Preparation method of lactobacillus and application thereof in fresh chilled meat preservation

Abstract

This invention discloses a method for preparing a strain of Lactobacillus saccharidus and its application in the preservation of chilled fresh meat, including the following steps: Step (1) Initial screening: Take 1.0 g of environmental sample, add 9 mL of sterile physiological saline, shake at 37℃ and 200 r / min for 30 min, sonicate for 10 min, then serially dilute to 10⁻⁴-10⁻⁶, take 100 μL and spread it on MRS solid medium, incubate at 37℃ for 48 h, pick single colonies and purify 3 times; Step (2) Antibacterial screening: Use 8 strains of putrefactive bacteria such as Enterobacter and Escherichia coli as indicator bacteria. The preparation method of this Lactobacillus saccharidus strain is based on natural sources, ensuring high safety and making it a suitable alternative to chemical preservatives. It exhibits significant inhibitory effects on major spoilage bacteria (such as Pseudomonas and Enterobacteriaceae) and pathogenic bacteria in chilled meat. Its low-temperature resistance (4℃) and acid and alkali resistance make it suitable for the storage environment of chilled meat. After application, it can reduce the total bacterial count, TVB-N content, pH value, and drip loss rate, delay color changes, and effectively extend the shelf life of chilled meat.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a method for preparing a strain of Lactobacillus saccharidus and its application in the preservation of chilled meat. Background Technology

[0002] Pork is one of the most widely consumed meats globally, and its quality and safety are directly related to human health. With the implementation of the "transfer of pork to meat" policy and the development of fresh food e-commerce, the demand for the storage and preservation of chilled meat is becoming increasingly prominent. Chilled meat needs to be stored at 0-4℃, but its shelf life is still limited by microbial spoilage. Traditional chemical preservatives are controversial in terms of safety, while lactic acid bacteria, as natural probiotics, have advantages such as antibacterial activity, safety, and high consumer acceptance, and have broad application prospects in food preservation. Summary of the Invention

[0003] The purpose of this invention is to provide a method for preparing a strain of Lactobacillus saccharidus and its application in the preservation of chilled fresh meat, so as to solve the existing problems mentioned in the background art.

[0004] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a strain of *Lactobacillus saccharidus* and its application in the preservation of chilled fresh meat, comprising the following steps:

[0005] Step (1) Initial screening: Take 1.0 g of environmental sample, add 9 mL of sterile physiological saline, shake at 37℃ and 200 r / min for 30 min, sonicate for 10 min, and then serially dilute to 10⁻ 4 -10⁻ 6 100 μL was spread onto MRS solid medium and incubated at 37°C for 48 h. Single colonies were picked and purified three times.

[0006] Step (2) Antibacterial screening: Using eight putrefactive bacteria such as Enterobacter and Escherichia coli as indicator bacteria, the diameter of the inhibition zone of the initial screening strains was determined by the perforation diffusion method, and strains with excellent antibacterial effects were screened.

[0007] Step (3) Secondary screening:

[0008] Low-temperature stress test: The strain was cultured to the logarithmic phase, centrifuged at 4000 rpm for 10 min, resuspended in physiological saline, and placed at 4℃. The OD value was measured from 0 to 10 days. Low-temperature acid and alkali tolerance test: The strain was cultured to the logarithmic phase, centrifuged at 10000 rpm for 10 min, resuspended in physiological saline at pH 4.0 and pH 7.5, and placed at 4℃. The OD value was measured from 0 to 3 days.

[0009] Step (4) Identification: The strain was identified as Lactobacillus saccharidus through morphological observation, physiological and biochemical tests and 16S rDNA sequencing.

[0010] Preferably, *Lactobacillus saccharidosis* is inoculated into MRS liquid medium, cultured at 37°C to the logarithmic phase, centrifuged at 10,000 rpm for 10 min, washed 2-3 times with sterile water, and the concentration adjusted to 10. 8 The bacterial suspension was prepared at CFU / mL. Fresh meat was cut into 50 g portions. The experimental group was sprayed with the bacterial suspension at a ratio of 4 mL / 100 g, while the blank group was left untreated. Both groups were sealed and stored at 4°C. The preservation effect was evaluated by detecting the total bacterial count, volatile basic nitrogen (TVB-N), pH, drip loss rate, and color change.

[0011] Preferably, the chilled fresh meat is pork tenderloin, which has significantly lower total bacterial count, TVB-N content, pH value and drip loss rate than the control group during storage, thus extending its shelf life.

[0012] Compared with the prior art, the beneficial effects of the present invention are: the preparation method of this *Lactobacillus saccharidus* strain is of natural origin, has high safety, and can replace chemical preservatives; it has a significant inhibitory effect on the main spoilage bacteria (such as *Pseudomonas*, *Enterobacter*, etc.) and pathogenic bacteria in chilled fresh meat; its low temperature resistance (4℃) and acid and alkali resistance characteristics are suitable for the storage environment of chilled fresh meat; after application, it can reduce the total bacterial count, TVB-N content, pH value and drip loss rate, delay color change, and effectively extend the shelf life of chilled fresh meat. Attached Figure Description

[0013] Figure 1 Results of low-temperature stress on lactic acid bacteria;

[0014] Figure 2 Results of low-temperature acid-base stress on lactic acid bacteria;

[0015] Figure 3 Colony morphology and Gram staining microscopic examination results of strain 11;

[0016] Figure 4 Results of the hemolysis test on chicken blood agar plates;

[0017] Figure 5 Colony count results;

[0018] Figure 6 Results of volatile basic nitrogen content;

[0019] Figure 7 Results of drip loss rate;

[0020] Figure 8 pH change results. Detailed Implementation

[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Please see Figures 1-8 This invention provides a technical solution: a method for preparing a strain of *Lactobacillus sucrose* and its application in the preservation of chilled meat.

[0023] Example 1: Screening and Identification of Strains

[0024] Preliminary screening: Take 1.0 g of environmental sample from the breeding farm and process it according to step (1) of claim 2 to obtain 20 purified strains.

[0025] Antibacterial screening: Using 8 putrefactive bacteria as indicator bacteria, the diameter of the inhibition zone was determined by the perforation diffusion method, and 8 strains, including strains 3, 11, and 12, with excellent antibacterial effects were screened out.

[0026] Secondary screening:

[0027] Low temperature stress: The OD value of strain 11 remained at around 0.5 for 10 days at 4℃, showing the strongest resistance to low temperatures;

[0028] Acid and alkali resistance: The absorbance of strain 11 decreased slowly under pH 4.0 conditions, indicating excellent acid tolerance.

[0029] Identification: Strain 11 is a Gram-positive short bacillus with physiological and biochemical characteristics consistent with the genus Lactobacillus. Its 16S rDNA sequencing shows 100% similarity to Lacticaseibacillus rhamnosus, and its hemolysis test shows incomplete α-type hemolysis.

[0030] Example 2: Application in chilled meat preservation

[0031] Preparation of bacterial suspension: Strain 11 was cultured to the logarithmic growth phase, and the concentration was adjusted to 10. 8 CFU / mL.

[0032] Sample processing: Pork tenderloin was cut into 50 g portions. The experimental group was sprayed with bacterial suspension (4 mL / 100 g) and stored at 4℃.

[0033] Indicator Testing:

[0034] Total bacterial count: at 6 days, the experimental group (3×10⁻⁶) 7 The CFU / g level was significantly lower than that of the control group (22×10). 7 CFU / g);

[0035] TVB-N: The TVB-N content in the experimental group was significantly lower than that in the control group at 9 days;

[0036] pH: The pH of the experimental group was 6.49 at 8 days, which was lower than that of the control group (6.61).

[0037] Drip loss rate: The drip loss rate of the experimental group during storage was significantly lower than that of the control group;

[0038] Color: The experimental group showed obvious color change after 5 days, which was better than the blank group (change after 3 days).

[0039] Working principle: When using the preparation method of Lactobacillus sucrose and its application in the preservation of chilled fresh meat, Lactobacillus sucrose strain 11 has excellent antibacterial activity, environmental adaptability and safety. In the preservation of chilled fresh meat, it can effectively inhibit the growth of spoilage microorganisms and delay quality deterioration, which has important practical application value. Moreover, the contents not described in detail in this specification are existing technologies known to those skilled in the art.

[0040] The terms “center,” “longitudinal,” “lateral,” “front,” “rear,” “left,” “right,” “vertical,” “horizontal,” “top,” “bottom,” “inner,” and “outer,” etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are merely simplified descriptions for the convenience of describing the present invention and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limiting the scope of protection of the present invention.

[0041] Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing a strain of *Lactobacillus sucrose*, characterized in that, Includes the following steps: Step (1) Initial screening: Take 1.0 g of environmental sample, add 9 mL of sterile physiological saline, shake at 37℃ and 200 r / min for 30 min, sonicate for 10 min, and then serially dilute to 10⁻ 4 -10⁻ 6 100 μL was spread onto MRS solid medium and incubated at 37°C for 48 h. Single colonies were picked and purified three times. Step (2) Antibacterial screening: Using eight putrefactive bacteria such as Enterobacter and Escherichia coli as indicator bacteria, the diameter of the inhibition zone of the initial screening strains was determined by the perforation diffusion method, and strains with excellent antibacterial effects were screened. Step (3) Secondary screening: Low-temperature stress test: The strain was cultured to the logarithmic phase, centrifuged at 4000 rpm for 10 min, resuspended in physiological saline, and placed at 4℃. The OD value was measured from 0 to 10 days. Low-temperature acid and alkali tolerance test: The strain was cultured to the logarithmic phase, centrifuged at 10000 rpm for 10 min, resuspended in physiological saline at pH 4.0 and pH 7.5, and placed at 4℃. The OD value was measured from 0 to 3 days. Step (4) Identification: The strain was identified as Lactobacillus saccharidus through morphological observation, physiological and biochemical tests and 16S rDNA sequencing.

2. The application of *Lactobacillus sucrose* according to claim 1 in the preservation of chilled meat, characterized in that: Inoculate *Lactobacillus saccharidosis* into MRS liquid medium, incubate at 37°C until the logarithmic growth phase, centrifuge at 10,000 rpm for 10 min, wash 2-3 times with sterile water, and adjust the concentration to 10. 8 The bacterial suspension was prepared at CFU / mL. Fresh meat was cut into 50 g portions. The experimental group was sprayed with the bacterial suspension at a ratio of 4 mL / 100 g, while the blank group was left untreated. Both groups were sealed and stored at 4°C. The preservation effect was evaluated by detecting the total bacterial count, volatile basic nitrogen (TVB-N), pH, drip loss rate, and color change.

3. The application of *Lactobacillus sucrose* according to claim 2 in the preservation of chilled meat, characterized in that: The chilled fresh meat was pork tenderloin. During storage, the total bacterial count, TVB-N content, pH value, and drip loss rate were all significantly lower than those of the control group, resulting in a longer shelf life.

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