CN115340965A - Lactobacillus paracasei PC724 and application thereof - Google Patents

Abstract

The invention discloses a strain of Lactobacillus paracasei with probiotic properties isolated from sour porridge, named as Lactobacillus paracasei PC724, which has been preserved in the General Microorganism Culture Collection Center of China Microbiology Culture Collection Management Committee, and the preservation number is CGMCC No. 23514. The Lactobacillus paracasei PC724 in the present invention has higher survival rate in gastrointestinal fluid, good bile salt tolerance, and stronger survival ability in human body. Lactobacillus paracasei PC724 has the probiotic characteristics of enhancing immune resistance, increasing the diversity of intestinal flora, and increasing the stability of intestinal flora. In addition, PC724 can be well applied to fermented dairy products as a probiotic.

Description

A strain of Lactobacillus paracasei PC724 and its application

【Technical field】

The invention relates to the technical field of microbes, in particular to a strain of Lacticaseibacillus paracasei PC724 isolated from sour porridge with probiotic properties, and also relates to its application in probiotic fermented milk products.

【Background technique】

Sour porridge is a traditional grain naturally fermented food in the Hetao area of my country (inner Mongolia, Shanxi, and parts of Shaanxi), which contains a large number of microorganisms and is rich in nutrition. Lactic acid bacteria (LAB) is a general term for a class of Gram-positive bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. Widely present in people's lives and abundantly present in fermented foods, it is recognized as a safe food-grade microorganism (generally recognized as safe, 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 in the body, maintain micro-ecological balance, control endotoxins, and inhibit the growth and reproduction of spoilage bacteria in the intestinal tract, but also the organic acids, special enzymes, exopolysaccharides, bacteriocins and other nutrients produced by lactic acid bacteria through fermentation Substances can stimulate tissue development, thereby promoting the nutritional status and immune response of the human body. Exploring the species diversity of LAB in sour porridge is beneficial to the development of lactic acid bacteria resources.

Lacticaseibacillus paracasei (Lacticaseibacillus paracasei) is a Gram-positive bacterium, facultatively anaerobic, and is a group of Lactobacillus casei in the genus Lactobacillus. It plays an important role in the fields of food and medical care, especially as a commercial starter. Fermentation characteristics and research value. Widely present in fermented foods and human mouth and intestines, it can metabolize various carbon sources and has good acid and bile salt resistance. Related studies have found that Lactobacillus paracasei can promote the balance of microbial flora and enzyme balance in the human body, and at the same time stimulate specific and non-specific immune mechanisms, which can prevent certain diseases, promote development, enhance physical fitness, delay aging and Life-extending prebiotic effects. It is a probiotic lactic acid bacteria that has been studied a lot at home and abroad in recent years.

【Content of invention】

The purpose of the present invention is to provide a strain of Lacticaseibacillus paracasei PC724 with certain probiotic properties isolated from sour porridge, which has been preserved in the General Microorganism Culture Collection Center of China Microbiology Culture Collection Management Committee, and the preservation number is CGMCC No. .23514.

The invention provides the application of Lacticaseibacillus paracasei PC724 in food additives.

Among them, the food additive is a dairy starter.

On the other hand, the present invention also provides the application of Lacticaseibacillus paracasei (Lacticaseibacillus paracasei) PC724 fermented milk, wherein, the fermented milk is fermented yogurt, drinking yogurt, fermented milk drink or fermented soybean milk.

The present invention provides a strain of Lacticaseibacillus paracasei PC724 isolated from the sour porridge of Wengengsum in Urad Zhongqi, Bayannaoer City, Inner Mongolia, which is confirmed by gastrointestinal fluid tolerance and bile salt tolerance tests. The strain has good probiotic properties, can enter the human intestinal tract in a living state, and exert health effects, which shows the potential of the strain as a probiotic development. It is a probiotic strain that can be widely used in general food and health care products.

The Lacticaseibacillus paracasei PC724 of the present invention can resist the adverse influence of the digestive tract environment and adhere and colonize well in the intestinal tract. Its live bacteria have the effect of enhancing immune resistance, increasing the diversity of intestinal flora, and increasing 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, thereby playing a role To prevent harmful bacteria from staying, and to produce lactic acid to lower the pH value of the intestines and inhibit the reproduction of harmful bacteria. In addition, the live bacteria used produce bacteriocins and peroxides, thereby inhibiting the reproduction of pathogenic bacteria and assisting the activity of intestinal villi responsible for absorbing nutrients.

The Lacticaseibacillus paracasei PC724 of the present invention has been confirmed to have excellent taste, mouthfeel score and odor score through sensory evaluation, and after being prepared into a brown milk drink, the obtained product is post-ripened at 4°C and reserved samples are measured at intervals of 7 days During the shelf life, the acidity and the number of viable bacteria of probiotics changed little, and the number of viable bacteria remained high during storage, which proves that the PC724 of the present invention has good probiotic properties and can be applied to commercial fermented dairy products.

The present invention relates to the following microorganisms:

Lacticaseibacillus paracasei PC724 was deposited in the General Microorganism Culture Collection Center of China Committee for the Collection of Microorganism Cultures on September 29, 2021, with the preservation number CGMCC No.23514.

【Description of drawings】

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

Figure 2: Colony morphology of Lactobacillus paracasei PC724 on the culture medium;

Figure 3: Changes in the survival rate of Lactobacillus paracasei PC724 in simulated artificial digestive tract.

【Detailed ways】

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

In the present invention, unless otherwise specified, "%" used to explain concentration is percentage by weight.

The present invention relates to the following culture medium:

MRS liquid medium: peptone (animal source) 10g, beef extract powder 8g, yeast extract powder 4g, glucose 20g, Tween-80 1ml, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triamine citrate 2g, manganese sulfate 0.05g , Magnesium sulfate 0.2g, distilled water 1L, adjust the pH to 6.5±0.2, sterilize at 121°C for 15min.

MRS solid medium: peptone (animal source) 10g, beef extract powder 8g, yeast extract powder 4g, glucose 20g, Tween-80 1ml, dipotassium hydrogen phosphate 2g, sodium acetate 5g, triamine citrate 2g, manganese sulfate 0.05g , Magnesium sulfate 0.2g, distilled water 1L, adjust the pH to 6.5±0.2, agar 15g, sterilize at 121°C for 15min.

The acquisition of embodiment 1 Lactobacillus paracasei PC724

The sour porridge sample collected from the sour porridge of Wengengsum in Urad Zhongqi, Bayannaoer City, Inner Mongolia was doubled to obtain dilution gradients of 10 ^-5 , 10 ^-6 , and 10 ^-7 . 200 μL of the porridge sample was evenly spread on the MRS solid medium plate, and cultured under anaerobic conditions at 37°C for 48-72h. Single clones with different shapes, sizes, and colors were picked and inoculated in MRS liquid medium, and cultured in a constant temperature incubator at 37°C for 24 hours. After the strains grow well, carry out Gram staining and microscopic examination, and save the isolates.

The Lacticaseibacillus paracasei (Lacticaseibacillus paracasei) PC724 obtained by the screening of the present invention has the following morphological characteristics: the thalline is short rod-shaped under the microscope, both ends are even and square, and the cells are arranged in single, in pairs or in chains (see Figure 1), no spores, no flagella, no movement.

It was confirmed by sequencing that the obtained strain was Lactobacillus paracasei.

The bacterium colony that this Lacticaseibacillus paracasei (Lacticaseibacillus paracasei) PC724 forms on the MRS medium is milky white, shiny, with neat edges, mostly convex lens-like protrusions, with a diameter between 0.5-1mm (see Figure 2).

Example 2 Tolerance Determination

1. Gastrointestinal fluid tolerance of the strain

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

After sterilizing PBS, adjust the pH value to 8.0 with 0.1mol/L NaOH, add 0.1% trypsin and 1.8% ox bile salt, filter and sterilize with a 0.22μm microporous membrane to make artificial simulated pancreatic juice.

Gastrointestinal fluid tolerance: Activate and culture the isolated and purified strain for two generations, wash the bacteria twice by centrifugation, collect the bacteria, take 0.5ml of re-screened bacteria suspension and add it to 4.5ml of pH 2.5 simulated artificial gastric juice, at 37°C After digesting for 3h, count and determine the number of viable bacteria by MRS solid medium pouring method at 0h and 3h respectively.

Then, take 0.5ml of artificial bacteria-containing gastric juice that has been digested for 3 hours and add it to 4.5ml of artificial pancreatic juice, continue to cultivate in a water bath at 37°C, and count and determine the number of viable bacteria by MRS agar medium pouring method at 4h and 8h respectively. 4 parallel samples were made.

Strain Survival Rate Calculation Formula

N ₁ - the number of viable bacteria after strain treatment;

N ₀ ――the initial number of viable bacteria of the strain.

The results show that Lactobacillus paracasei PC724 was digested in the artificial gastric juice of pH2.5 and the survival rate was 57.44% after 3 hours, and then continued to be digested in the artificial pancreatic juice of pH8.0 for 8 hours, and the final survival rate was 79.10% (as shown in Figure 3 shown). The results showed that Lactobacillus paracasei PC724 has good tolerance to gastrointestinal digestive juice, and can enter the human intestinal tract in the state of living bacteria to exert health effects. This property is the basis for probiotics. Lacticaseibacillus paracasei (Lacticaseibacillus paracasei) PC724 of the present invention is a probiotic with probiotic activity, and it can be reasonably expected that when it is used in a dry cell form or a fermented product form for humans or animals, it can have a positive effect on the intestinal tract of the host. The flora has a beneficial effect, and has the probiotic characteristics of enhancing immune resistance, increasing the diversity of intestinal flora, and increasing the stability of intestinal flora.

2. Strain tolerance to bile salts

After three generations of Lactobacillus paracasei PC724 activation culture, inoculated with 1% inoculum size into the MRS liquid containing 0.3% ox bile salt (add 0.2g/100mL sodium thioglycolate and 0.3 g/100mL ox bile salt to the medium) In the culture medium, the MRS medium without bovine bile salt 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 620nm, and three parallel measurements were made for each group, until the OD value increased by 0.3 units or more, the culture was stopped. Calculate the time required for the strain to grow in the MRS medium containing and without bile salts, and the OD value increases by 0.3 units, and the difference between the two is called the lag time (LT).

Experimental result is known, paracasei lactobacillus (Lacticaseibacillus paracasei) PC724 is cultivated in the culture medium that does not contain bile salt, and the time needed for OD _620nm value to increase 0.3 unit is 4.0h, needs in the culture medium that contains bile salt. The time is 4.4h, and the delay time is 0.4h. According to the conclusions of the bile salt tolerance experiment of Gilliland et al. (delay time of resistant strains ≤0.25h, tolerant strains 0.25h<delay time ≤0.67h, weakly tolerant strains 0.67h<delay time<1.00h, sensitive strains Bacteria paracasei PC724 with a delay time of 0.4 h belongs to the bile salt tolerance strain.

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

Application of Example 3 Lactobacillus paracasei PC724 in Brown Milk Beverage

The existing brown milk beverage is made of milk as the main raw material. After several hours of low-temperature heating and browning, lactic acid bacteria are added for fermentation, and auxiliary materials such as stabilizers, sweeteners, sour agents and essences are added after processing. Brown functional drink with burnt aroma and fermented aroma. Brown milk beverage is favored by consumers because of its various physiological effects such as tumor inhibition, anti-oxidation, and anti-aging, as well as good flavor. At present, for brown fermented milk beverages on the market, the starter basically selects Lactobacillus casei and Lactobacillus paracasei as the basic fermentation strains, and the fermentation time is basically more than 72h.

Preparation of brown milk beverage with Lactobacillus paracasei PC724:

After the distilled water is preheated to 60°C, add 10% skim milk and 2% white sugar in proportion to obtain a skim emulsion. Put the ingredients in a constant temperature water bath at 60°C for 15 minutes, then hydrate for 30 minutes, mix well and homogenize, and then heat at 95°C Browning under high temperature conditions for 3 hours, when cooled to 37°C, insert probiotic Lactobacillus paracasei PC724 (1×10 ⁷ CFU/g) and culture at 37°C for fermentation. The end point of fermentation is below pH 4.5, and the titrated acidity is 200 , ferment for about 60 hours to obtain the base material, and the required fermentation time is significantly shorter than that of the prior art. After the fermentation, the sensory evaluation of the base material was carried out, and the results are shown in Table 1.

Table 1 Sensory evaluation results of brown milk beverage

The sensory evaluation score is one of the important factors to determine whether a probiotic strain can be used as a commercial starter. Although many probiotics can be used as strains to prepare fermented beverages, they often have the disadvantage of low taste or mouthfeel score or poor smell, which makes the resulting product less flavorful and difficult to be accepted by consumers, so it cannot be applied to commercial fermentation agent.

Further preparation of brown milk beverage: preheat distilled water to 60°C, add apple juice and white sugar as flavoring agent, adjust pH to 3.7 with citric acid, prepare ingredients for 15 minutes, sterilize in 95°C water bath for 5 minutes, cool to 37°C, Add the base material, mix well and homogenize, store in a refrigerator at 4°C after subpackaging. After post-ripening, the retained samples at 4°C were measured at intervals of 7 days for the change of acidity and the number of viable probiotic bacteria during the shelf life. The results are shown in Table 2.

Table 2 Changes of acidity and viable count of probiotics in brown milk beverage during storage at 4°C

From the results, it can be seen that there is no post-acidification phenomenon after adding L. paracasei PC724 in the brown milk beverage; after the fermentation, the number of viable bacteria of PC724 reaches more than 10 ⁸ CFU/g and maintains a higher number of viable bacteria during storage, which proves that the present invention PC724 has good probiotic properties.

Claims (4)

1. The lactobacillus paracasei (Lactcaseibacillus paracasei) PC724 is preserved in China general microbiological culture collection center (CGMCC) at 29.9.2021 with the preservation number of CGMCC No.23514.

2. Use of paracasei (Lactcaseibacillus paracasei) PC724 according to claim 1 in food additives.

3. Use according to claim 2, characterized in that the food additive is a dairy starter.

4. Use of the Lactobacillus paracasei (Lactcasei paracasei) PC724 fermented milk product according to claim 1.

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