A strain of Lactobacillus paracasei PC724 and its application

Abstract

The invention discloses a strain of Lactobacillus paracasei with probiotic properties separated from sour porridge, named as Lactobacillus paracasei PC724, ​​which has been preserved in the General Microbial Culture Collection Center of China Microorganism Culture Collection Administration Committee, and the preservation number is CGMCC No.23514. Lactobacillus paracasei PC724 in the present invention has a higher survival rate in gastrointestinal fluid, good bile salt tolerance, and has a stronger survival ability in the human body. Lactobacillus paracasei PC724 has the probiotic characteristics of enhancing immune resistance, improving intestinal flora diversity, and increasing intestinal flora stability. In addition, PC724 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 in particular to a strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC724, ​​and also relates to its application in probiotic fermented dairy products. Background Art

[0002] Lactic acid bacteria (LAB) is a general term for Gram-positive bacteria that can produce a large amount of lactic acid using fermentable carbohydrates. They are widely present in people's lives and exist in large quantities in fermented foods. They are generally recognized as safe food-grade microorganisms (GRAS). The active use of lactic acid bacteria is closely related to human health. Lactic acid bacteria can not only regulate the normal intestinal flora, maintain the balance of microecology, control endotoxins, and inhibit the growth and reproduction of putrefactive bacteria in the intestine, but also stimulate tissue development through the organic acids, special enzyme systems, exopolysaccharides, bacteriocins and other nutrients produced by lactic acid bacteria through fermentation, thereby promoting the nutritional status and immune response of the human body. Exploring the species diversity of LAB in sour porridge is conducive to the development of lactic acid bacteria resources.

[0003] Lactobacillus paracasei ( Lacticaseibacillus paracasei ) is a Gram-positive bacterium, facultative anaerobic, and belongs to the Lactobacillus casei group in the genus Lactobacillus. It plays an important role in the fields of food and health care, especially as a commercial fermentation agent with good fermentation characteristics and research value. It is widely present in fermented foods and the human mouth and intestines, can metabolize a variety of carbon sources, and has good acid and bile resistance. Related studies have found that Lactobacillus paracasei can promote the balance of microbial flora and enzymes in the human body, and can also stimulate specific and non-specific immune mechanisms. It has the probiotic effects of preventing certain diseases, promoting development, strengthening physical fitness, delaying aging and prolonging life. It is a probiotic lactic acid bacterium that has been studied a lot at home and abroad in recent years. Summary of the invention

[0004] The present invention aims to provide a strain of Lactobacillus paracasei (Lactobacillus paracasei) isolated from sour porridge with certain probiotic properties. Lacticaseibacillus paracasei ) PC724, ​​has been deposited in the China General Microbiological Culture Collection Center with the deposit number CGMCC No. 23514.

[0005] The present invention provides Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of PC724 in food additives.

[0006] Among them, the food additive is dairy fermentation agent.

[0007] On the other hand, the present invention also provides Lactobacillus paracasei ( Lacticaseibacillus paracasei ) Application of PC724 in fermented dairy products, wherein the fermented dairy products are fermented yogurt, drinkable yogurt, fermented dairy beverage or fermented soy milk.

[0008] The present invention provides a strain of Lactobacillus paracasei isolated from sour porridge of Wengeng Sumu in Urad Middle Banner, Bayannur City, Inner Mongolia Autonomous Region ( Lacticaseibacillus paracasei ) PC724, ​​through gastrointestinal fluid tolerance and bile salt tolerance tests, confirmed that the strain has good probiotic properties, can enter the human intestine in a live state, and exert health effects, showing the potential of the strain as a probiotic. It is a probiotic that can be widely used in ordinary food and health products.

[0009] Lactobacillus paracasei of the present invention ( Lacticaseibacillus paracasei ) PC724 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, improving the diversity of intestinal flora, and increasing the stability of intestinal flora. When used in humans and animals, it can be densely packed and stay on the wall of the digestive tract in the intestine, thereby preventing harmful bacteria from staying, and producing lactic acid to lower the pH value in the intestine and inhibit the reproduction of harmful bacteria. In addition, the live bacteria used produce bacteriocins and peroxides, which can inhibit the reproduction of pathogens and help the activity of intestinal villi responsible for absorbing nutrients.

[0010] Lactobacillus paracasei of the present invention ( Lacticaseibacillus paracasei ) PC724 was confirmed to have excellent taste, mouthfeel and odor scores through sensory evaluation, and after being prepared into a brown milk beverage, the acidity and the number of live probiotic bacteria in the product after post-ripening at 4°C were measured every 7 days during the shelf life, and a high number of live bacteria was maintained during storage, demonstrating that the PC724 of the present invention has good probiotic properties and can be applied to commercial fermented dairy products.

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

[0012] Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC724, ​​deposited in the China General Microbiological Culture Collection Center on September 29, 2021, with the deposit number CGMCC No. 23514. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 :Electron micrograph of Lactobacillus paracasei PC724 (3000 times);

[0014] Figure 2 : Colony morphology of Lactobacillus paracasei PC724 on culture medium;

[0015] Figure 3 : Changes in the survival rate of Lactobacillus paracasei PC724 in the simulated artificial digestive tract. DETAILED DESCRIPTION

[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 to these examples.

[0017] In the present invention, unless otherwise specified, "%" used to explain concentrations refers to weight percentage.

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

[0019] MRS liquid culture medium: peptone (animal source) 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, triammine 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 culture medium: peptone (animal source) 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, triammine 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 Acquisition of Lactobacillus paracasei PC724

[0022] The sour porridge sample collected from Wengeng Sumu, Urad Middle Banner, Bayannur City, Inner Mongolia Autonomous Region was diluted in multiples to obtain 10 -5 , 10 -6 , 10 -7 Dilution gradient, take 200 μL of each gradient sour porridge sample and evenly spread it on the MRS solid culture medium plate, and culture it under anaerobic conditions at 37℃ for 48-72 hours. Pick out monoclones with different morphology, size, and color and inoculate them in MRS liquid culture medium, and culture them in a constant temperature incubator at 37℃ for 24 hours. After the strain grows well, perform Gram staining and microscopic examination, and preserve the isolated strain.

[0023] The Lactobacillus paracasei obtained by screening of the present invention ( Lacticaseibacillus paracasei ) PC724 has the following morphological characteristics: the bacteria appear as short rods under a microscope, with both ends flush and square, and the cells are arranged in single, paired or chain forms (see Figure 1 ), no spores, no flagella, and no movement.

[0024] The obtained strain was confirmed to be Lactobacillus paracasei by sequencing.

[0025] The Lactobacillus paracasei ( Lacticaseibacillus paracasei The colonies formed by PC724 on MRS medium are milky white, shiny, with neat edges, mostly convex lens-like protrusions, and the diameter is between 0.5-1 mm (see Figure 2 ).

[0026] Example 2 Tolerance Determination

[0027] 1. Strain tolerance to gastrointestinal fluid

[0028] Preparation method of simulated gastrointestinal fluid: After sterilizing PBS, adjust the pH value to 2.5 with 1 mol / L HCl, add 3.0 mg / ml pepsin, filter and sterilize with a 0.22 μm microporous filter membrane to prepare artificial simulated gastric fluid;

[0029] After PBS was sterilized, the pH value was adjusted to 8.0 with 0.1 mol / L NaOH, 0.1% trypsin and 1.8% ox bile salt were added, and the solution was sterilized by filtration with a 0.22 μm microporous filter membrane to prepare artificial simulated pancreatic juice.

[0030] Tolerance to gastrointestinal fluid: The isolated and purified strain was activated and cultured for two generations, centrifuged and washed twice, the bacteria were collected, 0.5 ml of the re-screened bacterial suspension was added to 4.5 ml of pH 2.5 simulated artificial gastric fluid, and digested at 37°C for 3 h. The number of live bacteria was determined by the MRS solid culture medium pouring method at 0 h and 3 h, respectively.

[0031] Then, 0.5 ml of artificial gastric juice containing bacteria that had been digested for 3 h was added to 4.5 ml of artificial pancreatic juice, and cultured in a water bath at 37°C. The number of live bacteria was determined by the MRS agar medium pouring method at 4 h and 8 h, respectively. Four parallels were made for each sample.

[0032] Strain survival rate calculation formula

[0033]

[0034] N 1 --The number of viable bacteria after strain treatment;

[0035] N 0 ——Initial viable count of the strain.

[0036] The results showed that the survival rate of Lactobacillus paracasei PC724 was 57.44% after digestion in artificial gastric juice at pH 2.5 for 3 hours, and then continued to digest in artificial pancreatic juice at pH 8.0 for 8 hours, and the final survival rate was 79.10% (e.g. Figure 3 The results show that Lactobacillus paracasei PC724 has good gastrointestinal digestive juice tolerance and can enter the human intestine in a live bacterial state to exert health effects. This property is the basis for probiotics. The Lactobacillus paracasei of the present invention ( Lacticaseibacillus paracasei ) PC724 is a probiotic with probiotic activity. It can be reasonably expected that when it is used in the form of dried cells or fermentation products for humans or animals, it can have a beneficial effect on the host's intestinal flora, and has the probiotic characteristics of enhancing immune resistance, improving the diversity of intestinal flora, and increasing the stability of intestinal flora.

[0037] 2. Bile salt tolerance of strains

[0038] After three generations of activation culture of Lactobacillus paracasei PC724, ​​the culture medium was inoculated with 1% inoculation into MRS liquid medium containing 0.3% ox bile salt (0.2 g / 100mL sodium thioglycolate and 0.3 g / 100mL ox bile salt were added to the culture medium), and the MRS medium without ox bile salt was used as a blank control, and the culture was placed at 37°C for anaerobically culture. During the culture period, samples were taken every hour to measure the OD value at 620nm, and three parallels were measured in each group until the OD value increased by 0.3 units or more, and the culture was stopped. The time required for the strain to grow in MRS medium containing bile salts and without bile salts, and the difference between the two was called the delay time (LT).

[0039] The experimental results show that Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC724 were cultured in medium without bile salts, OD 620nm The time required for the value to increase by 0.3 units is 4.0 h, the time required in the medium containing bile salts is 4.4 h, and the delay time is 0.4 h. According to the bile salt tolerance experiment conclusion of Gilliland et al. (resistant strain delay time ≤ 0.25h, tolerant strain 0.25h<delay time ≤ 0.67h, weakly tolerant strain 0.67h<delay time<1.00h, sensitive strain delay strain ≥1.00h), the delay time of 0.4 h of Lactobacillus paracasei PC724 belongs to the bile salt tolerant strain.

[0040] For living cells, bile salts can destroy cell membranes, and the factor that has the greatest impact on the survival of probiotics in the small intestine is bile salts. Therefore, tolerance to bile salts is one of the important indicators for evaluating probiotics. Lacticaseibacillus paracasei ) PC724 has good probiotic properties.

[0041] Example 3 Application of Lactobacillus paracasei PC724 in brown milk beverages

[0042] The existing brown milk beverage is a brown functional beverage with caramel and fermented aromas, which is made from cow's milk as the main raw material, first heated at low temperature for several hours to brown, then added with lactic acid bacteria for fermentation, and then added with stabilizers, sweeteners, acidulants and flavors. Brown milk beverages are favored by consumers because of their multiple physiological effects such as tumor inhibition, anti-oxidation, anti-aging and good flavor. At present, the brown fermented milk beverages on the market basically use Lactobacillus casei and Lactobacillus paracasei as the basic fermentation strains, and the fermentation time is basically more than 72 hours.

[0043] Preparation of brown milk beverage with Lactobacillus paracasei PC724:

[0044] After preheating distilled water to 60°C, 10% skim milk and 2% white sugar were added in proportion to obtain skim emulsion, which was then stirred in a constant temperature water bath at 60°C for 15 min, hydrated for 30 min, mixed evenly and homogenized, and then browned at 95°C for 3 h. When cooled to 37°C, the probiotic Lactobacillus paracasei PC724 (1×10 7 CFU / g) was cultured at 37°C for fermentation. The fermentation end point was below pH 4.5, the acidity was titrated to 200, and the base material was obtained by fermentation for about 60 hours. The required fermentation time was significantly shorter than that of the prior art. After the fermentation, the base material was subjected to sensory evaluation. The results are shown in Table 1.

[0045] Table 1 Sensory evaluation results of brown milk beverage

[0046]

[0047] The sensory evaluation score is one of the important factors in determining whether a probiotic strain can be used as a commercial fermentation agent. Although many probiotics can be used as strains to prepare fermented beverages, they often have defects such as low taste or mouthfeel scores or poor odor, which reduces the flavor of the resulting product and makes it difficult for consumers to accept it. Therefore, they cannot be used as commercial fermentation agents.

[0048] The brown milk beverage was further prepared: distilled water was preheated to 60°C, apple juice and white sugar were added as flavoring agents, citric acid was adjusted to pH 3.7, the material was dissolved for 15 min, sterilized in a water bath at 95°C for 5 min, cooled to 37°C, the base material was added in proportion, mixed evenly and homogenized, and stored in a refrigerator at 4°C after packaging. After ripening, samples were kept at 4°C and the changes in acidity and the number of viable probiotics during the shelf life were determined every 7 days. The results are shown in Table 2.

[0049] Table 2 Changes in acidity and viable probiotic counts of brown milk beverage during storage at 4℃

[0050]

[0051] The results show that there is no post-acidification phenomenon when Lactobacillus paracasei PC724 is added to brown milk beverage; the number of live bacteria of PC724 reaches 10 8 CFU / g and maintained a high number of viable bacteria during storage, proving that the PC724 of the present invention has good probiotic properties.

Claims

1. A strain of Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC724, ​​the Lactobacillus paracasei ( Lacticaseibacillus paracasei ) PC724 was deposited in the China General Microbiological Culture Collection Center on September 29, 2021, with the deposit number CGMCC No. 23514.

2. The Lactobacillus paracasei of claim 1 ( Lacticaseibacillus paracasei ) Application of PC724 in the preparation of brown milk beverages.

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