Heat-resistant paracasei sublactis and application thereof

Abstract

This invention relates to the field of microbial technology, specifically to a heat-resistant *Lactaseibacillus paracasei* and its applications. The preservation number for *Lactaseibacillus paracasei* R01NGW is GDMCC No:66026. This invention utilizes gradient high-temperature acclimatization with chicken manure and screening on a selective medium containing polylysine hydrochloride to obtain a strain that is both heat-resistant (survival ≥10 μL at 70℃). 8 This strain exhibits high CFU / mL concentration, strong antibacterial activity (inhibition zone 15mm-20mm), and resistance to gastric acid and bile salts. The screening medium utilizes a triple screening mechanism of "physical + chemical + biological targeted inhibition," achieving a 99.9% inhibition rate against Bacillus. This strain can be used in high-temperature feed processing, silage fermentation, probiotic preparations, and bio-antibacterial applications, providing a safe and efficient solution for the application of lactic acid bacteria in heat-resistant environments.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a heat-resistant Lactobacillus paracasei and its applications. Background Technology

[0002] Insufficient high-temperature tolerance of lactic acid bacteria: Traditional lactic acid bacteria usually have poor resistance and low survival rate in processing processes such as spray drying (≥70℃) and high-temperature sterilization in feed processing, resulting in unstable product quality and short shelf life, which seriously affects the application of lactic acid bacteria in feed.

[0003] The challenge of Bacillus interference in screening: In traditional lactic acid bacteria screening, Bacillus is heat-resistant and has strong nutrient competition, resulting in low enrichment efficiency of lactic acid bacteria. It is possible that the bacteria obtained in the final screening are Bacillus rather than lactic acid bacteria.

[0004] Natural antibacterial strains are scarce: the biocontrol of pathogens such as Salmonella and Staphylococcus urgently requires lactic acid bacteria that are heat-resistant, have strong antibacterial activity and probiotic function, but existing strains cannot meet the above requirements at the same time. Summary of the Invention

[0005] In view of the above-mentioned shortcomings in the prior art, the purpose of this invention is to provide a heat-resistant Lactobacillus paracasei and its application.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A heat-resistant Lacticaseibacillus paracasei is provided, with the accession number GDMCC No:66026 for Lacticaseibacillus paracasei R01NGW.

[0008] A microbial agent is provided, which contains Lacticaseibacillus paracasei R01NGW.

[0009] The invention provides the use of an agent containing Lacticaseibacillus paracasei R01NGW in the preparation of feed additives, probiotic preparations, or antibacterial preparations.

[0010] Furthermore, the feed additive is suitable for high-temperature processing at 70℃.

[0011] Furthermore, antibacterial agents are used to inhibit Salmonella or Staphylococcus.

[0012] A selective culture medium for screening Lacticaseibacillus paracasei R01NGW is provided, wherein each liter of the medium contains: 10.0 g tryptone, 15.0 g trehalose, 0.08 g polylysine hydrochloride, 5.0 g sodium acetate, pH 5.5; 0.2 g manganese sulfate, and 2.0 g dipotassium hydrogen phosphate.

[0013] A method for screening Lacticaseibacillus paracasei R01NGW is provided, comprising: treating fecal samples in a 70°C water bath and then inoculating them into the culture medium described in claim 6; subjecting them to five generations of high-temperature acclimatization at a gradient of 40°C-70°C, and identifying them by MRS streak plating and 16S rRNA gene sequencing.

[0014] The beneficial effects of this invention are as follows:

[0015] 1. This is the first report of Lacticaseibacillus paracasei R01NGW, which is resistant to high temperatures of 70℃, breaking through the bottleneck of traditional lactic acid bacteria for high-temperature applications;

[0016] 2. Develop feed-grade anti-spore-forming culture medium to replace antibiotics and improve screening efficiency;

[0017] 3. Lacticaseibacillus paracasei R01NGW combines heat resistance, antibacterial, and probiotic functions, filling the gap in natural biological antibacterial agents in high-temperature scenarios. Attached Figure Description

[0018] Figure 1 This image shows the morphology of colonies isolated by streaking on MRS agar medium.

[0019] Figure 2 The image shows the morphology of the bacterial strain under a microscope (Gram-positive bacillus, ×1000x).

[0020] Figure 3 The image shows the antibacterial test results of Lacticaseibacillus paracasei R01NGW (left: Staphylococcus aureus; right: Salmonella). Detailed Implementation

[0021] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0022] (I) Strain characteristics and preservation information

[0023] Strain name: Lacticaseibacillus paracasei GDMCC66026R01NGW.

[0024] Preservation information: Guangdong Provincial Microbial Culture Collection Center (GDMCC), Address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Preservation number: GDMCC No:66026, Preservation date: March 17, 2025.

[0025]

[0026] (II) Screening medium against Bacillus interference (modified MRS medium, 1L)

[0027] 1. Ingredients and Design Functions

[0028]

[0029]

[0030] 2. Triple screening pressure mechanism

[0031] Physical temperature screening: 70℃ water bath treatment kills non-thermoresistant bacteria and vegetative spores, while retaining thermoresistant lactic acid bacteria and spores.

[0032] Chemical screening: ε-PL inhibits the germination of Bacillus spores, and the acidic environment (pH 5.5) further inhibits their growth. Lactic acid bacteria proliferate normally through acid resistance and metabolic advantages, achieving a triple blockade of "killing and inhibiting germination".

[0033] This culture medium achieves effective inhibition of Bacillus through a triple mechanism of physical temperature screening, chemical environmental regulation (pH / carbon source), and biological inhibitor (ε-PL). At the same time, by optimizing the carbon source ratio and trace elements to enhance the metabolic advantages of lactic acid bacteria, it is suitable for the isolation of thermostable lactic acid bacteria in samples from high-temperature environments.

[0034] (III) Application Areas

[0035] High-temperature processed feed additives: improve the survival rate of lactic acid bacteria in spray drying, extend shelf life, and reduce pathogen contamination.

[0036] Fermented silage: Tolerates temperature fluctuations of 30-60℃, promotes lactic acid fermentation, and inhibits putrefactive bacteria.

[0037] Probiotic preparations: regulate the intestinal health of livestock and poultry, improve tolerance to gastric acid (pH 2.5) and bile salts (0.3%), and increase colonization efficiency by 30%.

[0038] Natural antibacterial agents: replace chemical antibacterial agents to inhibit pathogens such as Staphylococcus and Salmonella.

[0039] Example

[0040] (I) Strains Screening and Domestication Process

[0041] Sample pretreatment: Collect chicken manure, weigh 25g and add it to 250mL of sterile MRS broth, and incubate at 37℃ with shaking for 48h; then incubate at 70℃ for 10min to kill non-thermos-resistant bacteria.

[0042] Gradient high-temperature acclimatization: Inoculate into modified MRS medium, culture at 37℃ with shaking for 48 h, place in a 40℃ water bath for 20 min, and inoculate into modified MRS medium again for culture; repeat this process sequentially for acclimatization in water baths at 40℃, 50℃, 60℃, and 70℃ (5 generations per stage).

[0043] Isolation, purification and identification: The bacteria were plated on MRS plates and streaked to purify the milky white colonies. The strain was confirmed by 16S rRNA gene sequencing (primers 27F / 1492R) and physiological and biochemical identification.

[0044] (II) Key Implementation Examples

[0045] Example 1: After being acclimatized at 70℃ for 5 generations, the strain was confirmed to have 100% homology with Lacticaseibacillus paracasei by 16S rRNA gene sequencing.

[0046] Example 2: Using *Lactobacillus acidophilus* as a control, after a 20-minute water bath at 70°C, the survival rate of *Lactobacillus paracasei* was 1.2 × 10⁻⁶. 8 CFU / mL, control strain Lactobacillus acidophilus survival count <10 CFU / mL.

[0047] Example 3: The inhibition zones of heat-resistant Lactobacillus paracasei against Staphylococcus aureus and Salmonella were 20.00 mm and 15.35 mm, respectively, and the antibacterial effect was significantly better than that of commercially available probiotics.

[0048] Example 4: The Bacillus inhibition rate of the culture medium of the present invention is ≥99.9%, and it has good selectivity for screening heat-resistant lactic acid bacteria.

[0049] In summary, this invention has the following innovative design features:

[0050] 1. Screening method design: Through the triple mechanism of physical temperature screening, chemical environment regulation and biological targeted inhibition, the entire process of "killing vegetative cells → inhibiting metabolism → blocking germination" of Bacillus is blocked. At the same time, by optimizing the nutritional composition, the competitive advantage of lactic acid bacteria is strengthened, so as to achieve the heat-resistant screening target of inhibiting the growth of Bacillus and promoting the proliferation of lactic acid bacteria. (1) Physical temperature screening: 70℃ water bath treatment kills non-thermothermal bacteria and vegetative Bacillus, leaving only heat-resistant lactic acid bacteria and Bacillus spores (spores survive due to their high temperature resistance, but do not germinate). (2) Chemical environment screening: pH regulation: The pH of the culture medium is 5.5 to inhibit the metabolic activity of Bacillus, while providing a suitable acidic environment for lactic acid bacteria. Carbon source competition: Sodium acetate is used as a carbon source for lactic acid bacteria to promote acid production, while trehalose inhibits the utilization of carbon sources by Bacillus. (3) Bio-targeted inhibition: ε-PL (polylysine hydrochloride) specifically inhibits the germination of Gram-positive bacterial spores, blocks peptidoglycan synthase, and selectively inhibits the activation of Bacillus spores, while lactic acid bacteria are unaffected because their safety threshold is higher than the concentration used.

[0051] 2. Breakthrough in heat-resistant strains: For the first time, strains with a survival rate ≥10% were screened out after a 20-minute water bath at 70℃. 8 The CFU / mL Lactobacillus paracasei GDMCC 66026R01NGW breaks through the bottleneck of traditional high-temperature applications of lactic acid bacteria.

[0052] 3. Multifunctional application expansion: The strain has antibacterial properties (inhibition zone 15mm-20mm), resistance to gastric acid and bile salts (survival rate ≥82%), and integrates heat resistance, antibacterial and probiotic functions, filling the gap of natural biological antibacterial agents in high-temperature scenarios.

[0053] In summary, this invention, through gradient high-temperature acclimatization of chicken manure and screening in a selective culture medium containing polylysine hydrochloride, obtained a strain that is both heat-resistant (survival ≥10 at 70℃) and exhibits high survival rate. 8 This strain exhibits high CFU / mL concentration, strong antibacterial activity (inhibition zone 15mm-20mm), and resistance to gastric acid and bile salts. The screening medium utilizes a triple screening mechanism of "physical + chemical + biological targeted inhibition," achieving a 99.9% inhibition rate against Bacillus. This strain can be used in high-temperature feed processing, silage fermentation, probiotic preparations, and bio-antibacterial applications, providing a safe and efficient solution for the application of lactic acid bacteria in heat-resistant environments.

[0054] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0055] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. Contains Lactobacillus paracasei ( Lacticaseibacillus paracasei The application of R01NGW's microbial agent in the preparation of feed additives, probiotic preparations, or antibacterial preparations is characterized by, Feed additives are suitable for high-temperature processing at 70℃; antibacterial agents are used to inhibit Salmonella pullorum or Staphylococcus aureus in chickens. The Lactobacillus paracasei ( Lacticaseibacillus paracasei The accession number of R01NGW is GDMCCNo: 66026.

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