A Lactobacillus fermentum with hypoglycemic function and its application

Abstract

The present invention discloses a Lactobacillus fermentum with hypoglycemic function and its application. The Lactobacillus fermentum with high hypoglycemic ability was screened from the freshly brewed yogurt samples in Baoshan, Yunnan. The strain has been deposited in the China Center for Type Culture Collection, and the deposit number is CCTCC NO: M 20241504. During the fermentation process of the strain, it can inhibit α-glucosidase and α-amylase, and the inhibition rates are 43.91% and 59.44% respectively. The colony morphology of the Lactobacillus fermentum E003 is milky white, semi-transparent, relatively moist, smooth, with neat edges and obvious protrusions. It is used to prepare anti-sugar fermented milk, and the fermented milk can also inhibit α-glucosidase and α-amylase. At the same time, the fermented milk also has a variety of characteristic nutritional components; the fermentation broth of Lactobacillus fermentum E003 also has strong antibacterial ability, antioxidant activity and high GABA-producing ability.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to a Limosilactobacillus fermentum with hypoglycemic function and its application. Background Art

[0002] Hyperglycemia can lead to diabetes, which is a chronic disease that seriously affects the quality of human life and easily causes a series of complications such as kidney disease, cardiovascular disease, blindness, and disability. Patients need to take medicine for life to control blood sugar. Although there are many drugs for treating diabetes, they also have side effects.

[0003] Lactic acid bacteria have been recognized as beneficial flora to the human body and have various functions. The research on the hypoglycemic effect of lactic acid bacteria and the development of its health products has gradually become a hot topic. Lactic acid bacteria act gently, are stable in nature, and have obvious and lasting effects, opening up a new direction for the development of long-acting hypoglycemic products. Therefore, the hypoglycemic effect of lactic acid bacteria has great research value and application potential. However, at present, there is less research on the hypoglycemic effect of lactic acid bacteria dairy products in China, and the material basis and mechanism of its hypoglycemic effect are not yet clear. Summary of the Invention

[0004] Aiming at the deficiencies of the prior art, the present invention provides a Limosilactobacillus fermentum and its application. The Limosilactobacillus fermentum can inhibit α-glucosidase and α-amylase, and has a good hypoglycemic effect. In addition, the fermentation broth also has antioxidant function, antibacterial function, and high-yield γ-aminobutyric acid function.

[0005] To solve the above technical problems, the technical solution of the present invention is as follows:

[0006] The present invention provides a Limosilactobacillus fermentum with hypoglycemic function, named Limosilactobacillus fermentum E003, with the preservation number of CCTCC NO: M20241504, the preservation unit: China Center for Type Culture Collection, the preservation date: July 8, 2024, and the preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, within Wuhan University.

[0007] The present invention also provides the fermentation broth obtained by fermenting the Limosilactobacillus fermentum.

[0008] The present invention also provides the application of the Limosilactobacillus fermentum or the fermentation broth in the preparation of hypoglycemic or antioxidant drugs.

[0009] The present invention also provides the application of the Limosilactobacillus fermentum in the preparation of fermented milk.

[0010] In the application of preparing fermented milk, preferably, the inoculation amount of the Limosilactobacillus fermentum E003 is 1.0×10 8CFU / mL - 3.0×10 8 CFU / mL.

[0011] In the application of preparing fermented milk, preferably, the Lactobacillus fermentum E003 is used in combination with a commercial yogurt starter; preferably, the usage amount of the commercial yogurt starter is 0.01 - 0.03% (m / m); the fermentation time is 6h - 10h, and the fermentation temperature is 36.5°C - 39.5°C.

[0012] The characteristics of the present invention are as follows: The present invention screens a Lactobacillus fermentum with high blood glucose lowering ability from the freshly brewed yogurt samples in Baoshan, Yunnan. During the fermentation process of the strain, it can inhibit α-glucosidase and α-amylase, and the inhibition rates are 43.91% and 59.44% respectively. The colony morphology of the Lactobacillus fermentum E003 is milky white, semi-transparent, relatively moist, smooth, with neat edges and obvious protrusions. It is used to prepare anti-sugar fermented milk, and the fermented milk can also inhibit α-glucosidase and α-amylase. At the same time, the fermented milk also has various characteristic nutritional components; the fermentation broth of Lactobacillus fermentum E003 also has strong antibacterial ability, antioxidant activity and high GABA-producing ability.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] (1) The present invention isolates and screens a lactic acid bacterium with high blood glucose lowering ability from freshly brewed yogurt samples, and identifies the lactic acid bacterium through morphological identification and whole genome sequencing. It belongs to Limosilactobacillus fermentum in classification, which is Lactobacillus fermentum, and it is named Lactobacillus fermentum E003, which can effectively inhibit α-glucosidase and α-amylase.

[0015] (2) Using Lactobacillus fermentum E003 can produce fermented milk with good flavor, texture, high viable cell count and high inhibition rates of α-glucosidase and α-amylase.

[0016] (3) The fermentation broth of Lactobacillus fermentum E003 also has strong antibacterial ability, antioxidant activity and high GABA-producing ability. Specific embodiments

[0017] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be further described in detail below in conjunction with specific embodiments. The description of this part is only exemplary and explanatory, and should not have any restrictive effect on the protection scope of the present invention. The following comparison lactic acid bacteria applied in the examples: Lactobacillus plantarum B103 is derived from yak milk and is identified as Lactobacillus plantarum by 16s rDNA gene sequence analysis; Lactobacillus plantarum C039 is derived from Periplaneta americana and is identified as Lactobacillus plantarum by 16s rDNA gene sequence analysis; Lactobacillus fermentum D051 is derived from milk and is identified as Lactobacillus fermentum by 16s rDNA gene sequence analysis; Lactobacillus fermentum D021 is derived from pickled vegetables and is identified as Lactobacillus fermentum by 16s rDNA gene sequence analysis.

[0018] Example 1 Isolation and purification of strains

[0019] Take 10 g of freshly brewed yogurt sample collected from Baoshan City, Yunnan Province, and add it to 100 mL of sterile physiological saline and shake evenly. After gradient dilution of the sample, 100 μL is taken and spread on MRS solid medium containing 1% calcium carbonate, and cultured at 37 °C for 48 h. Select the colonies that form calcium dissolution circles on the solid medium, and repeatedly streak and purify on MRS solid medium until the colony morphology of the strain is consistent. Inoculate the purified lactic acid bacteria into MRS slant medium and store at 4 °C. Then mix the strain with 20% glycerol and transfer it into a cryopreservation tube. After uniform mixing, store at -80 °C.

[0020] (1) Morphological characteristics of strains

[0021] After Lactobacillus fermentum E003 is cultured on MRS agar medium for 24 h, the colony morphology is milky white, semi-transparent, relatively moist, smooth, with neat edges and obvious protrusions. Under an optical microscope, the strain cells of Lactobacillus fermentum E003 are rod-shaped, do not produce spores, and are Gram-positive.

[0022] (2) Molecular genetics identification of strains

[0023] Whole genome sequencing

[0024] Use a DNA extraction kit with TGUIDE S96 magnetic bead technology to extract the DNA of the screened strain. Use the Qubit method to determine its content, use the Nanodrop method to determine its DNA integrity, and use agarose gel electrophoresis to determine its DNA integrity. After the sample passes the detection, construct a library. After the library passes the quality inspection, perform the on-machine sequencing test. Through the PacBio third-generation sequencing technology, the genome size of Lactobacillus fermentum E003 is 2,096,565 bp, and the GC content is 51.52%.

[0025] Finally, through morphology and whole-genome sequencing, the screened strain was identified as Limosilactobacillus fermentum and named Limosilactobacillus fermentum E003, which was deposited in the China Center for Type Culture Collection with the deposit number: CCTCC NO: M 20241504. Example 2 Determination of the inhibitory rates of Limosilactobacillus fermentum E003 against α-glucosidase and α-amylase

[0026] (1) Growth curve of Limosilactobacillus fermentum E003

[0027] The seed liquid of Limosilactobacillus fermentum E003 activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V). 200 μL of the seed liquid was placed in a 96-well plate, and the absorbance value at 600 nm was measured every 2 h for a total of 24 h. Each time point was repeated 3 times, and the average value was taken. The growth curve was plotted with time as the abscissa and absorbance value as the ordinate.

[0028] As the fermentation time extended, the OD600nm value of Limosilactobacillus fermentum E003 showed an upward trend, and its growth conformed to an S-shaped curve. The 0 - 2 h was in the lag phase of growth, and the growth rate of the strain was relatively slow; 2 - 14 h was in the logarithmic phase of growth, and the growth rate of the strain increased sharply; 14 - 16 h was in the stationary phase of growth, and the OD600nm value of the strain basically stabilized; 16 - 24 h was in the decline phase of growth, and the OD600nm value of the bacteria decreased slightly.

[0029] (2) Determination of the α-glucosidase inhibitory rate of Limosilactobacillus fermentum E003

[0030] The seed liquid of Limosilactobacillus fermentum activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V), and cultured at 37 °C for 24 h. Then it was centrifuged at 8000 r / min for 15 min to obtain the supernatant of lactic acid bacteria fermentation. 50 μL of the sample solution was added to a 96-well plate respectively, and then 100 μL of α-glucosidase (0.1 U / mL) was added. After incubation at 37 °C for 10 min, 50 μL of PNPG (5 mmol / L) was added, and then cultured at 37 °C for 20 min. Finally, 1 mL of Na 2 CO 3 (0.1 mol / L) was added to terminate the reaction, and the absorbance value was measured at a wavelength of 405 nm. The inhibitory rate was calculated according to the formula as follows:

[0031]

[0032] In the formula:

[0033] A——OD405 value of the solution containing α-glucosidase but without the sample;

[0034] B——OD value of the solution without α-glucosidase and the test sample

[0035] C——OD value of the solution containing α-glucosidase and the test sample

[0036] D——OD value of the solution without α-glucosidase but containing the test sample

[0037] α-Glucosidase is located at the brush border of small intestinal epithelial cells and is a key enzyme that catalyzes the hydrolysis of oligosaccharides and increases the blood glucose content after meals. The enhancement of its activity easily leads to an increase in blood glucose content in diabetic patients. Inhibiting the activity of α-glucosidase is an effective way to treat type II diabetes. The α-glucosidase inhibitory rate of Lactobacillus fermentum E003 is the highest, with an inhibitory rate of 43.91%.

[0038] (3) Determination of the α-amylase inhibitory rate of Lactobacillus fermentum E003

[0039] The seed solution of Lactobacillus fermentum that had been activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V), cultured at a constant temperature of 37 °C for 24 h, centrifuged at 8000 r / min for 15 min to obtain the supernatant of lactic acid bacteria fermentation. 125 μL of the supernatant of lactic acid bacteria was added and mixed with an equal volume of 1 mg / mL α-amylase solution, incubated in a constant temperature water bath at 37 °C for 10 min, then the reaction solution was added to 250 μL of 1.5% soluble starch solution at 37 °C, reacted at 37 °C for 15 min, then 500 μL of DNS solution was added, reacted in a boiling water bath for 5 min and then quickly cooled to room temperature, diluted 20 times and allowed to stand for 30 min, and the absorbance value was measured at 540 nm. PBS solution (0.1 mol / L, pH = 6.8) was used as the blank control for the α-amylase solution and the test sample. And the inhibitory rate was calculated according to the formula as follows:

[0040]

[0041] In the formula:

[0042] A: is the sample group, containing the sample solution and the α-amylase solution

[0043] B: is the sample blank group, containing the sample solution and without the α-amylase solution

[0044] C: is the control group, without the sample solution and containing the α-amylase solution

[0045] D: is the blank group, without the sample solution and without the α-amylase solution

[0046] α - amylase is an important starch - hydrolyzing enzyme. It can cleave the glycosidic bonds inside starch, producing dextrin, oligosaccharides, glucose, etc. It can promote the hydrolysis and digestion of carbohydrates in food, facilitate the intake of sugars, and increase the levels of blood sugar and blood lipids. After humans eat, α - glucosidase and α - amylase synergistically promote the increase in blood sugar, thereby triggering hyperglycemic symptoms. Therefore, the α - amylase inhibitory activity of lactic acid bacteria is also an important indicator for evaluating its anti - sugar ability. The α - amylase inhibition rate of Lactobacillus fermentum E003 is the highest, reaching 59.44%. Therefore, Lactobacillus fermentum E003 or its fermentation broth can be used to lower blood sugar.

[0047] Example 3: Anti - sugar fermented milk prepared from Lactobacillus fermentum E003

[0048] (1) Preparation of anti - sugar fermented milk

[0049] The activated Lactobacillus fermentum E003 strain was inoculated into MRS broth at an inoculation amount of 1% and cultured (at 37 °C for 24 h) until the third generation. The seed liquid of Lactobacillus fermentum E003 was inoculated into milk at 1 - 3% (1.0×10 8 CFU / mL - 3.0×10 8 CFU / mL) (V / V) and a commercial direct - vat - set (Lactobacillus bulgaricus and Streptococcus thermophilus were purchased from Royal DSM Group, and the ratio of the two strains was 1:1) at an inoculation amount of 0.01%, and fermented at a constant temperature. The fermented milk after fermentation was placed in a 4 °C refrigerator for 12 h of after - ripening, and then sensory evaluation was carried out.

[0050] Process optimization of fermented milk

[0051] (1) Single - factor experiment on fermentation process

[0052] ① Effect of the inoculation amount of Lactobacillus fermentum E003 on the anti - sugar ability of fermented milk

[0053] The inoculation amounts were set as 1, 1.5, 2, 2.5, 3%, the fermentation temperature was 37 °C, and the fermentation time was 10 h to study the effect of the inoculation amount on the anti - sugar ability of fermented milk.

[0054] When the fermentation temperature was 37 °C, the fermentation time was 10 h, and the inoculation amounts of strain E003 were 1%, 1.5%, 2%, 2.5%, 3%, the anti - sugar ability of fermented milk showed a trend of first increasing and then decreasing. The anti - sugar ability was the highest when the inoculation amount was 2.5%. Therefore, 2%, 2.5%, and 3% were selected as the subsequent response surface optimization levels.

[0055] ② Effect of fermentation temperature on the anti - sugar ability

[0056] The fermentation temperatures were set as 33, 35, 37, 39, 41 °C, the inoculation amount was 2%, and the fermentation time was 10 h to study the effect of the fermentation temperature on the anti - sugar ability of fermented milk.

[0057] When the fermentation time is 10 h, the inoculation amount of strain CE is 2%, and the fermentation temperatures are 33 °C, 35 °C, 37 °C, 39 °C, and 41 °C, the antiglycation ability of the fermented milk shows a trend of first increasing and then decreasing. When the fermentation temperature is 39 °C, the antiglycation ability is the highest. Therefore, 37 °C, 39 °C, and 41 °C are selected as the subsequent response surface optimization levels.

[0058] ③ Influence of fermentation time on antiglycation ability

[0059] Set the fermentation time to 6, 8, 10, 12, and 14 h, the inoculation amount to 2%, and the fermentation temperature to 37 °C to study the influence of fermentation time on the antiglycation ability of fermented milk. The experimental results show that when the fermentation time is 10 h, the antiglycation ability is the highest. Therefore, 8 h, 10 h, and 12 h are selected as the subsequent response surface optimization levels.

[0060] ④ Sensory evaluation of fermented milk

[0061] Ten laboratory members with a certain understanding of the sensory characteristics of fermented milk were used to conduct a sensory evaluation of the fermented milk products. To avoid the influence of a cooler temperature on the taste, the samples were first placed at room temperature for 15 min during the sensory evaluation; the color and texture of the fermented milk were observed, and then the odor was smelled and the taste of the fermented milk was tasted. The sensory scoring table is shown in the table, and a 100-point scoring method was used. Finally, the score value of the fermented milk was calculated. The scoring criteria are shown in Table 1 for details.

[0062] Table 1 Sensory scoring table

[0063]

[0064] (2) Response surface optimization of the technological conditions for antiglycation fermented milk

[0065] On the basis of the single-factor experiment of antiglycation fermented milk, using the double response values of antiglycation ability and sensory score, the Box-Behnken in the Design-Expert software was used to conduct a response surface experiment design with 3 factors and 3 levels. Three factors, namely inoculation amount, fermentation temperature, and fermentation time, were selected respectively to optimize the best fermentation process of antiglycation fermented milk.

[0066] According to the results of the single-factor experiment, taking the inoculation amount of the strain % (A), fermentation temperature °C (B), and fermentation time h (C) as the investigation factors, and using the double response values of α-glucosidase inhibition rate and sensory score, a three-factor and three-level experiment was carried out using the Box-Behnken Design central composite experiment to obtain the best fermentation process conditions of the fermented milk. The variance analysis is shown in Table 2.

[0067] Table 2 Response surface experiment design and experimental results

[0068]

[0069]

[0070] ①Model establishment and significance test

[0071] Using Design-Expert 8.0.6 software to perform multiple regression fitting on Table 2, the quadratic equation models of the α-glucosidase inhibition rate (Y1) and sensory score (Y2) of the anti-sugar fermented milk with the strain ratio v / v (A), fermentation temperature °C (B), and fermentation time h (C) are as follows:

[0072] Y 1 = 44.54 - 1.33A - 1.55B + 0.45C + 0.23AB - 0.12AC + 1.11BC - 2.28A 2 - 3.68B 2 - 5.57C 2

[0073] Y 2 = 87.32 - 2.55A - 0.65B - 3.18C + 0.85AB + 0.90AC - 4.15BC - 4.84A 2 - 1.64B 2 - 10.13C 2

[0074] Analysis of variance results of the regression model: Both models are significant, and the lack-of-fit terms are not significant. R 1 2 = 0.9727, R 2 2 = 0.9904, R 2 Adj1＝ 0.9375, R 2 Adj2 = 0.9780, which also shows that the fitting degree between the actually measured values and the predicted values during the experiment is relatively good, and both the credibility and authenticity are very high. Therefore, this model can be used to optimize the process of fermented milk.

[0075] ②Verification of the regression model and determination of the optimal conditions

[0076] After the analysis by the design software, the optimal technological conditions for fermented milk were obtained, which were: inoculum amount 2.35%, fermentation temperature 38.55 °C, fermentation time 9.89 h. Under these conditions, the inhibition rate of α-glucosidase and the sensory score were 44.882% and 87.8796 points respectively. To verify the accuracy of the optimal technological conditions and also considering the actual situation, the technological conditions were adjusted to: inoculum amount 2.5%, fermentation temperature 39 °C, fermentation time 10 h. Verification tests were carried out under these technological conditions, and finally the inhibition rate of α-glucosidase of the fermented milk was 45.33%, and the sensory score was 86.3 points, which was relatively close to the theoretical value analyzed by the response surface method, proving that this model was relatively reliable and could be used in the experiments.

[0077] Example 4 Quality Analysis of Lactobacillus fermentum E003 Antisugar Fermented Milk

[0078] (1) Determination of Physicochemical Indexes of Fermented Milk

[0079] The protein content was determined by the Kjeldahl method with reference to the national standard GB 5009.5-2016; the fat content was determined by the alkali hydrolysis method with reference to the national standard GB 5009.6-2016; the acidity was determined with reference to the national standard GB 5009.239-2016; the viscosity was measured with a viscometer (No. 3 rotor, rotation speed 30 rpm, time 30 s); the water holding capacity was determined with reference to the method of S Guo.

[0080] The physicochemical indexes of the fermented milk are shown in Table 3. It can be seen from Table 3 that the protein, fat and acidity values of the fermented milk all meet the national standards. The acidity value is the main factor affecting the shelf life and taste of fermented milk. The pH value (4.44) of the fermented milk is beneficial to maintaining the stability of the taste of the fermented milk and effectively extending the market shelf life of the fermented milk. The water holding capacity can affect the viscosity and hardness of fermented dairy products and change the quality of fermented milk. The water holding capacity of the fermented milk is 72.64±5.85%, meeting the texture requirements of fermented milk. Viscosity is an important index for evaluating the texture and taste of fermented milk. The viscosity of the fermented milk is (4656.00±1287.51) MPa·s, reaching the viscosity value range of fermented milk reported in the research.

[0081] Table 3 Physicochemical Properties of Fermented Milk

[0082]

[0083] (2) Determination of Microbiological Indexes of Fermented Milk

[0084] Determination of microbial indicators: The total number of colonies was determined by referring to the relevant method in GB 4789.2-2016; the number of molds and yeasts was determined by referring to the first method in GB / T 4789.15-2016; the number of lactic acid bacteria was determined by referring to the relevant method in GB 4789.35-2016; the coliform group, Salmonella, and Staphylococcus aureus were determined by referring to GB / T 4789.3-2016, GB / T 4789.4-2016, and GB / T 4789.10-2016 respectively.

[0085] As shown in Table 4, the microbial indicators and the number of lactic acid bacteria in the fermented milk all meet the "National Food Safety Standard - Fermented Milk" (GB19302-2010), which is beneficial to the storage of the product while ensuring the product quality.

[0086] Table 4 Microbial indicators and viable number of lactic acid bacteria in fermented milk

[0087]

[0088]

[0089] Study on the composition of anti-glycation fermented milk prepared by Lactobacillus fermentum E003 in Example 5

[0090] Metabolite profile of fermented milk

[0091] The metabolites in the fermented milk were studied by LC-MS / MS. As shown in Table 5, compared with the fermented milk (BS-FM) prepared by the commercial starter alone, the fermented milk (CEBS-FM) prepared by the commercial starter and Lactobacillus fermentum E003 together had 36 differential metabolites, mainly including amino acids and peptides, organic acids, fatty acids, aromatic compounds and their derivatives. These differential metabolites indicate the differences in the substances of the fermented milk during the fermentation process of the screened strain and the commercial starters Lactobacillus bulgaricus and Streptococcus thermophilus, and these differences involve multiple fermentation processes such as strain hydrolysis, synthesis, growth and reproduction.

[0092] Table 5 Differential metabolites of CEBS-FM and BS-FM

[0093]

[0094]

[0095]

[0096]

[0097] Study on other probiotic functional factors produced by Lactobacillus fermentum E003 in Example 6

[0098] Determination of the AI-2 Production Ability of Lactobacillus fermentum E003

[0099] Vibrio harveyi BB170 (ATCC BAA-1117) was used as the indicator bacterium, which was purchased from the Guangdong Provincial Microbial Culture Collection Center (GDMCC). V. harveyi BB170 was streaked on Marine 17 agar medium and cultured at 30 °C for 24 h. It was inoculated into the Autoinducer Bioassay (AB) liquid medium and cultured at 30 °C for 12 h. The fermentation broths of different lactic acid bacteria (Lactobacillus plantarum B103, Lactobacillus fermentum E003, and Lactobacillus fermentum D051) cultured for 24 h and the fermentation broth of V. harveyi BB170 were centrifuged at 4000 rpm for 30 min, and the obtained supernatant was filtered through a 0.2 μm filter. Meanwhile, V. harveyi BB170 cultured overnight was diluted with AB medium at a ratio of 1:100 (v:v), and then mixed with the medium group (sterile MRS broth), different lactic acid bacteria groups, and AB liquid medium group (negative control) at a ratio of 1:50 (v:v). Then, 200 μL of each mixed sample was placed into a 96-well cell culture plate, with each sample in one well, and the fluorescence intensity of each sample was detected using the bioluminescence mode of a full-wavelength multifunctional microscope. The AI-2 content of each test group was calculated when the fluorescence value of the negative control was the lowest within 1 - 7 h. The content of AI-2 was the ratio of the fluorescence value of the experimental group to the fluorescence value of the negative control group, reaching the lowest within 1 - 7 h. It was the ratio of the fluorescence value of the experimental group to the fluorescence value of the medium. As is well known, the quorum sensing system is a mechanism for information exchange among microorganisms. As a signal molecule, AI-2 can conduct interspecies / intraspecies information exchange and has a certain regulatory effect on specific physiological functions and gene expression of microorganisms.

[0100] The amount of AI-2 produced by Lactobacillus fermentum E003 is higher than that of other lactic acid bacteria, indicating that it can promote the communication between microorganisms and facilitate the exertion of probiotic functions.

[0101] Determination of the GABA Production Ability of Lactobacillus fermentum E003

[0102] The determination was carried out according to the instruction manual of the γ-aminobutyric acid (GABA) content kit of Suzhou Grees Biotechnology Co., Ltd. The seed liquid of Lactobacillus fermentum E003 activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V), cultured at a constant temperature of 37 °C for 24 h, centrifuged at 8000 r / min for 15 min, and the supernatant was taken and the GABA content was determined with reference to the instruction manual. The calculation formula for GABA content: GABA (μg / ml –1 ) = 1960.8 × (ΔA + 0.0003) × D

[0103] Where: ΔA = Absorbance (sample) - Absorbance (CK); D is the dilution ratio.

[0104] As shown in Table 6, Lactobacillus fermentum E003 can produce GABA, with a content of 119.27 μg / ml –1 A, which is higher than that of Lactobacillus fermentum D051.

[0105] Table 6 GABA content of strains

[0106] Strain name <![CDATA[GABA content (μg / ml –1 )]]> Lactobacillus fermentum E003 119.27±7.69 Lactobacillus fermentum D051 64.91±5.84

[0107] Determination of the antibacterial ability of the fermentation broth of Lactobacillus fermentum E003

[0108] The seed liquid of Lactobacillus fermentum E003 activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V), cultured at a constant temperature of 37°C for 24 h, centrifuged at 8000 r / min for 15 min, and the supernatant was taken for the determination of antibacterial ability. Using Staphylococcus aureus and Escherichia coli as indicator bacteria, 100 μL of the indicator bacteria liquid (10 6 ~10 7 CFU / mL) was pipetted onto an LB agar plate and spread. Oxford cups (10 mm deep, 8 mm wide) were used to punch holes on the plate, and 100 μL of the lactic acid bacteria fermentation supernatant was added to the holes. After culturing at 37°C for 24 h, the size of the antibacterial zone was observed and measured.

[0109] An obvious antibacterial zone appeared on the Staphylococcus aureus plate of Lactobacillus fermentum E003, and the diameter of the antibacterial zone reached 16.9 ± 0.7 mm; no antibacterial zone appeared on the Escherichia coli plate. It shows that Lactobacillus fermentum E003 has a high antibacterial ability against Staphylococcus aureus, but has no antibacterial effect on Escherichia coli.

[0110] Determination of the antioxidant ability of the fermentation broth of Lactobacillus fermentum E003

[0111] (1) Determination of DPPH free radical scavenging ability

[0112] The seed liquid of lactic acid bacteria E003 activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at an inoculation amount of 1% (V / V), cultured at a constant temperature of 37°C for 24 h, centrifuged at 8000 r / min for 15 min, and the supernatant was taken for the determination of DPPH free radical scavenging ability. Take 2 mL of the fermentation supernatant and mix it well with 2 mL of 0.2 mmol / L DPPH-absolute ethanol solution, react in the dark for 30 min, and measure the absorbance at 517 nm. Calculate the DPPH free radical scavenging ability according to the formula, and the formula is as follows:

[0113]

[0114] In the formula:

[0115] A 1 represents the absorbance of the sample + DPPH - absolute ethanol solution;

[0116] A 2 represents the absorbance of the sample + absolute ethanol solution;

[0117] A 0 represents the absorbance of distilled water + DPPH - absolute ethanol solution.

[0118] The DPPH free radical scavenging rate of Lactobacillus fermentum E003 is 79.10%, which is higher than that of other strains, indicating that the strain has a high antioxidant capacity.

[0119] (2) Determination of ABTS free radical scavenging ability

[0120] Dissolve ABTS in absolute ethanol to prepare an ABTS stock solution with a concentration of 7 mM. At the same time, prepare a potassium persulfate solution with a concentration of 5 mM, mix the two in a volume ratio of 1:1, and react in the dark for 12 h to obtain the ABTS working solution. Dilute the ABTS working solution 20 times with PBS (pH = 7.4) before use. Inoculate the lactic acid bacteria seed solution activated for three generations in MRS liquid medium into MRS liquid medium at an inoculation amount of 1% (V / V), incubate at 37 °C for 24 h, and centrifuge at 8000 r / min for 15 min. Take 0.5 ml of the lactic acid bacteria fermentation supernatant and add it to 4.5 mL of the diluted ABTS working solution, shake well, react in the dark for 6 min, centrifuge at 12000 rpm for 2 min, and measure the absorbance of the supernatant at a wavelength of 734 nm. Calculate the scavenging ability according to the formula, and the formula is as follows:

[0121] ABTS free radical scavenging rate (%) = [1 - (A i - A j ) / A 0

[0122] In the formula:

[0123] A i represents the absorbance of the ABTS working solution + lactic acid bacteria fermentation supernatant;

[0124] A j represents the absorbance of the lactic acid bacteria fermentation supernatant + absolute ethanol solution;

[0125] A 0 represents the absorbance of the ABTS working solution + absolute ethanol solution.

[0126] ​The ABTA free radical scavenging rate of Lactobacillus fermentum E003 is 89.52%, which is higher than that of other strains, indicating that the strain has high antioxidant capacity.

[0127] The above are only the preferred specific embodiments of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the technical scope disclosed by the present invention, according to the technical solution and inventive concept of the present invention, making equivalent substitutions or changes, should be covered by the protection scope of the present invention.

Claims

1. A fermented lactobacillus having blood sugar lowering function, characterized in that: The fermented lactobacillus (Limosilactobacillusfermentum) is named as fermented lactobacillus E003, with a preservation number of CCTCC NO: M20241504, a preservation unit: China Center for Type Culture Collection, and a preservation address: No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University.

2. The fermented liquid obtained by fermenting Lactobacillus fermentum according to claim 1.

3. Application of the fermented lactobacillus according to claim 1 or the fermented liquid according to claim 2 in preparing hypoglycemic or antioxidant drugs.

4. Use of the fermented lactobacillus according to claim 1 in the preparation of fermented milk.

5. The use of Lactobacillus fermentum in preparing fermented milk according to claim 1, characterized in that: The inoculation amount of Lactobacillus fermentum E003 is 1.0×10 8 CFU / mL-3.0×10 8 CFU / mL.

6. The use of Lactobacillus fermentum in preparing fermented milk according to claim 1, characterized in that: The fermented lactobacillus E003 is used in combination with a commercial yogurt starter.

7. The use of Lactobacillus fermentum in preparing fermented milk according to claim 6, characterized in that: The usage amount of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 6h-10h, and the fermentation temperature is 36.5℃-39.5℃.

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