solid medium is supplemented with 2% (w/w) agar powder on the basis of the above MRS liquid medium.
Embodiment 1: Separation, purification and identification of TY-S07
In the embodiment of the present invention, experimental material sources and separation and purification are as follows: yak yogurt of natural fermentation in herdsmen's house in
Hongyuan County, Aba Zang and Qiang Autonomous Prefecture, Sichuan Province. The naturally fermented yak yogurt in the herdsman's house is taken with a sterile spoon, it is put into a 15 mL sterile screw-cap centrifuge tube containing an appropriate amount of sterile calcium carbonate and soluble starch (the mass ratio of calcium carbonate: soluble starch is 1:1), it is stirred uniformly, and after a screw-cap is screwed, it is placed in a refrigerator, and transported back to a laboratory for the purification and separation of lactic acid bacteria immediately. (1) Separation and purification
Under an aseptic condition, 1 mL of a sample is absorbed in 9 mL of sterile physiological saline, vortex is performed so that it is mixed uniformly, namely a sample diluents of 10° is obtained, then 10-fold gradient dilution is successively performed to 107, 100 pL of diluents under dilutions of 10°, 10°, and 107 are selected, and spread uniformly on an MRS plate, and inversely cultured at 37°C for 48 h. After the culture, the colony morphology on the MRS plate is observed, and a typical colony of the lactobacillus is picked to perform a plate streaking method to purify a strain, and this streaking operation is repeated, until the purified strain is obtained.
The bacterial colony morphology after the purification is as shown in Fig. 1, and after the PVÉ0S02581 strain is purified, a single bacterial colony is formed in the solid medium. The bacterial colony is larger, circular, semi-translucent, and non-bulged basically. The surface is not smooth, the edge is not regular, and the color is pale yellow. (2) Morphological structure observation
The purified strain is inoculated into a 5 mL sterile MRS liquid medium, and cultured at 37°C for 18 h. 1 mL of bacterial solution is taken and centrifuged at 12000 r/min for 1 min, after it is washed twice with sterile saline, an equal volume of the sterile saline is added to resuspend the bacteria, an inoculating loop is used to take a small amount and spread uniformly on a glass slide, after being fixed, Gram staining, microscopic examination, and photographing are performed. Observation is performed and cell morphology and Gram staining results are recorded. Gram-positive bacteria (G+) cells stained are blue-purple, and Gram-negative bacteria (G-) cells are red.
The result after Gram staining is shown in Fig. 2, the cells are all purple under a microscope, and are Gram-positive bacteria (G+), and the shape is rod-shaped, this shape is consistent with the characteristics of the lactobacillus. The morphological structure is uniform, it is indicated that the strain is pure. (3) PCR amplification of 16S rDNA sequence
A 25 pL reaction system is used for PCR amplification: 1 pL of a template, 1 uL of an upstream primer (the concentration is 10 pM, and the sequence is as shown in SEQ ID No.2), 1 uL of a downstream primer (the concentration is 10 uM, and the sequence is as shown in SEQ
ID No.3), 12.5 pL of 2xTagPCRMasterMix, and it is supplemented to 25 pL with sterile ultrapure water. PCR amplification conditions: pre-denaturation at 94 °C for 5 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, and a total of 35 cycles; and end extension at 72°C for 10 min. After the sequence is amplified, Sangon Bioengineering (Shanghai) Co., Ltd. is entrusted to sequence a PCR amplification product that is qualified by detection, the sequence as shown in SEQ ID No. 1 is obtained, and it is identified as the lactobacillus fermentum.
Embodiment 2: TY-S07 gastrointestinal tract endurance capacity determination
In the embodiment of the present invention, the lactobacillus fermentum TY-S07 (separated from the yak yoghurt of the natural fermentation in the herdsman's house in Hongyuan County of
Sichuan Province, preserved in CGMCC, and the preservation number is CGMCC No. 24628) separated in Embodiment 1 performs the gastrointestinal tract endurance capacity determination. (1) Endurance capacity determination of TY-S07 in pH 3.0 artificial gastric juice
The probiotics entering the human body should exert corresponding functional activities, so they should have the good gastrointestinal tract resistance. Before the probiotics enter a human PVÉ0S02581 intestinal tract to exert its functions, they must pass through a stomach (pH of the gastric juice is about 3.0, and the retention time is 1-3 h) firstly, the strong acidic stomach environment is not beneficial to the survival of the probiotics. Therefore, to exert the corresponding functional activities, a primary condition of the probiotics is that it is able to tolerate the action of the gastric juice in the human body.
TY-S07 is inoculated in the MRS liquid medium by 3% of the inoculation amount, and cultured at 37°C for 18 h, after three generations of the activation; 2 mL is taken and it is centrifuged at 8000 r/min for 10 min to collect a bacterial precipitate, it is washed for 2 times with sterile phosphate buffer (PBS) solution, and resuspended in an equal volume of sterile physiological saline, namely it is bacterial suspension; the prepared bacterial suspension and the artificial gastric juice (the mass fraction of NaCl is 0.2% and the mass fraction of a pepsin is 0.35%, the volume ratio is 1:10000, 1 mol/L of HCI is used to adjust pH=3.0, it is filtered and sterilized for later use) are mixed at a ratio of 1:9 (v/v), and then it is placed in a constant-temperature shaker after being mixed uniformly, and cultured at 37°C and 100 r/min for 3 h, and the viable counts at 0 h and 3 h are respectively measured by using a pouring plate method. The survival rate of the strain to tolerate pH 3.0 artificial gastric juice is calculated according to a formula (1):
Survivalrate/ % = 3hViable count (CFU /mL) count (CFU /mL) x100% (1) 0hViablecount/(CFU / mL)
Experimental results show that the survival rate of TY-S07 cultured in the artificial gastric juice of pH=3.0 for 3 h is 58.46%, and it has the better gastric juice tolerance. (2) Endurance capacity determination of TY-S07 in 0.3% bile salt
After the probiotics that survive in gastric juice treatment enter the intestinal tract, it may be suppressed and poisoned by bile salts in a small intestine. Therefore, the tolerance of the bacterial strains to the bile salts is also one of important indicators for screening of the probiotics, and the mass concentration of the human body bile salt is fluctuated in the range of 0.03%-0.3%.
TY-S07 is inoculated in the MRS liquid medium by 3% of the inoculation amount, and cultured at 37°C for 18 h, after three generations of the activation; 2 mL is taken and it is centrifuged at 8000 r/min for 10 min to collect a bacterial precipitate, it is washed for 2 times with sterile PBS solution, and resuspended in an equal volume of sterile physiological saline containing 0.0% and 0.3% bile salt, it is placed in a constant-temperature shaker after being mixed uniformly, and cultured at 37°C and 100 r/min for 2 h, and the viable counts in 0.0% and 0.3% bile salt culture solution are respectively measured by using the pouring plate method. The survival rate of the strain in the bile salt is calculated according to a formula (2): . 1g(0.3%Bilesalt viable count) 5920602581
Survivalrate/% = ———————— x 100% (2) 1g(0.0%Bile salt viabk count)
Experimental results show that the growth efficiency of TY-S07 in the bile salt with the concentration of 0.3% is 12.06%, and it has the better bile salt tolerance.
Embodiment 3: EPS yield test
In the embodiment of the present invention, a EPS yield test is performed on the lactobacillus fermentum TY-S07 (separated from the yak yoghurt of the natural fermentation in the herdsman's house in Hongyuan County of Sichuan Province, preserved in CGMCC, and the preservation number is CGMCC No. 24628) separated in Embodiment 1. (1) Colony drawing performance test
EPS is a mucus that is secreted by microorganisms outside cells in the growth and metabolism process or a capsule that is adhered to the cell surface. Therefore, the colonies of high EPS-generating strains should have the better drawing performance, and this is also a common method for screening the high EPS-generating lactic acid bacteria according to a phenotype.
In the embodiment of the present invention, steps of the colony drawing performance test are as follows: the bacterial strain is inoculated in the MRS liquid medium by 3% of the inoculation amount, and cultured at 37°C for 18 h, after three generations of the activation, the inoculating loop is used to dip bacterial solution in the MRS solid medium for scratching, and it is cultured at 37°C for 48 h; and the single colonies obtained by scratching are picked with a toothpick, and the drawing lengths of the colonies are compared. 118 strains of the lactobacillus fermentum in a probiotics storehouse of Chongqing Tianyou
Dairy Co., Ltd. perform the drawing performance test according to the above method, herein the drawing length of TY-S07 is the longest, and its drawing condition is as shown in Fig. 3. (2) EPS content determination (1) Drawing of glucose standard curve 0.2 mg/mL of glucose is used as standard solution, 0.0 mL, 0.1 mL, 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL, and 1.0 mL of the standard solution is taken, and water is replenished to 2.0 mL; 200 uL of glucose solution with different concentrations is pipetted, 100 uL of phenol solution with the mass fraction of 5% is added, and after being shaken uniformly, 500 pL of concentrated H2SO4 is slowly added, to prevent droplets from splashing; and after standing in the dark for 30 min, reaction solution is put into a 96-well plate, the concentration of the glucose is used as an abscissa, an OD490nm value is used as an ordinate, and a standard curve is drawn, and the standard curve thereof is as shown in Fig. 4.
(2) Determination of EPS content in strain fermentation supernatant
In the embodiment of the present invention, a phenol-sulfuric acid method is adopted fo PPÉ0S02581 measure the content of EPS in the strain fermentation supernatant, and it is specially as follows: 200 pL of the above EPS is pipetted in a clean test tube, 100 pL of phenol solution with the mass fraction of 5% is added, and after being shaken uniformly, 500 pL of concentrated H:SO4 is slowly added, to prevent droplets from splashing; and after standing in the dark for 30 min,
OD490nm is measured, and the EPS content is calculated according to the standard curve.
In the embodiment of the present invention, the EPS contents in 34 strains of the lactobacillus fermentum fermentation supernatants are measured in total, experimental results are as shown in Fig. 5, and the EPS content in the TY-S07 strain fermentation supernatant is higher, and reaches (318.0+10.9) mg/L, it is indicated that the TY-S07 strain has the high
EPS-generating capacity.
Embodiment 4: Optimization of fermentation condition
In the embodiment of the present invention, the lactobacillus fermentum TY-S07 (separated from the yak yoghurt of the natural fermentation in the herdsman's house in Hongyuan County of
Sichuan Province, preserved in CGMCC, and the preservation number is CGMCC No. 24628) separated in Embodiment 1 performs a test that acquires different yields of EPS under different fermentation conditions. (1) EPS content determination under different nitrogen source conditions
The ability of the same strain of lactic acid bacteria to generate EPS has a difference under the different fermentation conditions, a peptone is formed after a protein is decomposed by an acid, an alkali or a protease, is rich in organic nitrogen compounds, and may provide a nitrogen source for microbial growth.
The peptone in the MRS liquid medium is respectively replaced with a tryptone, a proteose peptone, a casein peptone, a bacterial peptone and a soy peptone in equivalent amount (10 g/L); the strain is inoculated in the MRS liquid medium in which the peptone is replaced according to 3% of the inoculation amount, and cultured at 37°C for 18 h, and after three generations of activation, it is centrifuged at 4°C and 10,000 r/min for 15 min, precipitated bacteria are removed, and a strain fermentation supernatant is retained; 3-fold volume of 95% ethanol is added to the supernatant, it is standing at 4°C for 24 h, and then centrifuged for 15 min, the supernatant is poured off, and after a precipitate is dissolved with an appropriate amount of distilled water, it is placed in a dialysis bag for 24 h dialysis at 4°C; a sevage reagent [V(chloroform):
V(n-butanol)=4:1] with the volume fraction of 25% is added to liquid obtained by the dialysis, and it is placed on a shaker at a room temperature and shaken for 30 min, so that the protein is absorbed in an organic phase, then it is centrifuged at 8000 r/min for 1 min, and an aqueous phase is retained; and it is repeated for 4-5 times, until the protein is completely removed, EPS solution is obtained. 5920602581
A phenol-sulfuric acid method is adopted to measure the content of EPS in the strain fermentation supernatant, and it is specially as follows: 200 pL of the above EPS is pipetted in a clean test tube, 100 pL of phenol solution with the mass fraction of 5% is added, and after being shaken uniformly, 500 pL of concentrated H2SO4 is slowly added, to prevent droplets from splashing; and after standing in the dark for 30 min, OD4ç0nm is measured, and the EPS content is calculated according to the standard curve.
The EPS yields of TY-S07 under the different nitrogen source conditions are as shown in
Fig. 6. In the original MRS liquid medium (without replacing the peptone), the EPS yield of
TY-S07 is (318.0+10.9) mg/L; after the peptone in the MRS liquid medium is replaced with the same amount of the proteose peptone, the EPS yield thereof is (469.0+13.0) mg/L, it is increased by 47.5%; after the the peptone in the MRS liquid medium is replaced with the same amount of the soy peptone, the EPS yield thereof is (444.7+1.6) mg/L, and it is increased by 39.8%. (2) Viable counts under different nitrogen source conditions
In the embodiment of the present invention, in order to find out the reason that the
EPS-generating capacity is improved after the nitrogen source is replaced, the viable count is performed. It is specifically as follows: the peptone in the MRS liquid medium is respectively replaced with a tryptone, a proteose peptone, a casein peptone, a bacterial peptone and a soy peptone in equivalent amount (10 g/L); the strain is inoculated in the MRS liquid medium in which the peptone is replaced according to 3% of the inoculation amount, and cultured at 37°C for 18 h, and after three generations of activation, viable bacteria are counted by using a pouring method.
Viable count results of the different nitrogen sources are as shown in Table 1, and its viable counts of TY-S07 are all on an order of magnitude under the different nitrogen source conditions, it is indicated that its growth ability is not changed, but the EPS-generating capacity is improved.
Table 1: TY-S07 viable counts under different nitrogen source conditions peptone peptone peptone peptone
Viable count/lg
Embodiment 5: EPS monosaccharide composition analysis
In the embodiment of the present invention, the monosaccharide composition analysis is performed on EPS generated by the lactobacillus fermentum TY-S07 (separated from the yak yoghurt of the natural fermentation in the herdsman's house in Hongyuan County of Sichuan
Province, preserved in CGMCC, and the preservation number is CGMCC No. 24628) separated in Embodiment 1. 5920602581
The peptone in the MRS liquid medium is replaced with the proteose peptone in the equivalent amount (10 g/L), and EPS solution is obtained by the method in Embodiment 4, and then EPS powder is obtained after freeze-drying; 10 mg of the EPS powder is weighed, 2 mL of 2 M trifluoroacetic acid is added, and after a bottle is sealed, it is hydrolyzed in an oven at 120°C for 2 h; after being cooled, a rotary evaporator is used to evaporate and remove the trifluoroacetic acid, then a small amount of methanol is added and it is evaporated to dryness so as to remove the residual trifluoroacetic acid, and it is repeated for 3 times; and finally a small amount of water is added to dissolve, a completely acid-hydrolyzed monosaccharide sample is obtained, the monosaccharide composition is measured by an ion chromatograph, and the ion chromatographic analysis conditions are shown in Table 2 below.
Table 2: Ion chromatography analysis condition
CT ese
Chromatographic Dionex CarboPacTM PA10 a 1 TE
Mobile phase Mobile phase À is water, and mobile phase B is 200 mmol/L
LT Em
Column 30°C ee
Detector Electrochemical detector, corrugated gold electrode, sugars,
HE dia
In the embodiment of the present invention, the monosaccharide composition of EPS generated by TY-S07 after medium optimization is analyzed, and its EPS is mainly composed of glucose, galactose, and fructose according to a molar ratio of 11.5:2.7:1.0.
Embodiment 6: Determination of cholesterol lowering capacity
In the embodiment of the present invention, the lactobacillus fermentum TY-S07 (separated from the yak yoghurt of the natural fermentation in the herdsman's house in Hongyuan County of
Sichuan Province, preserved in CGMCC, and the preservation number is CGMCC No. 24628) separated in Embodiment 1 performs the determination of cholesterol lowering capacity, and is compared with lactobacillus TY-G02, TY-G03, and TY-G04.
In the embodiment of the present invention, the preparation of an MRS-CHOL medium is as follows: cholesterol is dissolved in absolute ethanol to obtain 10.0 mg/mL of cholesterol solution, it is sterilized with 0.45 um of a microporous filter membrane, and added to the sterile
MRS liquid medium according to 1% (v /v), to obtain the MRS-CHOL medium containing 01 (0802581 mg/mL of the cholesterol solution. (1) Drawing of cholesterol standard curve 5 test tubes are taken out and numbered according to 1-5, 0.0 mL, 0.1 mL, 0.2 mL, 0.3 mL, 0.4 mL, and 0.5 mL of the MRS-CHOL medium are respectively added in sequence, the above test tubes are filled to 0.5 mL with a glacial acetic acid; 0.2 mL of o-phthalaldehyde working solution (1 mg/mL, dissolved in absolute ethanol) is respectively added to each test tube, and it is shaken and mixed uniformly; after standing for 10 min, 4.0 mL of a mixed acid (the glacial acetic acid and a concentrated sulfuric acid are mixed according to a volume ratio of 1:1) is respectively added, and mixed uniformly; and after standing at a room temperature for 10 min, reaction solution is put into a 96-well plate, a standard curve is drawn by using the concentration of the cholesterol as an abscissa and using an ODssonm Value as an ordinate, and the cholesterol standard curve obtained is as shown in Fig. 7. (2) Determination of cholesterol removal rate
The strain is cultured at 37°C for 18 h, after three generations of activation, it is added to 5 mL of the MRS-CHOL medium according to 3% of the inoculation amount, and cultured at 37°C for 24 h; at the same time, according to an o-phthalaldehyde colorimetry method, a sample of 0 h is measured rapidly, the medium which is just inoculated with the bacteria is centrifuged at 9000 r/min for 10 min, 0.5 mL of a supernatant is taken, 0.2 mL of o-phthalaldehyde working solution is added, and after being fully shaken, it is standing for 10 min, 4.0 mL of a mixed acid is added, it is standing at a room temperature for 10 min, reaction solution is placed in a 96-well plate, and its ODssonm Value is measured. After the strain is cultured for 24 h, the ODssonm value of fermentation broth is determined according to the above o-phthalaldehyde method. Finally, the cholesterol content in the fermentation broth is determined according to a fitting equation of the cholesterol standard curve, and the cholesterol removal rate wis calculated according to a formula (3):
Cholestero 1 removal rate / % = ra x100% (3)
Herein: A is the cholesterol content in the supernatant after the strain fermentation; and B is the cholesterol content in the supernatant before the fermentation of each test strain.
In the embodiment of the present invention, the cholesterol removal rates of TY-S07 before and after the optimization of the medium and lactobacillus TY-G02, TY-G03, and TY-G04 are measured, experimental result are as shown in Fig. 8, the cholesterol removal rate of TY-S07 without being optimized is (11.7+0.2)%, which is significantly higher than that of lactobacillus
TY-G02, TY-G03, and TY-G04, it is indicated that TY-S07 itself has the higher cholesterol removal rate; and the cholesterol removal rate of TY-S07 after the optimization is (17.8:40.5)%, (0602581 which is 52.1% higher than that before the optimization, and it is significantly improved.
Embodiment 7: Determination of antioxidant capacity
In the embodiment of the present invention, the lactobacillus fermentum TY-S07 (separated from the yak yoghurt of the natural fermentation in the herdsman's house in Hongyuan County of
Sichuan Province, preserved in CGMCC, and the preservation number is CGMCC No. 24628) separated in Embodiment 1 performs the determination of antioxidant capacity, and is compared with lactobacillus TY-G02, TY-G03, and TY-G04.
The strain is inoculated in the MRS liquid medium by 3% of the inoculation amount, and cultured at 37°C for 18 h, after three generations of activation, it is centrifuged at 4°C and 10,000 r/min for 15 min, the precipitated bacteria are removed, and a strain fermentation supernatant is taken; 1 mL of anhydrous ethanol solution of 0.2 mmol/L DPPH is added, and after being fully mixed uniformly, it is reacted at a room temperature for 30 min in the dark; then it is centrifuged at 6000 r/min for 10 min, and the supernatant is taken for measurement.
Sample solution is replaced with an equal volume of the MRS liquid medium, an OD5171m value is measured according to the above same operation steps, and ODs171 (blank) in a formula (4) is obtained. In addition, a mixture of an equal volume of normal saline and absolute ethanol is used as blank to adjust zero. The scavenging rate of a DPPH free radical is calculated according to the formula (4):
DPPH free radical scavenging rate/ % = a Ma! x 100% (4).
The embodiment of the present invention measures the DPPH free radical scavenging rates of TY-S07 before and after the optimization of the medium and lactobacillus TY-G02, TY-G03, and TY-G04, and experimental results are as shown in Fig. 9, the DPPH free radical scavenging rate of TY-S07 without being optimized is (31.2+0.6)%, which is significantly higher than that of lactobacillus TY-G02, TY-G03 and TY-G04, it is indicated that TY-S07 has the higher DPPH free radical scavenging rate; and the DPPH free radical removal rate of TY-S07 after the optimization is (28.0+0.3)%, although it is not as good as the removal rate before the optimization, but there is no significant difference between the two, and it is also significantly higher than that of lactobacillus TY-G02, TY-G03, and TY- G04.
Combined with all above embodiment tests, it may be known that Lactobacillus fermentum
TY-S07 in the technical scheme has the good gastrointestinal tract resistance. The colony drawing performance of the strain is stronger, and it has the higher EPS yield; and the strain has the higher removal rate of the cholesterol and DPPH free radicals, and has the potential of cholesterol lowering and anti-oxidation. "°PÉ0S02581
Finally, it should be noted that the above embodiments are only used to describe the technical schemes of the present invention and are not limited. Although the present invention is described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that modifications or equivalent replacements may be made to the technical schemes of the present invention without departing from the spirit and scope of the technical schemes of the present invention, and they should all be included in a scope of the claims of the present invention.
SEQUENCE LISTING LUS02pRho581 <110> Chongqing Tianyou Dairy Co., Ltd. <120> Lactobacillus fermentum TY-S07 and application thereof <130> 2022-4-22 <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 1123 <212> DNA <213> Lactobacillusfermentum <400> 1 ggacgggcgg gtgctataca tgcaagtcga acgcgttggc ccaattgatt gatggtactt 60 gcacctgatt gattttggtc gccaacgagt ggeggacggg tgagtaacac gtaggtaacc 120 tacccagaag cgggggacaa catttggaaa cagatgctaa taccgcataa caacgttgtt 180 cgcatgaaca acgcttaaaa gatggcttct cgetatcact tctggatgga cctgeggtge 240 attagcttgt tggtggggta acggcctacc aaggegatga tgcatagccg agttgagaga 300 ctgatcggcc acaatgggac tgagacacgg cccatactcc tacgggagec agcagtageg 360 aatcttccac aatgggcgca agcctgatgg agcaacaccg cgtgagtgaa gaagggtttc 420 gectcgtaaa gctctettgt taaagaagaa cacgtatgag agtaactgtt catacgttga 480 cggtatttaa ccagaaagtc acggctaact acgtgccagc agccgcggta atacgtaggt 540 gecaagcett atccggattt attgggcgta aagagagtge aggcggtttt ctaagtctga 600 tetgaaagcc ttcgecttaa ccggagaagt gcatcggaaa ctggataact tgagtgcaga 660 agagggtagt ggaactccat gtgtagcget ggaatgegta gatatatgga agaacaccag 720 tggcgaaggc gectacctgg tctgcaactg acgctgagac tcgaaagcat gggtagegaa 780 caggattaga taccctggta gtccateccg taaacgatga gtgctatgtg ttggaggett 840 LUSOPÉR (2581 tecgcecttc agteccggag ctaacgeatt aagcactccg cctggggagt acgaccgcaa 900 agttgaaact caaaagaatt gacggggccc gcacaagcgg tggagcatet ggtttaattc 960 gaagctacgc gaagaacctt accagtcttg acatcttgcg ccaaccctag agataggegt 1020 ttccttcggg acgcatgaca gtgtgeatgg gtcgtcetca getegtgteg tgagatgttg 1080 ggttaagtcc cgecacgageg caaacccttg gttactagtt get 1123
<210> 2
<211> 20
<212> DNA
<213> Artificial Sequence
<400> 2 agagtttgat cmtggctcag 20 <210> 3
<211> 19
<212> DNA
<213> Artificial Sequence
<400> 3 ggttaccttg ttacgactt 19
Claims (10)
1. A Lactobacillus fermentum TY-S07, wherein its preservation number is CGMCC
No.24628.
2. The Lactobacillus fermentum TY-S07 according to claim 1, wherein its 16SrDNA sequence is as shown in SEQ ID No. 1.
3. A fermentation composition for preparation of an exopolysaccharides (EPS), wherein it comprises the Lactobacillus fermentum TY-S07 according to claim 1 or 2.
4. The fermentation composition according to claim 3, further comprising a proteose peptone and/or a soy peptone.
5. A preparation method for EPS, comprising: using the Lactobacillus fermentum TY-S07 according to claim 1 or 2 or the fermentation composition according to claim 3 or 4 to prepare.
6. The preparation method according to claim 5, wherein the Lactobacillus fermentum TY-S07 according to claim 1 is used for inoculation and fermentation, and the inoculation amount thereof is 2%-4%.
7. EPS prepared by the preparation method according to claim 5 or 6.
8. A drug or food, comprising the Lactobacillus fermentum TY-S07 according to claim 1 or 2 or EPS according to claim 7 and an auxiliary material; and the auxiliary material is an edible carrier and/or diluent, or the auxiliary material is a pharmaceutically acceptable carrier and/or diluent.
9. The drug or food according to claim 8, wherein the edible or pharmaceutically acceptable carrier and/or diluent is suitable for 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 particle dispersion dosage form or a solid dispersion dosage form.
10. An application of Lactobacillus fermentum TY-S07 according to claim 1 or 2 or the fermentation composition according to claim 3 or 4 or the EPS according to claim 7 as or in preparing a preparation, and the preparation is a preparation for anti-oxidation or a preparation LU502581 for reducing a cholesterol.
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