Phytobacterium plantarum with good probiotic function and characteristics of fermented yoghurt thereof

Abstract

The invention discloses a plant lactobacillus with a good probiotic function and characteristics of fermented yoghurt of the plant lactobacillus, and belongs to the technical field of microorganisms. The phytobacterium plantarum g1-2 disclosed by the invention is relatively good in physiological property and probiotic property, and fermented yoghurt is fine and smooth in tissue state and long in fermentation aroma, which is specifically embodied in that (1) different carbon sources can be relatively well utilized, and the utilization capacities are remarkably different; (2) the tolerance to acid stress is good; and (3) the compound has better tolerance to simulated gastrointestinal tracts. And (4) the adhesive ability to HT-29 cells is relatively good. And (5) the yogurt fermented by using the fermented milk is fine and smooth in texture and long in fermentation aroma. Therefore, the phytobacterium plantarum g1-2 has a huge application prospect in fermentation of stirred yogurt and other dairy products.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus plantarum with good probiotic function and its yogurt fermentation characteristics, belonging to the field of microbial technology. Background Art

[0002] In the dairy industry, yogurt is popular among consumers due to its unique taste and nutritional value. Traditional yogurt preparation mainly relies on specific lactic acid bacteria for fermentation. These lactic acid bacteria can not only produce organic acids such as lactic acid during fermentation, but also endow yogurt with unique flavors and textures. With the increasing demand of consumers for healthy foods, the development of yogurt products with probiotic functions has become an important research direction in the dairy industry.

[0003] Lactiplantibacillus plantarum has a variety of probiotic functions, including inhibiting the growth of pathogenic bacteria, enhancing intestinal barrier function, regulating the immune system, promoting nutrient absorption, etc. These functions are mainly due to the mechanisms by which Lactiplantibacillus plantarum can produce a variety of antibacterial substances, regulate the intestinal pH value, interact with intestinal epithelial cells, etc. Literature research shows that Lactiplantibacillus plantarum can utilize a variety of carbon sources, including monosaccharides (glucose, fructose), disaccharides (such as sucrose, lactose, maltose, etc.), and polysaccharides (such as arabinose, xylose, and galactose polymers). This property makes it have good fermentation performance in fermented vegetables, pickles, and yogurt. The strong acid tolerance of lactic acid bacteria enables them to pass through the gastric acid environment well and colonize in the intestine to play their probiotic role. Therefore, the acid tolerance of lactic acid bacteria is regarded as one of the evaluation indicators of their growth ability. At the same time, the pH of the growth environment of lactic acid bacteria affects their growth and metabolism, weakening their absorption of nutrients, and the intracellular enzyme activity is also affected by pH. In addition, when lactic acid bacteria are used as a special starter in the fermentation of dairy products such as cow's milk and camel milk, they can exhibit good fermentation performance in an acidic environment and produce flavor substances. In addition, the gastrointestinal tolerance of lactic acid bacteria is often one of the important evaluation indicators of their probiotic properties. Probiotic lactic acid bacteria with good gastrointestinal tolerance can be made into probiotic powders, functional probiotic foods, and functional dairy products for consumption. In addition, the most ideal property of probiotics is to remain viable in the host gastrointestinal tract, colonize in the intestine, and adhere to the intestinal mucosa without being cleared by Escherichia coli. The ability to adhere to epithelial cells and mucosal surfaces is one of the important properties used for probiotics. Based on this, it is particularly important to conduct in vitro cell experiments to study the adhesion of lactic acid bacteria to human colon cancer cells HT-29. Therefore, applying Lactiplantibacillus plantarum to yogurt fermentation can not only retain the flavor and texture of traditional yogurt but also endow yogurt with more health benefits.

[0004] However, not all Lactiplantibacillus plantarum strains possess excellent probiotic functions and fermentation performance. During the process of fermenting carbohydrates to produce lactic acid, Lactiplantibacillus plantarum is faced with an acid stress environment. Different strains have different abilities to utilize carbohydrates, which affects their application potential in fermented foods. Therefore, it is necessary to further screen and optimize strains with high carbohydrate utilization ability to improve their performance and application scope in the food industry. In addition, Lactiplantibacillus plantarum needs to have certain acid tolerance to ensure that it can successfully pass through the acidic gastric digestive tract and enter the intestine. Research shows that different strains have different tolerances to acid stress. For example, L. plantarum BG24 exhibits good acid tolerance. However, there is still room for improvement to increase its survival rate in the gastric acid environment. When passing through the gastrointestinal tract, Lactiplantibacillus plantarum is affected not only by low pH and high concentrations of bile salts but also by various digestive enzymes such as pepsin and trypsin. Although some strains such as L. plantarum BG24 show tolerance to bile salts, it is still necessary to further study and screen strains that can tolerate these environmental stresses to increase their survival rate in the gastrointestinal tract. Lactiplantibacillus plantarum is widely used in fermented foods, but its fermentation characteristics may vary. Research shows that different strains have different growth and acid production characteristics in fermented milk, which affects the quality of fermented milk. Therefore, it is necessary to further study the fermentation characteristics of different strains to optimize the fermentation process and improve product quality. The ability to adhere to intestinal cells is the basis for probiotics to exert their probiotic effects. Research shows that the adhesion ability of Lactiplantibacillus plantarum to the surface of the intestinal mucosa provides it with a competitive advantage. However, the adhesion abilities of different strains vary, and it is necessary to further study and improve the adhesion ability of the strains to enhance their colonization and probiotic effects in the intestine. In practical applications, it is necessary to screen Lactiplantibacillus plantarum strains with better probiotic functions and stable fermentation performance to ensure the quality and safety of yogurt products. Through traditional isolation, screening, and identification methods, Lactiplantibacillus plantarum strains with specific probiotic functions can be obtained. These strains can grow stably, produce abundant lactic acid and other beneficial substances during the yogurt fermentation process.

[0005] In summary, having high carbohydrate utilization ability, acid tolerance, gastrointestinal tolerance, and being able to colonize in the intestine are important probiotic functions of strains. Screening a Lactiplantibacillus plantarum strain with good probiotic functions and capable of fermenting to produce high-quality yogurt has important research value and market prospects. Summary of the Invention

[0006] The present invention provides a Lactiplantibacillus plantarum with good probiotic function, characterized in that the Lactiplantibacillus plantarum g1-2 was deposited at the Guangdong Provincial Microbial Culture Collection Center on November 11, 2024, with the deposit number GDMCC No: 65467 and the deposit address being the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0007] In one embodiment, the Lactiplantibacillus plantarum g1-2 is derived from the milk curd collected from Emin County, Tacheng Prefecture, Xinjiang. The colonies of this strain on MRS medium are round, with a smooth surface, small size, relatively moist, and white.

[0008] In one embodiment, the Lactiplantibacillus plantarum g1-2 can utilize different carbon sources (fructose, trehalose, cellobiose, mannitol, and sorbose) well, and there are significant differences in the utilization ability.

[0009] In one embodiment, the Lactiplantibacillus plantarum g1-2 has good tolerance to acid stress.

[0010] In one embodiment, the Lactiplantibacillus plantarum g1-2 has good tolerance to simulated gastrointestinal tract.

[0011] In one embodiment, the Lactiplantibacillus plantarum g1-2 has good adhesion ability to HT-29 cells.

[0012] The present invention also provides a microbial preparation containing the Lactiplantibacillus plantarum g1-2.

[0013] In one embodiment, in the microbial preparation, the cell count of Lactiplantibacillus plantarum g1-2 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

[0014] The present invention also provides a product containing the Lactiplantibacillus plantarum g1-2 or the microbial preparation.

[0015] In one embodiment, the product includes food, medicine, or health products.

[0016] In one embodiment, the food includes but is not limited to yogurt.

[0017] The present invention also provides a method for preparing a yogurt with a cheesy flavor, which is obtained by inoculating the Lactiplantibacillus plantarum g1-2 into dairy products for fermentation.

[0018] In one embodiment, the dairy products include but are not limited to cow's milk.

[0019] In one embodiment, fermentation is carried out at 42 °C for 16 - 24 h. After fermentation, demulsification is performed, and then after-ripening is carried out at 2 °C - 10 °C for at least 72 h.

[0020] In one embodiment, the fermentation-related indexes of the yogurt are as follows:

[0021] (a) Raw milk indexes: protein 3.23; fat 3.71; total solids 12.72; pH 6.68; acidity 12.40;

[0022] (b) Indexes after batching: protein 2.94; fat 3.64; total solids 19.68; pH 6.56; acidity 14.00;

[0023] (c) Indexes after demulsification at the end of fermentation: protein 1.53; fat 1.82; total solids 11.39; pH 4.17; acidity 86.7; viscosity 3975; the tissue state after fermentation is relatively tender; the taste is normal, with the fermentation aroma of ordinary yogurt;

[0024] (d) Indexes after 72 h of after-ripening: pH 4.13; acidity 91.6; viscosity 10048; the tissue state after fermentation is delicate; the fermentation aroma is long, with a cheesy flavor.

[0025] The present invention also provides the application of the Lactiplantibacillus plantarum g1-2 or the microbial preparation in the preparation of fermented foods.

[0026] Advantages and effects of the present invention:

[0027] The Lactiplantibacillus plantarum g1-2 of the present invention has good physiological characteristics and probiotic characteristics, and the yogurt fermented by it has a delicate tissue state and a long fermentation aroma, specifically manifested in:

[0028] (1) It can utilize different carbon sources (fructose, trehalose, cellobiose, mannitol and sorbose) well, and there are significant differences in the utilization ability. Its utilization ability for the above carbon sources is better than that of glucose;

[0029] (2) It has good tolerance to acid stress;

[0030] (3) It has good tolerance to the simulated gastrointestinal tract;

[0031] (4) It has good adhesion ability to HT-29 cells;

[0032] (5) The yogurt fermented with it has a delicate tissue state, a long fermentation aroma, and a buttery flavor.

[0033] Therefore, Lactiplantibacillus plantarum g1-2 has great application prospects in the application of fermented stirred yogurt and other dairy products.

[0034] Biological material preservation

[0035] A strain of Lactiplantibacillus plantarum g1-2, classified and named Lactiplantibacillus plantarum, was deposited in the Guangdong Provincial Microbial Culture Collection Center on November 11, 2024, with the deposit number GDMCC No: 65467, and the deposit location is the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou. Brief description of the drawings

[0036] Figure 1 It is the ability of the strain to utilize different carbon sources;

[0037] Figure 2 It is the acid stress tolerance ability of the strain;

[0038] Figure 3 It is the gastrointestinal tolerance ability of the strain simulated;

[0039] Figure 4 It is the adhesion ability of the strain to HT-29 cells. Specific implementation examples

[0040] The present invention will be further described below in conjunction with specific embodiments.

[0041] The glucose involved in the following examples was purchased from Urumqi Kehua Weiye Biotechnology Co., Ltd., and the yeast powder, etc. were purchased from Shaanxi Tongshengchang Biotechnology Co., Ltd.

[0042] The culture media, simulated gastric juice and intestinal juice involved in the following examples are as follows:

[0043] MRS solid medium (g / L): glucose 20 g / L, peptone 10 g / L, yeast powder 5 g / L, beef extract powder 10 g / L, anhydrous sodium acetate 2 g / L, diammonium citrate 2 g / L, dipotassium hydrogen phosphate 2.6 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 1 g / L, agar 20 g / L, distilled water 1 L.

[0044] MRS liquid medium (g / L): Glucose 20 g / L, peptone 10 g / L, yeast extract 5 g / L, beef extract powder 10 g / L, anhydrous sodium acetate 2 g / L, diammonium citrate 2 g / L, dipotassium hydrogen phosphate 2.6 g / L, magnesium sulfate 0.5 g / L, manganese sulfate 0.25 g / L, Tween 80 1 g / L, distilled water 1 L.

[0045] Simulated gastric juice: ① 0.9% w / v normal saline adjusted to pH 3.0 with hydrochloric acid; ② Pepsin was dissolved in sterilized normal saline (0.9% w / v, adjusted to pH 3.0 with hydrochloric acid), and the final concentration was 3 g / L. Filtered through a 0.22 μm sterile filter membrane and used freshly prepared.

[0046] Simulated intestinal fluid: ① 0.9% w / v normal saline adjusted to pH 8.0 with NaOH; ② Trypsin was dissolved in sterilized normal saline (0.9% w / v, adjusted to pH 8.0 with NaOH) to a final concentration of 1 g / L, and bile salts were added to a final concentration of 0.3%. Filtered through a 0.22 μm sterile filter membrane and used freshly prepared.

[0047] The culture medium for HT-29 cells is DMEM medium.

[0048] The Lactiplantibacillus plantarum g1-2 involved in the following examples is the Lactiplantibacillus plantarum XJU-LP-g1-2 in the preservation certificate, and the preservation number is GDMCC No: 65467.

[0049] Example 1: Isolation, screening and strain identification of Lactiplantibacillus plantarum

[0050] (1) Isolation and screening

[0051] Take 2 mL of yogurt collected from Emin County, Xinjiang and enrich it in 45 mL of MRS liquid medium for 12 h. Then take out 0.5 mL of the sample, perform gradient dilution with 0.9% sterilized normal saline, and take 10 -6 、10 -7 、10 -8 dilution solutions of 100 μL, spread them by dilution coating, with 3 parallel samples for each gradient. Place them in an anaerobic incubator at 37 °C for 18 - 24 h. Select the suspected lactic acid bacteria colonies according to the odor, shape, size, luster, color, etc. of the colonies, and perform streak purification on MRS solid medium 2 - 3 times until pure colonies are obtained.

[0052] (2) Identification

[0053] The purified single colony was inoculated into 5 mL of MRS liquid medium and cultured for 24 h. The bacterial liquid was centrifuged at 8000 r / min for 3 min, the supernatant was discarded, the bacterial sludge was washed twice with sterile water, and resuspended in 50 μL of sterile water as the bacterial liquid template. Primers 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used. The PCR reaction system was as follows: TaqMaster Mix (2×) 12.5 μL; double-distilled water 10.5 μL; template 1 μL; forward and reverse primers 0.5 μL each. The amplification program was: 98 °C for 5 min; 94 °C for 30 s; 55 °C for 30 s; 72 °C for 1 min, 35 cycles, 72 °C for 10 min.

[0054] After the PCR amplification products were detected by agarose gel electrophoresis with a mass fraction of 1.8%, the PCR products with correct electrophoresis bands were sent to Xinjiang Youkang Biotechnology Co., Ltd. for sequence determination. The obtained sequences were aligned online at the National Center for Biotechnology Information (NCBI) in the United States, and strains with a similarity of more than 98% were defined as the same species. After analysis, the screened strain was Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum g1-2 (Lactiplantibacillus plantarum XJU-LP-g1-2), which was deposited in the Guangdong Provincial Culture Collection of Microorganisms on November 11, 2024, with the deposit number GDMCC No: 65467.

[0055] Example 2: Analysis of the utilization ability of Lactiplantibacillus plantarum for different carbon sources

[0056] The growth status of Lactiplantibacillus plantarum g1-2 in carbon source media such as fructose, trehalose, cellobiose, mannitol, and sorbitol was determined. After the carbon-free MRS liquid medium was sterilized at 121 °C for 15 min, the measured sugars were filtered and sterilized with a 0.22 μm water-based sterile microporous filter and then added to the carbon-free MRS liquid medium at a ratio of 0.5% (wt / vol, g / mL). The strain preserved in glycerol was inoculated into the MRS liquid medium at an inoculation amount of 2% and cultured at 37 °C for 24 h for activation, and cultured continuously for 3 generations. The strain cultured for 3 generations was centrifuged at 8000 r / min at 4 °C for 10 min to collect the bacteria, washed twice with sterile PBS (7.2 - 7.4) buffer, and then the bacteria were resuspended in the PBS buffer. The prepared media with different carbon sources were added to 96-well plates, and 3 parallels were set. With a final concentration of 1×10 7Inoculate Lactiplantibacillus plantarum into the culture media with different carbon sources (the concentration of carbon source is 0.5 g / mL) at an inoculum concentration of CFU / mL, incubate at 37 °C for 24 h, and then use a microplate reader to measure the OD 600 nm value. Use the MRS culture medium with a conventional carbon source (glucose) to culture Lactiplantibacillus plantarum g1-2 as a control. The results show that the OD value of the bacterial solution in the MRS culture medium with the conventional carbon source is 0.66, while the OD values of the bacterial solutions in the culture media with other carbon sources are all above 1 ( Figure 1 ), indicating that Lactiplantibacillus plantarum g1-2 has good utilization ability for fructose, trehalose, cellobiose, mannitol, and sorbitol.

[0057] Example 3: Analysis of acid stress tolerance of Lactiplantibacillus plantarum

[0058] Prepare MRS liquid media with pH values of 2, 3, 4, 5, 6, and 7 respectively. Inoculate the strains into the MRS liquid media with different pH values at an inoculum concentration of 2% (v / v), and measure the OD 600 nm value of the strains at 0 h, 16 h, and 24 h of incubation at 37 °C. Set 3 replicates for each strain. The results show that Lactiplantibacillus plantarum g1-2 can survive in an environment with a pH of 2 and has good acid tolerance ( Figure 2 ).

[0059] Example 4: Analysis of the tolerance ability of Lactiplantibacillus plantarum to the simulated gastrointestinal tract

[0060] Inoculate the strains preserved in glycerol into the MRS liquid medium at an inoculum concentration of 2% (v / v), incubate at 37 °C for 24 h for activation. After continuous culturing for 3 generations, centrifuge at 8000 r / min for 10 min to collect the bacterial cells, discard the supernatant, wash twice with 0.9% sterile physiological saline, and resuspend in an equal volume of simulated gastric juice (the inoculation concentration of Lactiplantibacillus plantarum is 1×10 9 CFU / mL), mix well with a vortex mixer, and perform plate viable count after incubation at 37 °C for 3 h. Similarly, resuspend the bacterial suspension in simulated intestinal fluid, mix well with a vortex mixer, and perform plate viable count after incubation at 37 °C for 4 h. The survival rate is the percentage value of the number of viable bacteria on the plate after culturing in gastrointestinal fluid to the number of viable bacteria on the plate before culturing in gastrointestinal fluid. The results show that ( Figure 3 ) the survival rates of the strains in simulated gastric juice and intestinal fluid are both relatively high. Among them, the survival rate in gastric juice is as high as 73.85%, and the survival rate in intestinal fluid is 61.27%.

[0061] Example 5: Analysis of the adhesion ability of Lactiplantibacillus plantarum to HT-29 cells

[0062] HT-29 cells were cultured in an incubator at 37 °C and 5% CO2, and the culture medium was changed every other day. The cells were subcultured for 3 - 4 passages. When the cells reached 80% confluence in the culture dish, the cell surface was washed with an appropriate amount of PBS. Then, the PBS was discarded, and trypsin was added for digestion. After complete digestion, the digestion was terminated with the culture medium. The cell suspension was centrifuged at 1000 r / min for 5 min, the supernatant was discarded, and an appropriate amount of culture medium was added to resuspend the cells by pipetting. 1 mL of the cell suspension was transferred into a 6-well plate containing a coverslip and cultured in an incubator at 37 °C and 5% CO2 for 24 h to allow the cells to adhere to the coverslip. Preparation of the bacterial suspension: The strain was activated for 2 generations at 37 °C. The activated bacterial suspension was centrifuged (8000 r / min, 4 °C, 6 min), the bacterial cells were collected, washed three times with PBS, and the concentration of the bacterial suspension was adjusted to 1×10 7 CFU / mL with DMEM medium. 2 mL of the bacterial suspension was added to the above 6-well plate. After the adherent cells and the bacterial suspension were co-cultured at 37 °C for 3 h, the co-culture medium was discarded, washed with PBS, fixed with methanol for 10 - 20 min, then stained with the Gram staining method, and observed under a 100× oil immersion objective lens. At the same time, the viable cell count on the plate was performed. The formula for calculating the adhesion rate is (the number of viable bacteria before culture - the number of viable bacteria after culture) / the number of viable bacteria before culture × 100%. The results showed that ( Figure 4 ), the adhesion rate of the strain was relatively high, at 88.41%.

[0063] Example 6: Analysis of the characteristics of yogurt fermented by Lactiplantibacillus plantarum

[0064] The strain was inoculated into cow's milk at an addition amount of 0.1% (1×10 9 CFU / mL) to ferment stirred yogurt. Fermentation was carried out at 42 °C. The raw milk indexes (protein, fat, total solids, pH, acidity), indexes after batching (protein, fat, total solids, pH, acidity), indexes after breaking the emulsion at the end of fermentation (protein, fat, total solids, pH, acidity, taste), and indexes after 72 h of post-ripening (protein, fat, total solids, pH, acidity, taste) of the stirred yogurt were measured respectively.

[0065] Fermentation process: First, conduct a sensory inspection on the raw milk. Check its color, smell, and taste through vision, smell, and taste to determine whether it is normal. Then, conduct physical and chemical index tests, including protein, fat, total solids, pH, and acidity, etc. Then, mix the raw milk and the ingredients evenly. Ensure that the temperature is appropriate during mixing, usually between 40°C and 50°C, to avoid caking or denaturation of the ingredients. Measure the relevant indexes after adding the ingredients, including protein, fat, total solids, pH, and acidity. After that, inoculate the strain into fresh milk. After inoculation, ferment at 42°C for 22 hours. Keep the temperature constant during the fermentation process. After the fermentation is completed, conduct demulsification treatment on the fermented milk to improve its tissue state and facilitate subsequent emptying. Measure the relevant indexes after demulsification at the end of fermentation, including protein, fat, total solids, pH, acidity, and viscosity, etc. The fermented milk after demulsification needs to be post-ripened to promote the generation of flavor substances and improve the texture and water-holding capacity of the product. Cool the fermented milk to 2°C - 10°C and keep it for 72 hours. Measure the relevant indexes after 72 hours of post-ripening, including protein, fat, total solids, pH, acidity, and viscosity, etc.

[0066] Table 1 Data analysis of preparing fermented yogurt with Lactiplantibacillus plantarum g1-2

[0067]

[0068] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A Lactiplantibacillus plantarum g1-2, which has been deposited in the Guangdong Microbial Culture Collection Center with the deposit number GDMCC No: 65467.

2. A microbial preparation containing the Lactiplantibacillus plantarum g1-2 described in claim 1.

3. The microbial agent according to claim 2, wherein In the microbial agent, the cell count of Lactiplantibacillus plantarum g1-2 is not less than 1×10 9 CFU / mL or 1×10 9 CFU / g.

4. A product containing the Lactiplantibacillus plantarum g1-2 described in claim 1 or the microbial preparation described in claim 2.

5. The product according to claim 4, wherein The product includes food, medicine, or health products.

6. The product according to claim 5, wherein, The food includes, but is not limited to, yogurt.

7. A method for preparing a yogurt with a cheesy flavor, characterized in that, Obtained by inoculating the Lactiplantibacillus plantarum g1-2 described in claim 1 into dairy products for fermentation.

8. The method according to claim 7, wherein The dairy products include, but are not limited to, cow's milk.

9. The method according to claim 8, wherein Ferment at 42°C for 16 - 24 h, perform demulsification after fermentation, and then ripen at 2°C - 10°C for at least 72 h.

10. Use of the Lactiplantibacillus plantarum g1-2 described in claim 1 or the microbial preparation described in claim 2 in the preparation of fermented foods.

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