A strain of Lactobacillus paracasei PC804 and its applications

Abstract

The present invention discloses a Lactobacillus paracasei with probiotic characteristics isolated from pickled vegetables, named Lactobacillus paracasei PC804, which has been deposited in the General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, with the deposit number of CGMCC No. 23917. The Lactobacillus paracasei PC804 in the present invention has a high survival rate in gastrointestinal fluid, good bile salt tolerance, and strong survival ability in the human body. Lactobacillus paracasei PC804 has probiotic characteristics of enhancing immune resistance, improving the diversity of intestinal flora, and increasing the stability of intestinal flora. In addition, PC804 can be well applied to fermented dairy products as a probiotic.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a Lactobacillus paracasei strain ( Lacticaseibacillus paracasei ) PC804 with probiotic characteristics isolated from pickled vegetables, and also relates to its application in probiotic fermented dairy products. Background Art

[0002] Pickled vegetables are a traditional fermented vegetable product. There is rich diversity of lactic acid bacteria in pickled vegetables, including Lactiplantibacillus plantarum ( Lactiplantibacillus plantarum ), Lactobacillus mucosae ( Limosilactobacillus fermentium ), Lactobacillus paracasei ( Lacticaseibacillus casei ), etc. Studying its lactic acid bacteria has important value.

[0003] Lactic acid bacteria (LAB) refers to a general term for a group of non-spore-forming, Gram-positive bacteria that ferment sugars and whose main product is lactic acid. They are widely present in people's lives and are abundant in fermented foods. Lactic acid bacteria not only play an important role in the sensory characteristics of foods, but also have probiotic functions such as regulating the intestinal flora, enhancing immunity, and promoting nutrient absorption. A large number of studies have found that lactic acid bacteria present in fermented foods can promote the absorption of nutrients by organisms, reduce cholesterol in the body, and improve the body's immunity by increasing non-specific (cell function, natural killer cell activity) and specific (cytokine production, antibody production, lymphocyte proliferation, delayed-type hypersensitivity) host immune responses. Lactic acid bacteria have a rather long history of safe use, and lactic acid bacteria commonly used in fermented products have long been considered generally recognized as safe (GRAS) strains.

[0004] Lactobacillus paracasei ( Lacticaseibacillus paracasei ) is a facultative anaerobic, Gram-positive, heterofermentative lactic acid bacterium, which is widely present in the human oral cavity, intestine, fermented dairy products (especially cheese) and plant raw material fermentates (pickled vegetables, wine and feed). It has the characteristics of the genus Lactobacillus casei and can decompose proteins to produce small molecular peptides through the action of extracellular enzymes, playing an important role in blood pressure reduction, anti-tumor, antioxidant, cholesterol reduction, etc. At the same time, Lactobacillus paracasei has no toxic side effects, no drug resistance and is easily degraded by the human body, and has probiotic functions such as regulating intestinal balance, enhancing non-specific immunity, and promoting absorption. Summary of the Invention

[0005] The purpose of the present invention is to provide a Lactobacillus paracasei strain ( Lacticaseibacillus paracasei ) PC804 with certain probiotic characteristics isolated from pickled vegetables, which has been deposited in the China General Microbiological Culture Collection Center of the China Committee for Culture Collection of Microorganisms, and the deposit number is CGMCC No. 23917.

[0006] The present invention provides the application of Lactobacillus paracasei PC804 Lacticaseibacillus paracasei in food additives.

[0007] Among them, the food additive is a dairy starter.

[0008] On the other hand, the present invention also provides the application of Lactobacillus paracasei PC804 Lacticaseibacillus paracasei in fermented dairy products, wherein the fermented dairy product is fermented yogurt, drinking yogurt, fermented milk beverage or fermented soy milk.

[0009] The Lactobacillus paracasei PC804 of the present invention Lacticaseibacillus paracasei can resist the adverse effects of the digestive tract environment and adhere and colonize well in the intestine. Its live bacteria have the effects of enhancing immune resistance, increasing the diversity of intestinal flora, and enhancing the stability of intestinal flora. When used in humans and animals, it can densely stay on the wall of the digestive tract in the intestine, so as to prevent harmful bacteria from staying, and produce lactic acid to lower the pH value in the intestine and inhibit the reproduction of harmful bacteria. Moreover, the live bacteria used produce bacteriocin and peroxide, so as to be able to inhibit the reproduction of pathogenic bacteria and help the activities of intestinal villi responsible for absorbing nutrients.

[0010] The Lactobacillus paracasei PC804 of the present invention Lacticaseibacillus paracasei has been confirmed by sensory evaluation to have excellent taste and texture scores and odor scores. After being prepared into a brown milk beverage, the acidity and viable count of probiotics in the product samples stored at 4 °C every 7 days after ripening change little during the shelf life, and a high viable count is maintained during storage, proving that PC804 of the present invention has good probiotic characteristics and can be applied to commercial fermented dairy products.

[0011] The present invention relates to the following microorganisms:

[0012] Lactobacillus paracasei Lacticaseibacillus paracasei PC804, which was deposited at the China General Microbiological Culture Collection Center on November 15, 2021, with the deposit number of CGMCC No. 23917. Description of the Drawings

[0013] Figure 1 : Electron micrograph of Lactobacillus paracasei PC804 (3000 times);

[0014] Figure 2 : Colony morphology of Lactobacillus paracasei PC804 on the medium;

[0015] Figure 3 : Lactobacillus paracasei Survival rate change of PC804 in the simulated artificial digestive tract. Detailed implementation mode

[0016] The present invention will be further described below in conjunction with embodiments. The following embodiments are only illustrative, and the present invention is not limited by these examples.

[0017] In the present invention, unless otherwise specified, "%" for explaining concentration is weight percentage.

[0018] The present invention relates to the following culture media:

[0019] MRS liquid medium: Peptone (animal origin) 10 g, Beef extract powder 8 g, Yeast extract powder 4 g, Glucose 20 g, Tween-80 1 ml, Dipotassium hydrogen phosphate 2 g, Sodium acetate 5 g, Triammonium citrate 2 g, Manganese sulfate 0.05 g, Magnesium sulfate 0.2 g, Distilled water 1 L, adjust pH to 6.5±0.2, sterilize at 121°C for 15 min.

[0020] MRS solid medium: Peptone (animal origin) 10 g, Beef extract powder 8 g, Yeast extract powder 4 g, Glucose 20 g, Tween-80 1 ml, Dipotassium hydrogen phosphate 2 g, Sodium acetate 5 g, Triammonium citrate 2 g, Manganese sulfate 0.05 g, Magnesium sulfate 0.2 g, Distilled water 1 L, adjust pH to 6.5±0.2, Agar 15 g, sterilize at 121°C for 15 min.

[0021] Example 1 Obtaining of Lactobacillus paracasei PC804

[0022] The pickled vegetable samples collected from pickled vegetables in Dayi County, Sichuan Province were serially diluted to obtain dilution gradients of 10 -5 、10 -6 、10 -7 200 μL of each gradient pickled vegetable sample was evenly spread on the MRS solid medium petri dish and anaerobically cultured at 37°C for 48 - 72 h. Monoclonal colonies with different morphologies, sizes, and colors were picked and inoculated into the MRS liquid medium, and cultured in a 37°C constant temperature incubator for 24 h. After the strains grew well, Gram staining and microscopic examination were performed. The isolated strains were preserved, and the genomic DNA of the strains was extracted for subsequent determination and analysis.

[0023] The tested strains stored frozen were inoculated into the MRS liquid medium and cultured at 37°C for 24 h. After subculturing 2 - 3 generations, 2 mL of the cell culture at the late logarithmic growth phase was placed in a sterile EP tube and centrifuged. The cells were collected after centrifugation at 8000×g for 3 min (4°C), the supernatant was discarded, and the genomic DNA of the strains was extracted using the special CTAB freeze-thaw method for lactic acid bacteria.

[0024] The whole genome of PC804 was sequenced using the PacBio SMRT RSII third-generation sequencing platform to confirm that the obtained strain was Lactobacillus paracasei ( Lacticaseibacillus paracasei ). Lacticaseibacillus paracasei )

[0025] The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 screened in the present invention has the following morphological characteristics: The cells are short rod-shaped under the microscope, with both ends flush and square, and the cells are arranged singly, in pairs or in chains (as shown in Figure 1 ), without spores and flagella, and immobile.

[0026] The colonies formed by the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 on MRS medium are milky white, shiny, with neat edges, mostly convex lens-shaped protrusions, and the diameter is between 0.5 - 1 mm (as shown in Figure 2 ).

[0027] Example 2 Tolerance determination

[0028] 1. Tolerance of the strain to gastrointestinal fluids

[0029] Preparation method of simulated gastrointestinal fluids: After sterilizing PBS, adjust the pH value to 2.5 with 1 mol / L HCl, add 3.0 mg / ml pepsin, and filter and sterilize with a 0.22 μm microporous filter membrane to prepare artificial simulated gastric juice;

[0030] Preparation method of artificial simulated pancreatic juice: After sterilizing PBS, adjust the pH value to 8.0 with 0.1 mol / L NaOH, add 0.1% trypsin and 1.8% bile salts, and filter and sterilize with a 0.22 μm microporous filter membrane to prepare artificial simulated pancreatic juice.

[0031] Tolerance to gastrointestinal fluids: Activate and culture the isolated and purified strain for two generations, centrifuge and wash the bacteria twice, collect the bacteria, take 0.5 ml of the rescreened bacterial suspension and add it to 4.5 ml of artificial simulated gastric juice with pH 2.5, digest at 37°C for 3 h, and simultaneously count the viable bacteria at 0 h and 3 h by the MRS agar medium pouring method.

[0032] After that, take 0.5 ml of the artificial bacteria-containing gastric juice that has been digested for 3 h and add it to 4.5 ml of artificial intestinal juice, continue to culture in a water bath at 37°C, and count the viable bacteria at 4 h and 8 h by the MRS agar medium pouring method. Each sample is done in 4 parallels.

[0033] Calculation formula for the survival rate of the strain

[0034]

[0035] N 1 ―― viable cell count after strain treatment;

[0036] N 0 ―― initial viable cell count of the strain.

[0037] The results showed that Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 was digested in artificial gastric juice at pH 2.5 for 3 hours (survival rate 57.94%), and then continued to be digested in artificial digestive juice at pH 8.0 for 8 hours, with a final survival rate of 30.68% (as Figure 3 shown).

[0038] The results indicated that Lactobacillus paracasei PC804 had good tolerance to gastrointestinal digestive juices and could enter the human intestine in a viable state to exert health effects. The above characteristics are the basis for being a probiotic. The Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 of the present invention is a probiotic with probiotic activity, and it can be expected that when it is used in humans or animals in the form of dried cell morphology or fermentation product form, it can have a beneficial effect on the host's intestinal flora and has probiotic characteristics such as enhancing immune resistance, increasing the diversity of intestinal flora, and increasing the stability of intestinal flora.

[0039] 2. Bile salt tolerance of the strain

[0040] After the isolated and purified strain was activated and cultured for three generations, it was inoculated into MRS medium containing 0.3% bile salt (0.2 g / 100 mL sodium thioglycolate and 0.3 g / 100 mL bile salt were added to the medium) and MRS medium without bile salt at an inoculation amount of 1% respectively. The latter was used as a blank control and placed in anaerobic culture at 37°C. During the culture period, samples were taken every hour to measure the OD value at 620 nm, and three parallels were measured for each group. The culture was stopped until the OD value increased by 0.3 units or more. And the time required for the OD value to increase by 0.3 units when the strain grew in MRS medium with and without bile salt was calculated, and the difference between the two was called the lag time (LT).

[0041] The results showed that Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 was cultured in medium without bile salt, and OD 620nmThe time required for the value to increase by 0.3 units is 4 h, and the time required in the medium containing bile salts is 4.5 h, with a lag time of 0.5 h. According to the experimental conclusions on bile salt tolerance by Gilliland et al. (the lag time of resistant strains ≤ 0.25 h, the lag time of tolerant strains 0.25 h < lag time ≤ 0.67 h, the lag time of weakly tolerant strains 0.67 h < lag time < 1.00 h, and the lag time of sensitive strains ≥ 1.00 h), Lactobacillus paracasei PC804 with a lag time of 0.4 h belongs to bile salt tolerant strains.

[0042] For live cells, bile salts can damage cell membranes, and bile salts are the most influential factor on the viability of probiotics in the small intestine. Therefore, bile salt tolerance is one of the important indicators for evaluating probiotics. Thus, this Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 isolated from pickled vegetables has good probiotic characteristics.

[0043] Example 3 Application of Lactobacillus paracasei PC804 in brown milk beverage

[0044] Existing brown milk beverages are fermented milk beverages made from skim milk powder and reducing sugar as raw materials. After hydration, they are subjected to high-temperature heat treatment, cooling, long-term fermentation, and then formulated and homogenized. They have a rich and fragrant taste, are nutritious, and have the function of regulating the gastrointestinal tract. The special color of brown milk beverages is based on the mechanism of the Maillard reaction, where proteins and amino acids in milk react with reducing sugar under high-temperature conditions to give the product a special brown color. Brown milk beverages have a unique taste and are rich in nutrition. It has been found that brown lactic acid bacteria beverages have good antioxidant and anti-aging effects.

[0045] Using the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC804 of the present invention to make brown milk beverage:

[0046] After preheating distilled water to 60 °C, add 10% skim milk and 2% granulated sugar in proportion, melt the materials in a 60 °C constant temperature water bath for 15 min, then hydrate for 30 min, mix evenly and homogenize, and then carry out browning at 95 °C for 3 h. When cooled to 37 °C, inoculate Lactobacillus paracasei PC804 (1×10 7 CFU / g) and culture at 37 °C for fermentation. The fermentation end point is when the pH is below 4.5 and the titratable acidity is 200. Fermentation takes about 108 hours to obtain the base material. After fermentation, sensory evaluation is carried out, and the results are shown in Table 1.

[0047] Table 1 Sensory evaluation results of brown milk beverage

[0048]

[0049] The sensory evaluation score is one of the important factors for whether a probiotic can be used as a commercial starter culture. Although many probiotics can be used as strains for preparing fermented beverages, there are often defects such as low taste or texture scores or poor odor, which result in a decline in the flavor of the resulting product and make it difficult to be accepted by consumers, so they cannot be applied as commercial starter cultures.

[0050] Further prepare a brown milk beverage: After preheating distilled water to 60 °C, add apple juice and granulated sugar in proportion, adjust the pH to 3.7 with citric acid, mix the materials for 15 min, sterilize in a water bath at 95 °C for 5 min, cool to 37 °C, add the base material in proportion, homogenize after mixing evenly, and store in a 4 °C refrigerator after packaging. After ripening, samples were taken at 4 °C and the changes in acidity and viable probiotic counts during the shelf life were measured every 7 days. The results are shown in Table 2.

[0051] Table 2 Changes in acidity and viable probiotic counts of brown milk beverage during storage at 4 °C

[0052]

[0053] From the measurement results in Table 2, it can be seen that there is no post-acidification phenomenon in the brown milk beverage added with Lactobacillus paracasei PC804; the viable count of PC804 reaches more than 10 7 CFU / g and remains at a relatively high viable count during storage, proving that the Lactobacillus paracasei PC804 obtained in the present invention has good probiotic characteristics.

Claims

1. A Lactobacillus paracasei Lacticaseibacillus paracasei ) PC804, which was deposited with the China General Microbiological Culture Collection Center on November 15, 2021, and the deposit number is CGMCC No. 23917.

2. Use of the Lacticaseibacillus paracasei PC804 according to claim 1 in the preparation of a brown milk beverage.

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