CN115261264A - Lactobacillus paracasei PC804 and application thereof - Google Patents

Lactobacillus paracasei PC804 and application thereof Download PDF

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CN115261264A
CN115261264A CN202210795191.XA CN202210795191A CN115261264A CN 115261264 A CN115261264 A CN 115261264A CN 202210795191 A CN202210795191 A CN 202210795191A CN 115261264 A CN115261264 A CN 115261264A
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paracasei
lactobacillus
lactobacillus paracasei
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intestinal flora
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张文羿
王记成
姚国强
刘文俊
何秋雯
白梅
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Inner Mongolia Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/50—Fermented pulses or legumes; Fermentation of pulses or legumes based on the addition of microorganisms
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L11/00—Pulses, i.e. fruits of leguminous plants, for production of food; Products from legumes; Preparation or treatment thereof
    • A23L11/60—Drinks from legumes, e.g. lupine drinks
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/165—Paracasei
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Abstract

The invention discloses a paracasei separated from pickle and having probiotic characteristics, which is named as paracasei PC804 and is preserved in China general microbiological culture collection center of China Committee for culture Collection of microorganisms with the preservation number of CGMCC No.23917. The Lactobacillus paracasei PC804 has high survival rate in gastrointestinal fluids, good bile salt tolerance and strong survival ability in human bodies. The Lactobacillus paracasei PC804 has the probiotic characteristics of enhancing immune resistance, improving the diversity of intestinal flora and increasing the stability of the intestinal flora. In addition, the PC804 serving as the probiotics can be well applied to fermented dairy products.

Description

Lactobacillus paracasei PC804 and application thereof
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of microorganisms, in particular to a strain of paracaseobacter paracasei (Lactcaseibacillus paracasei) PC804 with probiotic characteristics, which is separated from pickle, and also relates to application of the strain in probiotic fermented dairy products.
[ background ] A method for producing a semiconductor device
The pickle is a traditional fermented vegetable product, and the lactobacillus in the pickle is rich in diversity, comprises lactobacillus plantarum (LacctiIBacillus plantium), lactobacillus mucilaginosus (Lactobacillus fermentum), lactobacillus casei (Lactobacillus casei) and the like, and has important value in researching the lactobacillus of the pickle.
Lactic Acid Bacteria (LAB) are a generic term for a group of spore-free, gram-positive bacteria that ferment sugars, the main product of which is Lactic acid, and are widely present in human life and abundantly present in fermented foods. The lactobacillus not only has important effect on the sensory characteristics of the food, but also has the probiotic functions of regulating intestinal flora, enhancing immunity, promoting nutrient absorption and the like. A great deal of research shows that lactic acid bacteria in fermented food can promote the absorption of organism to nutrient substances, reduce cholesterol in vivo, and improve organism immunity by increasing nonspecific (cell function, natural killer cell activity) and specific (cell factor production, antibody production, lymphocyte proliferation, delayed type hypersensitivity) host immune response. Lactic acid bacteria have a long history of safe use and have long been Generally Recognized As Safe (GRAS) species in fermentation products.
Lactobacillus paracasei (Lactcaseibacterium paracasei) is a facultative anaerobic, gram-positive, heterotypic fermenting lactic acid bacterium, widely found in human mouth, intestines, fermented dairy products (especially cheese) and plant material fermentates (kimchi, wine and feed). It has the characteristics of lactobacillus casei, can decompose protein to generate small molecular peptide by the action of extracellular enzyme, and plays an important role in reducing blood pressure, resisting tumor, resisting oxidation, reducing cholesterol and the like. Meanwhile, the paracasei cheese lactobacillus has no toxic or side effect, no drug resistance and easy degradation by human bodies, and has the probiotic functions of regulating intestinal balance, enhancing non-specific immunity, promoting absorption and the like.
[ summary of the invention ]
The invention aims to provide a lactobacillus paracasei (Lactcaseibacillus paracasei) PC804 with certain probiotic characteristics, which is separated from acid porridge and is preserved in the China general microbiological culture Collection center with the preservation number of CGMCC No.23917.
The invention provides an application of Lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 in food additives.
Wherein the food additive is a dairy starter.
On the other hand, the invention also provides application of lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 fermented milk product, wherein the fermented milk product is fermented yogurt, drinking yogurt, fermented milk beverage or fermented soybean milk.
The Lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 can resist the adverse effect of the digestive tract environment and well adhere and colonize in the intestinal tract. The viable bacteria have the effects of enhancing immunity, improving intestinal flora diversity and increasing intestinal flora stability, and can be densely stayed on the wall of the intestinal digestive tract when being used for human and animals, thereby playing the role of preventing harmful bacteria from staying, generating lactic acid to reduce the pH value in the intestine and inhibiting the propagation of harmful bacteria. In addition, the used live bacteria produce bacteriocin and peroxide, thereby inhibiting the propagation of pathogenic bacteria and helping the activity of intestinal villi which is responsible for absorbing nutrient components.
The Lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 is confirmed to have excellent taste and mouthfeel scores and odor scores through sensory evaluation, and after the brown milk beverage is prepared, the acidity and the number of live probiotics in a shelf life are measured to be less in change every 7 days after the obtained product is subjected to after-ripening and is kept at 4 ℃, and the higher number of the viable bacteria is kept during storage, so that the PC804 has good probiotic characteristics and can be applied to commercial fermented milk products.
The present invention relates to the following microorganisms:
the Lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 is preserved in China general microbiological culture Collection center (CGMCC) at 11 month and 15 month 2021, and the preservation number is CGMCC No.23917.
[ description of the drawings ]
FIG. 1: cheese bacillus paracasei PC804 electron microscope picture (3000 times)
FIG. 2: colony morphology of Lactobacillus paracasei PC804 on medium
FIG. 3: survival rate change condition of paracasei PC804 in simulated artificial digestive tract
[ detailed description ] embodiments
The present invention will be further illustrated with reference to the following examples, which are illustrative only and the present invention is not limited to these examples.
In the present invention, "%" used for explaining the concentration is weight percent unless otherwise specified.
The present invention relates to the following media:
MRS liquid culture medium: 10g of peptone (animal source), 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 1ml of Tween-80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triamine citric acid, 0.05g of manganese sulfate, 0.2g of magnesium sulfate and 1L of distilled water, adjusting the pH to 6.5 +/-0.2, sterilizing at 121 ℃ for 15 min.
MRS solid medium: 10g of peptone (animal source), 8g of beef extract powder, 4g of yeast extract powder, 20g of glucose, 1ml of Tween-80, 2g of dipotassium hydrogen phosphate, 5g of sodium acetate, 2g of triamine citric acid, 0.05g of manganese sulfate, 0.2g of magnesium sulfate and 1L of distilled water, adjusting the pH to 6.5 +/-0.2, 15g of agar, sterilizing at 121 ℃ for 15 min.
Example 1 acquisition of Lactobacillus paracasei PC804
Diluting sauerkraut sample collected from Mali county of Sichuan province by multiple times to obtain 10-5、10-6、10-7Diluting the gradient, sucking 200 μ L of gradient sauerkraut sample, uniformly spreading on MRS solid culture medium plate, and anaerobically culturing at 37 deg.C for 48-72h. Selecting monoclonals with different shapes, sizes and colors, inoculating the monoclonals into an MRS liquid culture medium, and culturing the monoclonals in a constant-temperature incubator at 37 ℃ for 24 hours. After the strain grows well, gram staining and microscopic examination are carried out. Isolates were stored and strain genomic DNA was extracted for subsequent assay analysis.
Inoculating a frozen and preserved test strain into an MRS liquid culture medium, culturing for 24h at constant temperature of 37 ℃, subculturing for 2-3 generations, taking 2mL of thallus culture at the last logarithmic growth stage, placing the thallus culture in a sterile EP tube for centrifugation, collecting thallus after centrifugation for 3min (4 ℃) at 8000 Xg, discarding supernatant, and extracting genome DNA of the strain by adopting a CTAB freeze-thawing method special for lactobacillus.
The Lactceibacillus paracasei PC804 whole genome was determined by using PacBio SMRT RSII third generation sequencing platform, and the resulting strain, lactobacillus paracasei (Lactceibacillus paracasei), was confirmed.
The paracaseobacter paracasei (Lactcaseibacillus paracasei) PC804 obtained by screening has the following morphological characteristics: the thallus is in the shape of short rod under microscope, with two ends flush and square, the cells are arranged in single, paired or chain form (as shown in figure 1), and have no spore, no flagellum and no movement.
The colony formed by the Lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 on the MRS culture medium is milk white, glossy, regular in edge, and convex lens-shaped in most cases, and the diameter of the colony is between 0.5 and 1mm (as shown in figure 2).
Example 2 tolerance assay
1. Bacterial strain gastrointestinal fluid tolerance
The preparation method of the simulated gastrointestinal fluid comprises the following steps: sterilizing PBS, adjusting pH to 2.5 with 1mol/L HCl, adding pepsin 3.0mg/ml, filtering with 0.22 μm microporous membrane for sterilization, and making into artificial gastric juice;
the preparation method of the artificial simulated pancreatic juice comprises the following steps: sterilizing PBS, adjusting pH to 8.0 with 0.1mol/L NaOH, adding 0.1% trypsin and 1.8% ox bile salt, filtering with 0.22 μm microporous membrane for sterilization, and making into artificial simulated pancreatic juice.
Gastrointestinal fluid tolerance: and performing activated culture on the separated and purified strain for two generations, performing centrifugal bacteria washing twice, collecting bacteria, adding 0.5ml of re-screened bacteria suspension into 4.5ml of simulated artificial gastric juice with the pH value of 2.5, digesting for 3 hours at 37 ℃, and simultaneously counting and determining the number of the viable bacteria by using an MRS agar culture medium pouring method for 0 hour and 3 hours respectively.
Then, 0.5ml of digested 3h artificial bacteria-containing gastric juice is added into 4.5ml artificial intestinal juice, water bath culture is continued at 37 ℃, viable count is determined by pouring MRS agar culture medium for 4h and 8h respectively, and each sample is divided into 4 parallels.
Formula for calculating survival rate of strain
Figure RE-GDA0003839495050000041
N1Viable count after strain treatment;
N0the initial viable count of the strain.
The results showed that the L.paracasei (Lactcaseibacterium paracasei) PC804 was digested in the artificial gastric juice at pH2.5 for 3 hours (survival rate 57.94%), followed by continued digestion in the artificial digestive juice at pH8.0 for 8 hours with a final survival rate of 30.68% (as shown in FIG. 3).
The result shows that the Lactobacillus paracasei PC804 has good gastrointestinal digestive juice tolerance, can enter the intestinal tract of a human body in a living state and has a health effect. The above properties are the basis for probiotics. The paracaseobacter paracasei (Lactcaseibacterium paracasei) PC804 is a probiotic having probiotic activity, and is expected to have beneficial effects on the intestinal flora of a host when used in a dry cell form or a fermentation product form in humans or animals, and has probiotic characteristics of enhancing immune resistance, improving the diversity of the intestinal flora, and increasing the stability of the intestinal flora.
2. Bile salt tolerance of strains
After the strain obtained by separation and purification is activated and cultured for three generations, the strain is respectively inoculated into MRS culture medium containing 0.3% of bovine bile salt (0.2 g/100mL of sodium thioglycolate and 0.3g/100mL of bovine bile salt are added into the culture medium) and without the bovine bile salt by the inoculation amount of 1%, and the MRS culture medium is used as a blank control and is placed at 37 ℃ for anaerobic culture. During the culture period, samples were taken at intervals of one hour to determine OD values at 620nm, and triplicate measurements were taken in each group, and the culture was stopped until OD values increased by 0.3 unit or more. And the time required for the strain to grow in MRS medium with and without bile salts to increase the OD by 0.3 units was calculated, the difference being called the Lag Time (LT).
The results showed that Lactobacillus paracasei (Lactcaseibacillus paracasei) PC804 was cultured in a medium without bile salts, OD620nmThe time required for a value increase of 0.3 unit was 4h, the time required in medium containing bile salts was 4.5h and the delay time was 0.5h. According to the conclusion of the experiment of the bile salt tolerance of Gilliland et al (the delay time of a resistant strain is less than or equal to 0.25h, the delay time of a tolerant strain is more than or equal to 0.25h and less than or equal to 0.67h, the delay time of a weakly tolerant strain is more than 0.67h and less than 1.00h, and the delay time of a sensitive strain is more than or equal to 1.00 h), the Lactobacillus paracasei PC804 with the delay time of 0.4h belongs to the bile salt tolerant strain.
In the case of living cells, bile salts can damage cell membranes, and the factors that most affect the viability of probiotics in the small intestine are bile salts, so that the tolerance to bile salts is one of the important indicators for evaluating probiotics. Therefore, the strain of lactobacillus paracasei (lactuca paracasei) PC804 isolated from kimchi has good probiotic properties.
Example 3 use of Lactobacillus paracasei PC804 in Brown milk beverage
The existing brown milk beverage is a fermented milk beverage which is prepared by taking skim milk powder and reducing sugar as raw materials, hydrating, carrying out high-temperature heat treatment, cooling, fermenting for a long time, blending, homogenizing and the like, and has the advantages of strong taste and rich nutrition and the function of regulating gastrointestinal tracts. The special color of the brown milk beverage is based on the mechanism of Maillard reaction, and the protein and amino acid in the milk react with reducing sugar under high temperature to make the product have special brown color. The brown milk beverage has unique taste and rich nutrition, and researches show that the brown lactobacillus beverage has better effects of resisting oxidation, delaying senescence and the like.
The cheese bacillus paracasei (Lactcaseibacterium paracasei) PC804 of the invention is used for preparing brown milk beverage:
preheating distilled water to 60 deg.C, adding 10% skimmed milk and 2% white sugar at a certain ratio, melting in a water bath at 60 deg.C for 15min, hydrating for 30min, mixing, homogenizing, browning at 95 deg.C for 3 hr, cooling to 37 deg.C, inoculating Lactobacillus paracasei PC804 (1 × 10)7CFU/g) is cultured at 37 ℃, fermentation is carried out, the fermentation end point is below pH4.5, the acidity is titrated to 200, and fermentation is carried out for about 108 hours, so as to obtain the base material. Sensory evaluation was performed after the fermentation was completed, and the results are shown in table 1.
TABLE 1 Brown milk beverage sensory evaluation results
Figure BDA0003735482790000061
The sensory evaluation score is one of the important factors for determining whether the probiotics can be used as a commercial leavening agent. Although many probiotics can be used as strains for preparing fermented drinks, the defects of low taste or mouthfeel score or poor smell often exist, so that the obtained product has reduced flavor and is difficult to be accepted by consumers, and therefore, the probiotics cannot be applied to commercial leavening agents.
Further preparation of brown milk drink: preheating distilled water to 60 deg.C, adding apple juice and white sugar in proportion, adjusting pH to 3.7 with citric acid, dissolving material for 15min, sterilizing in 95 deg.C water bath for 5min, cooling to 37 deg.C, adding base material in proportion, mixing well, homogenizing, packaging, and storing in 4 deg.C refrigerator. The acidity and viable count changes of the probiotics in shelf life were measured every 7 days after the postmaturation at 4 ℃ with samples left, and the results are shown in table 2.
TABLE 2 changes in acidity and viable count of probiotics during storage of brown milk beverages at 4 ℃
Figure BDA0003735482790000071
From the measurement results in table 2, it was found that addition of lactobacillus paracasei PC804 to brown milk beverages did not cause post-acidification; the viable count of the PC804 after the fermentation is finished reaches 107Above CFU/g and maintaining a high viable count during storage, the obtained Lactobacillus paracasei PC804 has good probiotic properties.

Claims (4)

1. The lactobacillus paracasei (Lactcaseibacillus paracasei) PC804 is preserved in the China general microbiological culture collection center at 11-15 th of 2021 with the preservation number of CGMCC No.23917.
2. Use of the Lactobacillus paracasei (Lactcaseibacterium paracasei) PC804 of 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 (Lactcaseibacillus paracasei) PC804 fermented milk product according to claim 1.
CN202210795191.XA 2022-07-07 2022-07-07 Lactobacillus paracasei PC804 and application thereof Pending CN115261264A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116286519A (en) * 2023-03-13 2023-06-23 广东悦创生物科技有限公司 Lactobacillus paracasei KS3 and application thereof in preparation of anti-aging and digestion-aiding foods and medicines
CN116751725A (en) * 2023-07-27 2023-09-15 内蒙古农业大学 Anti-phage fermented lactobacillus mucilaginosus and application thereof
CN116814481A (en) * 2023-06-09 2023-09-29 内蒙古农业大学 Probiotic Lactobacillus paracasei PC646 derived from fermented mare milk and artificial intelligent screening method thereof
CN116832068A (en) * 2023-06-09 2023-10-03 内蒙古农业大学 Lactobacillus paracasei PC646 with broad-spectrum antibacterial activity and application thereof

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116286519A (en) * 2023-03-13 2023-06-23 广东悦创生物科技有限公司 Lactobacillus paracasei KS3 and application thereof in preparation of anti-aging and digestion-aiding foods and medicines
CN116286519B (en) * 2023-03-13 2023-11-28 广东悦创生物科技有限公司 Lactobacillus paracasei KS3 and application thereof in preparation of anti-aging and digestion-aiding foods and medicines
CN116814481A (en) * 2023-06-09 2023-09-29 内蒙古农业大学 Probiotic Lactobacillus paracasei PC646 derived from fermented mare milk and artificial intelligent screening method thereof
CN116832068A (en) * 2023-06-09 2023-10-03 内蒙古农业大学 Lactobacillus paracasei PC646 with broad-spectrum antibacterial activity and application thereof
CN116814481B (en) * 2023-06-09 2024-04-09 内蒙古农业大学 Probiotic Lactobacillus paracasei PC646 derived from fermented mare milk and artificial intelligent screening method thereof
CN116832068B (en) * 2023-06-09 2024-04-09 内蒙古农业大学 Lactobacillus paracasei PC646 with broad-spectrum antibacterial activity and application thereof
CN116751725A (en) * 2023-07-27 2023-09-15 内蒙古农业大学 Anti-phage fermented lactobacillus mucilaginosus and application thereof

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