A strain of fermented Lactobacillus mucilaginosus producing γ-aminobutyric acid and its application

Abstract

The invention discloses a strain of fermented mucus lactobacillus producing γ-aminobutyric acid and its application. The invention provides a strain of fermented mucus lactobacillus producing high γ-aminobutyric acid, named as fermented mucus lactobacillus B41. Limosilactobacillus fermentum B41, the deposit number is CCTCC NO: M 20242081, the deposit unit is: China Center for Type Culture Collection, and the deposit address is: Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province. The strain can produce high γ-aminobutyric acid, and the fermented mucus lactobacillus can be used to produce fermented milk rich in γ-aminobutyric acid.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a fermented mucus lactobacillus producing gamma-aminobutyric acid and application thereof. Background Art

[0002] γ-aminobutyric acid (GABA) is a non-protein amino acid that is commonly found in animals, plants, and microorganisms. It is a major inhibitory neurotransmitter that has the effects of calming the nerves, improving sleep, treating diabetes, and improving liver and kidney function. In recent years, with the increase of life pressure and the emergence of various diseases, the phenomenon of "sub-health" has become increasingly common. Appropriate intake of GABA can prevent and alleviate some diseases, so the demand for GABA is also increasing. GABA can be obtained through chemical synthesis, biosynthesis, and natural extraction. However, the content of GABA in natural extraction is limited, and chemical synthesis does not meet consumers' demand for green consumption. In contrast, the preparation of GABA by microbial methods has the advantages of being safe, pollution-free, and inexpensive.

[0003] At present, the biosynthesis of GABA is carried out through genetic engineering, metabolic engineering, enzyme purification and immobilization, which requires a high level of technology, so finding new methods has become the research focus of this application. Studies have shown that quorum sensing (QS) is a cell-to-cell communication mechanism that can regulate a variety of biological functions of strains. For example, exopolysaccharides, bacteriocins and biofilms are widely used to improve the biosynthesis of microorganisms. It has been reported in the literature that lactic acid bacteria can be used to metabolize and produce GABA. The fermented mucus Lactobacillus provided in this application can not only produce high γ-aminobutyric acid, but also produce other functional factors and have high antioxidant properties. Summary of the invention

[0004] The present invention aims to provide a fermented mucus lactobacillus with high γ-aminobutyric acid production and its application. The present invention isolates a fermented mucus lactobacillus from goat milk samples collected from Jianchuan, Dali, Yunnan. The strain produces high γ-aminobutyric acid and can significantly improve the environmental tolerance of the strain.

[0005] In a first aspect of the present invention, a fermented Lactobacillus mucosa producing γ-aminobutyric acid is provided, wherein the fermented Lactobacillus mucosa ( Limosilactobacillus fermentum ) named Lactobacillus fermentans B41 Limosilactobacillus fermentum B41, the deposit number is CCTCC NO: M 20242081, the depository is China Center for Type Culture Collection, and the deposit address is Wuhan University, Wuhan, China.

[0006] The second aspect of the present invention provides a microbial agent containing the fermented Lactobacillus mucilaginosus producing γ-aminobutyric acid.

[0007] The third aspect of the present invention provides the use of the fermented mucus lactobacillus or the microbial agent in the preparation of γ-aminobutyric acid.

[0008] As a further description of the above scheme: when fermenting mucus lactobacillus or microbial agents to prepare γ-aminobutyric acid, 10-30 μmol / L of exogenous AI-2 is added; after adding exogenous AI-2, the γ-aminobutyric acid content is 402.91±23.23 ug / mL.

[0009] The fourth aspect of the present invention provides the use of the fermented mucus lactobacillus or the microbial agent in the preparation of fermented milk containing γ-aminobutyric acid.

[0010] As a further description of the above scheme: the inoculum amount of the fermented mucus lactobacillus is 2.0×10 8 CFU / mL-3.1×10 8 CFU / mL.

[0011] As a further description of the above scheme: the fermented mucus lactobacillus is used in combination with a commercial yogurt starter; the usage of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 22h-26h, and the fermentation temperature is 36.5℃-37.5℃.

[0012] The characteristics of the present invention are as follows: The present invention screened a fermentation mucus lactobacillus strain from goat milk samples in Jianchuan, Dali, Yunnan. The strain has acid resistance, and the fermentation liquid has antioxidant function. It can also produce high γ-aminobutyric acid during the fermentation process, adding 10-30 μmol / L exogenous AI-2; after adding exogenous AI-2, the γ-aminobutyric acid content exceeds 400ug / mL. The production of γ-aminobutyric acid by the strain is regulated by the LuxS / AI-2 quorum sensing system; the proteins that specifically regulate the synthesis of γ-aminobutyric acid are LuxS and ArgD.

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

[0014] The present invention isolates and screens a lactic acid bacterium with high γ-aminobutyric acid production from goat milk, identifies the lactic acid bacterium through morphological identification and whole genome sequencing, and finds that the lactic acid bacterium belongs to Limosilactobacillus fermentum , which is fermented mucus Lactobacillus, and was named fermented mucus Lactobacillus B41.

[0015] The γ-aminobutyric acid content of the high-γ-aminobutyric acid-producing lactic acid bacteria strain of the present invention increases with the increase of the cell density and the intensity of the AI-2 signal molecule; after exogenous addition of AI-2, the cell density, the AI-2 signal molecule and the γ-aminobutyric acid all increase significantly, indicating that the synthesis of γ-aminobutyric acid of the strain is regulated by the LuxS / AI-2 quorum sensing system.

[0016] The fermented mucus lactobacillus B41 provided by the present invention is combined with a commercial starter to prepare fermented milk rich in gamma-aminobutyric acid, and the content of gamma-aminobutyric acid in the obtained fermented milk is significantly increased.

[0017] The microbial deposit information is as follows:

[0018] Classification and nomenclature: Limosilactobacillus fermentum B41;

[0019] Deposit number: CCTCC NO: M 20242081;

[0020] Depository: China Center for Type Culture Collection;

[0021] Deposit date: September 25, 2024;

[0022] Deposit address: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The colony morphology of fermenting Lactobacillus mucilaginosus B41 on MRS agar medium;

[0024] Figure 2 The colony morphology of Lactobacillus mucilaginosus B41 under electron microscope;

[0025] Figure 3 This is the gene circle map of Lactobacillus mucilaginosus B41;

[0026] Figure 4 is the growth curve of fermentative Lactobacillus mucilaginosus B41;

[0027] Figure 5 This is a graph showing the content of γ-aminobutyric acid produced by fermentation of Lactobacillus mucilaginosus B41;

[0028] Figure 6 This is a graph showing the content of AI-2 produced by fermentation of Lactobacillus mucilaginosus B41;

[0029] Figure 7 The effect of exogenous addition of AI-2 on the γ-aminobutyric acid content produced by fermentation of Lactobacillus mucilaginosus B41;

[0030] Figure 8 Venn diagram of differentially expressed proteins

[0031] Fig. 9 Statistical graph of differentially expressed proteins;

[0032] Fig.10 KEGG pathway enrichment analysis for fermentative Lactobacillus mucilaginosus B41;

[0033] Fig.11 (A) WB analysis of the expression of key proteins (LuxS and ArgD) in the F200 control group (1-1, 1-2, and 1-3) and the F200 group (F-1, F-1, and F-3); (B) Relative intensity of LuxS and ArgD; (C) Effects of furanone treatment on the cell density, GABA content, and AI-2 content of fermented Lactobacillus mucilaginosus B41;

[0034] Fig.12 The molecular mechanism of GABA synthesis in fermentative Lactobacillus mucilaginosus B41;

[0035] Fig.13 To determine the acid resistance of fermented Lactobacillus mucilaginosus B41;

[0036] Fig.14 The osmotic pressure resistance of fermented Lactobacillus mucilaginosus B41;

[0037] Fig.15 The DPPH free radical scavenging ability of fermented Lactobacillus mucilaginosus B41;

[0038] Fig.16 is the ABTS+ free radical scavenging rate of fermented Lactobacillus mucilaginosus B41;

[0039] Fig.17 This is the determination of the antibacterial ability of fermented Lactobacillus mucilaginosus B41, A is the inhibition zone against Pseudomonas aeruginosa, B is the inhibition zone against Staphylococcus aureus, C is the inhibition zone against Escherichia coli, and D is the inhibition zone against Salmonella;

[0040] Fig.18 is the γ-aminobutyric acid content of fermented milk;

[0041] Fig.19 Sensory scoring of fermented milk. DETAILED DESCRIPTION

[0042] In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is further described in detail below in conjunction with specific embodiments. The description in this section is only exemplary and explanatory and should not have any limiting effect on the scope of protection of the present invention. The fermented mucus Lactobacillus A78 used in the present invention is isolated from sauerkraut, and the strain identified by the whole gene sequencing method is fermented mucus Lactobacillus; the fermented mucus Lactobacillus B130 is derived from pickled papaya, and the strain identified by the whole gene sequencing method is fermented mucus Lactobacillus.

[0043] Example 1 Isolation and purification of bacterial strains

[0044] Take 10mL of goat milk sample collected from Jianchuan, Dali, Yunnan, add it to 100mL of sterile saline and shake it evenly. After gradient dilution, take 100uL and spread it on 1% CaCO3-MRS solid medium and culture it at 37℃ for 48h. Select the colonies that form calcium-dissolving circles on CaCO3-MRS solid medium and repeatedly streak them on MRS solid medium for purification until the colony morphology of the strain is consistent. Pick a single colony and inoculate it in MRS liquid medium at 37℃ for 24h for preservation and identification.

[0045] Identification of bacterial species

[0046] (1) Morphological characteristics of the strain

[0047] like Figure 1 As shown in the figure, after culturing Lactobacillus mucilaginosus B41 on MRS agar medium for 24 h, the colony morphology was round, milky white, moist, smooth, with neat edges and obvious protrusions. Figure 1 Under an optical microscope, the cells of Lactobacillus mucilaginosus B41 are rod-shaped, do not produce spores, and are Gram-positive. Figure 2 .

[0048] (2) Molecular genetic identification of bacterial species

[0049] Whole genome sequencing: The DNA extraction kit using TGUIDE S96 magnetic bead technology was used to extract the DNA of the screened strains. The Qubit method was used to determine its content, the Nanodrop method was used to determine its DNA integrity, and the agarose electrophoresis method was used to determine its DNA integrity. After the sample was tested, the library was constructed, and the sequencing test was performed on the machine after the library passed the quality inspection. Through PacBio third-generation sequencing technology, it was found that the genome size of fermentation mucus Lactobacillus B41 was 2,053,816 bp and the GC content was 51.62%. Using the assembled and predicted genomic information, such as tRNA, rRNA, repeated sequences, GC content, and gene function information, the software Circos v0.66 was used to draw a genome circle map. The gene circle map is shown in the figure below. Figure 3 shown.

[0050] Finally, the screened strain was determined to be fermented mucus Lactobacillus through morphology and whole genome sequencing, and was named fermented mucus Lactobacillus B41 Limosilactobacillus fermentum B41, and preserved in the China Center for Type Culture Collection with the accession number: CCTCC NO: M 20242081.

[0051] Example 2 Growth curve of fermented Lactobacillus mucilaginosus B41

[0052] The fermented Lactobacillus mucilaginosus B41 seed liquid activated for three generations in MRS liquid medium was inoculated into the sterilized MRS liquid medium at a 1% inoculation volume (V / V), and 200 uL was placed in a 96-well plate, and its absorbance at 600 nm was measured every 2 hours for a total of 24 hours. Each time point was repeated 3 times, and the average value was taken to draw a growth curve with time as the horizontal axis and absorbance as the vertical axis.

[0053] like Figure 4 As shown in the figure, the growth of fermentative Lactobacillus mucilaginosus B41 conforms to an S-shaped curve. The strain experienced a lag phase (0-2 h), a logarithmic phase (2-10 h), a stable phase (10-16 h), and a decay phase (16-24 h) within 24 h.

[0054] Example 3 Determination of γ-aminobutyric acid produced by fermentation of Lactobacillus mucilaginosus B41 and content of AI-2

[0055] (1) Determination of γ-aminobutyric acid produced by fermentation of Lactobacillus mucilaginosus B41

[0056] The determination was performed according to the instructions of the γ-aminobutyric acid (GABA) content kit of Suzhou Grace Biotechnology Co., Ltd. The fermented Lactobacillus mucosa B41 seed liquid activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at a 1% inoculation rate (V / V), cultured at 37°C for different time periods, centrifuged at 8000r / min for 15min, and the supernatant was taken and the GABA content was determined according to the instructions. The GABA content calculation formula is: GABA (μg / mL) = 1960.8 × (ΔA + 0.0003) × D;

[0057] Where: ΔA = absorbance (sample) - absorbance (CK); D is the dilution ratio.

[0058] like Figure 5 As shown, within 24 h, the content of γ-aminobutyric acid in fermented Lactobacillus mucilaginosus B41 increased with the increase of culture time, and the content was the highest at 24 h.

[0059] (2) Determination of AI-2 content produced by fermentation of Lactobacillus mucilaginosus B41

[0060] Using Vibrio harveyi ( V. harveyi )BB170 (ATCC BAA-1117) was used as the indicator bacteria, which was purchased from Guangdong Microbiological 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 autogenous bioassay (AB) liquid medium and cultured at 30°C for 12 h. V. harveyi The fermentation broth of BB170 was centrifuged at 4000 rpm for 30 min, and the resulting supernatant was filtered through a 0.2 μm filter. At the same time, the overnight cultured 100 μL of AB medium was diluted at a ratio of 1:100 (v:v). V. harveyi BB170 was mixed with the culture medium group (sterile MRS broth), different culture time groups, and AB liquid culture medium group (negative control) at a ratio of 1:50 (v:v). Then, 200 μL of each mixed sample was placed in a 96-well cell culture plate, each sample was placed in one well, and the fluorescence intensity of each sample was detected using the bioluminescence mode of the full-wavelength multifunctional microscope. When the fluorescence value of the negative control was the lowest within 1-7 h, the AI-2 content of each experimental group was calculated. The AI-2 content is the ratio of the fluorescence value of the experimental group to the fluorescence value of the negative control group.

[0061] like Figure 6 As shown in the figure, AI-2 produced by fermentation of Lactobacillus mucilaginosus B41 showed a trend of first increasing and then decreasing within 24 hours, especially at the end of the logarithmic phase, AI-2 was the highest.

[0062] result Figure 5 and Figure 6 The present application found that the γ-aminobutyric acid content of the strain increased with the increase of AI-2 content. AI-2 is an important signal molecule of the LuxS / AI-2 quorum sensing system. It is speculated that the γ-aminobutyric acid synthesis of fermentative Lactobacillus mucilaginosus B41 is regulated by the LuxS / AI-2 quorum sensing system.

[0063] (3) Effect of exogenous addition of AI-2 on the γ-aminobutyric acid content of fermented Lactobacillus mucilaginosus B41

[0064] In order to further verify the effect of LuxS / AI-2 quorum sensing system on γ-aminobutyric acid synthesis, the exogenous signal molecule (S)-4,5-dihydroxy-2,3-pentanedione (AI-2) purchased from Shanghai Saiers Biochemical Technology Co., Ltd. was added for verification.

[0065] Exogenous AI-2 was added to the culture medium to make the concentration of AI-2 in the culture medium 0, 10, 20, 40 and 80 μmol / L. The fermented Lactobacillus mucosa B41 seed liquid activated for three generations in MRS liquid medium was inoculated into the MRS liquid medium containing exogenous AI-2 at an inoculum rate of 1% (V / V), cultured at 37°C for 24 h, centrifuged at 8000 r / min for 15 min, and the supernatant was taken and the GABA content was determined according to the instructions.

[0066] Depend on Figure 7It can be seen that with the increase of exogenous AI-2 concentration in the culture medium, the content of GABA showed a trend of first increasing and then decreasing; when the concentration was 20 μmol / L, the content of signal GABA was the highest (403.91±23.23 μg / mL).

[0067] (4) Comparison of GABA content of fermented Lactobacillus mucilaginosus B41 with other strains

[0068] The determination was performed according to the instructions of the γ-aminobutyric acid (GABA) content kit of Suzhou Grace Biotechnology Co., Ltd. The fermented Lactobacillus mucosa seed liquid activated for three generations in MRS liquid medium was inoculated into MRS liquid medium at a 1% inoculation rate (V / V), cultured at 37°C for different time periods, centrifuged at 8000r / min for 15min, and the supernatant was taken and the GABA content was determined according to the instructions. The GABA content calculation formula is: GABA (μg / mL) = 1960.8 × (ΔA + 0.0003) × D;

[0069] Where: ΔA = absorbance (sample) - absorbance (CK); D is the dilution ratio.

[0070] As shown in Table 1, the GABA content of fermented Lactobacillus mucilaginosus B41 was higher than that of other strains.

[0071] Table 1 GABA content of strains

[0072]

[0073] Example 4 Mining and verification of key proteins for high γ-aminobutyric acid production by fermentation of Lactobacillus mucilaginosus B41

[0074] (1) Identification of differentially expressed proteins (DEPs) in Lactobacillus mucilaginosus B41 using 4D-DIA quantitative proteomics

[0075] The samples from the 2h (early exponential period; LuxS / AI-2-QS system not activated, low GABA content) and 10h (late exponential period; LuxS / AI-2-QS system activated, high GABA content) groups were combined in equal amounts as pool samples for HPRP fractionation and analyzed by LC-MS / MS (timsTOF_DDA mode) to obtain the DDA library database. Then, each sample was analyzed by LC-MS / MS (timsTOF DIA mode) and the above DDA database was used for qualitative and quantitative analysis.

[0076] 4D-DIA quantitative proteomics analysis was performed on the samples of the 2h and 10h groups to study the mechanism of LuxS / AI-2 QS system regulating GABA biosynthesis in fermentation bacteria B41. The results showed that a total of 1873 and 1849 proteins were identified in the 2h and 10h incubation groups, of which 1809 proteins were shared between the two groups ( Figure 8 ). A total of 657 significant DEPs (fold change >1.5 or <0.67, P < 0.05) were identified between the 2h and 10h incubation groups, of which 335 were upregulated and 322 were downregulated ( Fig. 9 ). These results indicate that the activation of the LuxS / AI-2 QS system affects the expression of the B41 protein in fermented milk.

[0077] (2) KEGG pathway enrichment analysis reveals key proteins

[0078] KEGG pathway enrichment analysis was performed to determine the metabolic pathways associated with DEPs in the fermentative Lactobacillus mucilaginosus B41 in the 2h and 10h incubation groups after the activation of the LuxS / AI-2 QS system. As shown in the figure, these differentially expressed proteins were enriched in the cysteine ​​and methionine metabolic pathways ( Fig.10 A). Notably, LuxS was significantly upregulated in these pathways (P<0.5). LuxS plays a key role in the production of AI-2, which plays multiple roles in bacterial communication, especially in the LuxS / AI-2 QS system. In addition, the differentially expressed proteins were also enriched in the GABA biosynthesis pathway ( Fig.10 B), ArgD is a key protein in GAB synthesis. Based on these results, it is speculated that fermentative Lactobacillus mucilaginosus B41 may promote GABA synthesis by regulating signaling molecules through the QS system.

[0079] (3) Verification of key proteins in the GABA biosynthesis pathway in fermentative Lactobacillus mucilaginosus B41

[0080] Furanone was added to the culture medium to make the concentration of furanone in the culture medium 200 μg / mL. The fermented Lactobacillus mucosa B41 seed liquid activated for three generations in MRS liquid medium was inoculated into the MRS liquid medium containing furanone at a 1% inoculation rate (V / V), cultured at 37°C for 24 h, centrifuged at 8000r / min for 15 min, the supernatant was taken and the GABA content was determined according to the instructions, and the bacterial cells were taken for key protein verification.

[0081] To verify the effect of LuxS / AI-2-QS system on GABA biosynthesis in fermented milk B41, the expression levels of LuxS and ArgD in the F200 control group and F200 group were determined. Fig.11As shown in A and B, the expression of LuxS and ArgD in the F200 control group was upregulated. After the addition of furanone, the expression of the F200 group was downregulated. In addition, the addition of furanone reduced the cell density, GABA content and AI-2 content of the fermented Lactobacillus mucilaginosus B41, as shown in Fig.11 C. These results indicate that furanone treatment inhibited the LuxS / AI-2QS system and GABA synthesis-related proteins and phenotypes, suggesting that GABA synthesis is regulated by the LuxS / AI-2 QS system.

[0082] (4) Molecular mechanism of GABA production mediated by LuxS / AI-2 QS system in fermented Lactobacillus mucilaginosus B41

[0083] This application also clarifies the mechanism by which the LuxS / AI-2 QS system regulates GABA biosynthesis in fermentative Lactobacillus mucilaginosus B41 ( Fig.12 ).like Fig.12 As shown, upregulation of LuxS promotes the production of AI-2 and its secretion into the extracellular space. Thereafter, AI-2 accumulates in the extracellular space and reaches a concentration threshold in the late exponential growth phase. Extracellular AI-2 enters the cell by binding to the receptor, then regulates the expression of ArgD and promotes the synthesis of GABA. When furanone enters the cell, it inhibits the LuxS / AI-2-QS system. Downregulation of LuxS inhibits the synthesis of AI-2, leading to the inhibition of GABA synthesis. In summary, the results of this study indicate that the synthesis of GABA in fermentative Lactobacillus mucilaginosus B41 is regulated by the LuxS / AI-2-QS system, thus providing a theoretical basis for the large-scale production of GABA.

[0084] Example 5 Probiotic properties of fermented Lactobacillus mucilaginosus B41

[0085] (1) Determination of acid resistance of strains

[0086] 1 mol / L HCl solution and NaOH solution were prepared respectively, and used to adjust the pH value in the MRS broth medium to 2.0, 3.0, 4.0, 5.0, 6.0 and 7.0, respectively. Lactic acid bacteria strains in the logarithmic growth period were taken with a pipette and inoculated into MRS liquid culture medium with different pH values ​​at an inoculum size of 6% (V / V). The plates were fully mixed, and the plates were cultured in a constant temperature incubator at 37°C for 24 h. The absorbance was measured at a wavelength of 600 nm using a microplate reader.

[0087] The strain must have good acid resistance to be suitable for fermentation and inoculation. Fig.13As shown in the figure, with the increase of pH, the growth of the strain first increased and then decreased. Compared with other strains, the growth of fermented mucus Lactobacillus B41 at low pH (2-5) was better than that of other strains (p < 0.05), which shows that fermented mucus Lactobacillus B41 has good acid resistance.

[0088] (2) Determination of strain osmotic pressure tolerance

[0089] MRS liquid culture medium with NaCl mass fractions of 0%, 2%, 4%, 6%, 7%, 8% and 9% was prepared and sterilized in an autoclave. The lactic acid bacteria strains in the logarithmic growth phase were taken with a pipette and inoculated into MRS liquid culture medium with different NaCl mass fractions at an inoculum volume of 6% (V / V). The culture was statically cultured in a 37℃ constant temperature incubator for 24 h, and the absorbance was measured at a wavelength of 600 nm using an ELISA reader.

[0090] Lactic acid bacteria have a certain degree of adaptability to osmotic pressure. When the osmotic pressure rises or falls sharply, it will affect the growth of microorganisms and even cause the death of microorganisms. Fig.14 As shown, compared with other strains, the osmotic pressure resistance of fermentative Lactobacillus mucilaginosus B41 is weaker and is not suitable for fermenting high-salt foods.

[0091] (3) Determination of antioxidant capacity of strains

[0092] Too many reactive oxygen free radicals can cause oxidative stress, which can cause a series of diseases in the body such as cardiovascular disease, atherosclerosis, diabetes, etc. Therefore, studying whether the strain has antioxidant capacity is extremely important for its application in the pharmaceutical and food industries.

[0093] ① Preparation of bacterial supernatant and bacterial cells

[0094] After the MRS liquid culture medium was sterilized and cooled, the target bacteria were inoculated into it at a ratio of 1%, and cultured at 37°C for 24 h. After culture, the bacterial solution was mixed and centrifuged at 3000 r / min for 5 min. The supernatant and bacterial cells were collected and stored at 4°C for later use.

[0095] ②DPPH free radical scavenging ability

[0096] Pipette 100 uL of lactic acid bacteria sample solution into a 96-well plate, add 100 uL of DPPH anhydrous ethanol solution (0.05 mmol / L), mix the solution, react at room temperature in the dark for 30 min, and finally use pure water as a blank. After the reaction is completed, vibrate the microplate reader at 37°C for 20 s and measure the absorbance at 517 nm.

[0097] Calculation formula: DPPH free radical scavenging rate (%) = [(A0-As) / A0] × 100

[0098] Wherein, A0: absorbance of distilled water + DPPH anhydrous ethanol solution; As: absorbance of sample solution + DPPH anhydrous ethanol.

[0099] like Fig.15 As shown, the DPPH free radical scavenging rate of the fermentation supernatant and bacteria of Lactobacillus mucilaginosus B41 reached more than 80%, which was significantly higher than that of other strains (p < 0.05).

[0100] ②ABTS + Free radical scavenging ability

[0101] ABTS + Preparation of diluted working solution (λ=734 nm, absorbance 0.7-0.8): Accurately weigh 0.192 g of ABTS + Standard samples were dissolved in distilled water and accurately diluted to the scale line of a 50 mL volumetric flask; at the same time, 0.067 g of potassium persulfate was accurately weighed and dissolved to 50 mL in the same way. After 1:1 mixing, it was stored in a dark environment for 12 h and diluted 30-35 times with PBS buffer (5 mmol / L, pH=7.4) for use. 25 uL of lactic acid bacteria sample solution was placed in a 96-well plate, and then 190 uL of ABTS dilution working solution was added. The reaction was carried out at room temperature for 6 min. After the reaction was completed, the absorbance value was measured at a wavelength of 734 nm using an ELISA reader at 37°C for 20 s. 25 uL of ultrapure water was transferred as a blank control.

[0102] Calculation formula: ABTS + Free radical scavenging rate (%) = [(A0-A1) / A0] × 100

[0103] Where: A0 is the absorbance of blank control; A1 is the absorbance of ABTS + The absorbance of working solution + PBS.

[0104] like Fig.16 As shown, the fermentation supernatant and ABTS of Lactobacillus mucilaginosus B41 + The free radical scavenging rate was significantly higher than that of other strains (p<0.05).

[0105] (4) Determination of antibacterial ability of strains

[0106] Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella were used as indicator strains for the determination of the antibacterial ability of fermented Lactobacillus mucilaginosus B41. The activated indicator strain suspension was inoculated into the unsolidified agar medium at a volume fraction of 3% (v / v), mixed and poured into the plate. After the plate was placed in the clean bench for 30 minutes, the Oxford cup (10 mm deep, 8 mm wide) was pulled out with tweezers, and 200 μL of fermented Lactobacillus mucilaginosus B41 fermentation supernatant (CFS) was added to the well, and 200 μL of sterilized sterile MRS broth was added to the control group. The plate was placed at 37°C for 24 hours, and the size of the inhibition zone was observed and measured, and repeated three times.

[0107] like Fig.17 As shown, compared with the control sterile MRS broth, fermented Lactobacillus mucoides B41 CFS showed obvious inhibition zones on Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella plates, with the diameters of the inhibition zones reaching 12.50±0.15 mm, 16.27±1.00 mm, 16.74±0.52 mm and 16.62±1.30 mm, respectively. This indicated that fermented Lactobacillus mucoides B41 CF had a high antibacterial ability and could inhibit the growth of Pseudomonas aeruginosa, Staphylococcus aureus, Escherichia coli and Salmonella strains.

[0108] Example 6 Application of fermented Lactobacillus mucilaginosus B41 in the preparation of fermented milk rich in γ-aminobutyric acid

[0109] 1. Preparation of fermented milk rich in γ-aminobutyric acid

[0110] The activated Lactobacillus mucilaginosus B41 strain was inoculated into MRS broth at a 1% inoculation rate and cultured (37°C, 24h) to the third generation. The Lactobacillus mucilaginosus B41 seed liquid domesticated in milk was inoculated into 1000 mL of milk with a protein content of 3.5% at a 4% (V / V) rate and a commercial direct-injection starter (Lactobacillus bulgaricus and Streptococcus thermophilus 1:1) at a 0.01% inoculation rate. The concentration of Lactobacillus mucilaginosus B41 was 2.6×10 8 CFU / mL, fermented at 37℃ for 24 h, and the fermented milk was placed in a 4℃ refrigerator for 12 h, and then the γ-aminobutyric acid content, physical and chemical indicators, texture indicators and sensory evaluation of the fermented milk were determined. Fermented Lactobacillus mucilaginosus B130 was compounded with a commercial direct-injection starter to prepare fermented milk according to the same steps as above, and the γ-aminobutyric acid content, physical and chemical indicators, texture indicators and sensory evaluation of the fermented milk of Lactobacillus mucilaginosus B130 were determined. Control group A was fermented milk prepared by fermentation of only Lactobacillus mucilaginosus B41, and control group B was fermented milk fermented by commercial starter alone.

[0111] 2. Determination of γ-aminobutyric acid content in γ-aminobutyric acid-rich fermented milk

[0112] like Fig.18 As shown in the figure, compared with the control group, the addition of Lactobacillus fermentans B41 increased the production of GABA in fermented milk (201.53 ± 7.83 μg / mL). The GABA content of fermented milk prepared by mixing Lactobacillus fermentans B41 with commercial starter was significantly higher than that of fermented milk prepared by fermenting Lactobacillus fermentans B130 and commercial starter and fermented milk prepared by commercial starter alone. The reason for this result is that Lactobacillus fermentans B41 is a lactic acid bacterium with high GABA production and is suitable for the preparation of fermented milk.

[0113] 3. Determination of physical and chemical indicators in fermented milk rich in γ-aminobutyric acid

[0114] The hardness of yogurt was determined by TA-XTPlus texture analyzer. The pH value of yogurt was determined by pH meter. The protein content was determined by Kjeldahl method according to GB5009.5-2016. The fat content was determined by alkaline hydrolysis method according to GB5009.6-2016. The sensory evaluation was conducted by 10 professionally trained employees (5 males and 5 females) based on color, smell, taste and texture.

[0115] Protein content, fat content, hardness, pH value and sensory evaluation have a significant impact on the final quality of fermented dairy products. As shown in Table 2, the protein content, hardness and pH value of milk fermented by Lactobacillus mucilaginosus B41 were higher than those of the control group. Fig.19 As shown in the results of sensory evaluation, the color and taste scores of fermented milk were similar. In addition, the tissue state and odor scores of fermented milk with the addition of Lactobacillus mucilaginosus B41 were higher than those of the control group. The above results further verified the suitability of Lactobacillus mucilaginosus B41 with high GABA production in fermented milk production.

[0116] Table 2 Basic physical and chemical indicators of fermented milk

[0117]

[0118] Note: Different lowercase letters in the same row indicate significant differences (P<0.05).

[0119] 4. Determination of microbial indicators of fermented milk

[0120] The count of molds and yeasts was determined with reference to the first method of GB / T 4789.15-2016. The count of coliform bacteria, Salmonella and Staphylococcus aureus were determined with reference to GB / T 4789.3-2016, GB / T 4789.4-2016 and GB / T 4789.10-2016 respectively. The count of lactic acid bacteria was determined with reference to the relevant methods of GB 4789.35-2016.

[0121] As shown in Table 3, the lactic acid bacteria microbial indicators meet the standards, and the number of lactic acid bacteria is higher than 1×10 6 CFU / g.

[0122] Table 3 Microbial indexes of fermented milk and number of viable lactic acid bacteria

[0123]

[0124] Note: Different lowercase letters in the same row indicate significant differences (P<0.05).

[0125] The above-mentioned implementation cases are preferred implementations of the present invention, but the implementations of the present invention are not limited to the above-mentioned implementation cases. Any other changes made without departing from the spirit and principle of the present invention shall be equivalent replacement modes and shall be included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus mucilaginosus that produces γ-aminobutyric acid ( Limosilactobacillus fermentum ) B41, characterized in that, The fermented mucus lactobacillus has a preservation number of CCTCC NO: M 20242081, a preservation unit of which is China Center for Type Culture Collection, and a preservation address of which is Wuhan University, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent containing the fermented Lactobacillus mucilaginosus producing γ-aminobutyric acid according to claim 1.

3. Use of the fermented mucus lactobacillus producing γ-aminobutyric acid according to claim 1 or the microbial agent according to claim 2 in the preparation of γ-aminobutyric acid, antioxidants and antibacterial agents, characterized in that: The bacteria are Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella or Escherichia coli.

4. The use according to claim 3, characterized in that When fermenting mucus lactobacillus or microbial agents to prepare gamma-aminobutyric acid, 10-30 μmol / L of (S)-4,5-dihydroxy-2,3-pentanedione is added.

5. Use of the fermented Lactobacillus mucilaginosus producing γ-aminobutyric acid as claimed in claim 1 or the microbial agent as claimed in claim 2 in the preparation of fermented milk containing γ-aminobutyric acid.

6. The use according to claim 5, characterized in that The inoculation amount of the fermented mucus lactobacillus is 2.0×10 8 CFU / mL-3.1×10 8 CFU / mL.

7. The use according to claim 5, characterized in that The fermented mucus lactobacillus is used in combination with a commercial yogurt starter; the usage of the commercial yogurt starter is 0.01-0.03% (m / m); the fermentation time is 22h-26h, and the fermentation temperature is 36.5°C-37.5°C.

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