Digestion associated lactobacillus CZ24, microbial inoculum, microbial inoculum preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses digestion associated lactobacillus CZ24, a microbial agent, a microbial agent preparation method and application, the digestion associated lactobacillus CZ24 is preserved in China General Microbiological Culture Collection Center on September 18, 2024, and the preservation number is CGMCC NO.31987. The digestion associated lactobacillus CZ24 has a DNA sequence of SEQ ID NO.1 in a sequence table; the digestion associated lactobacillus CZ24 fermentation liquor has the characteristic of inhibiting the growth of harmful microorganisms, can regulate and control fence factors, and can remarkably prolong the shelf life of food, and the sensory score of the food is good.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and in particular to a digestive-associated lactobacillus CZ24, a bacterial agent, a bacterial agent preparation method and applications thereof. Background Art

[0002] During food processing and preservation, sterilization and antibacterial methods are critical steps in ensuring food safety and extending its shelf life. Currently, commonly used sterilization and antibacterial methods in the food industry include high-temperature sterilization, pasteurization, room-temperature sterilization, and the addition of preservatives. High-temperature sterilization typically involves sterilization at 121°C, a temperature high enough to kill most bacteria, molds, and yeasts, including spores. To further enhance food preservation, small amounts of preservatives, antioxidants, and other additives are often added during or after sterilization. Pasteurization occurs at temperatures of 61-63°C for 30 minutes, or 72-75°C for 10-15 minutes. These conditions can kill some pathogens, but they cannot completely eliminate them. Preservatives (such as potassium sorbate and sodium benzoate) are often added. Room-temperature sterilization typically involves sterilization at room temperature, relying primarily on physical methods (such as ultraviolet light and filtration) or chemical methods (such as preservatives).

[0003] However, high-temperature sterilization will cause the taste, color and nutritional value of food to decline, especially heat-sensitive vitamins and proteins. Pasteurization and room-temperature sterilization cannot kill most bacteria, molds and yeasts, and there are certain food safety risks. They also require the use of large amounts of preservatives, and long-term intake may have adverse effects on health.

[0004] Therefore, how to achieve sterilization and antibacterial while ensuring food safety and food quality is a major challenge faced by the food industry. Currently, no perfect solution has been found to the above problems. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a digestive-associated Lactobacillus CZ24, a bacterial agent, a bacterial agent preparation method and an application thereof, and to verify the application and effect of the strain in food production.

[0006] To achieve the above object, the present invention provides the following technical solutions: a digestive-associated Lactobacillus CZ24, the digestive-associated Lactobacillus ( Companilactobacillus alimentarius ) CZ24 was deposited in the General Microbiology Center of China Culture Collection Administration on September 18, 2024, with the deposit number: CGMCC N.31987; The digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 has the DNA sequence of SEQ ID NO.1 in the sequence listing.

[0007] A bacterial agent, comprising the digestive-associated lactobacillus ( Companilactobacillus alimentarius )CZ24.

[0008] The preparation method of the above bacterial agent is as follows: inoculate the digestive associated lactobacillus CZ24 into MRS medium, culture at 37℃ for 24h, inoculate the obtained bacterial liquid into fermentation medium at an inoculum rate of 3%, and culture in a 37℃ incubator to obtain digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 bacterial agent; Fermentation medium: peanut sprout juice 50 ml / L, glucose 10.00 g / L, lactose 10.00 g / L, soy peptone 25.00 g / L, K2HPO4 2.29 g / L and MgSO4·7H2O 0.80 g / L.

[0009] Furthermore, the digestive-associated lactobacillus or the bacterial agent is used in food production.

[0010] Compared with the prior art, the beneficial effects of the present invention are: the fermentation broth of digestive-associated Lactobacillus CZ24 has the characteristics of inhibiting the growth of harmful microorganisms, can regulate the barrier factor, and can significantly extend the shelf life of food, and the sensory score of the food is good. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is a plate colony morphology of the digestive-associated Lactobacillus CZ24; Figure 2 This is a Gram-stained micrograph of the digestive-associated Lactobacillus CZ24; Figure 3 A phylogenetic tree was constructed for the 16S rDNA sequence of Lactobacillus pepticus CZ24; Figure 4 This is a diagram showing the inhibition zone produced by digestive-associated Lactobacillus CZ24 in Escherichia coli; Figure 5 This is a diagram showing the inhibition zone produced by the digestive-associated Lactobacillus CZ24 in Bacillus subtilis; Figure 6 This is a diagram showing the inhibition zone produced by digestive-associated Lactobacillus CZ24 on Staphylococcus aureus. DETAILED DESCRIPTION

[0012] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0013] See also Figures 1-6 The present invention provides a digestive-associated lactobacillus CZ24, which is isolated from kimchi and collected in May 2024 in Chongqing, China. Companilactobacillus alimentarius ) CZ24 was deposited in the General Microbiology Center of China Culture Collection Administration on September 18, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Its deposit number is: CGMCC NO.31987.

[0014] Example 1: Isolation, purification and identification of digestive-associated Lactobacillus CZ24 1. Isolation and Purification of Strain Lactobacillus spp. CZ24 was isolated and purified from kimchi. A small amount of kimchi juice was added to MRS liquid medium containing antibiotics and incubated for 24 hours. After incubation, sterilized calcium carbonate was added to the MRS liquid medium at a concentration of 2%. The medium was cooled to approximately 46°C in a constant-temperature water bath and shaken to mix the calcium carbonate thoroughly, taking care not to create bubbles. After mixing, the MRS liquid medium containing 2% calcium carbonate was mixed with an appropriate amount of agar and heated until the agar was completely dissolved. The mixture was then immediately poured into a Petri dish and allowed to solidify to obtain MRS solid medium. Under sterile conditions, 200 μL of the sample was spread onto the surface of a solidified MRS solid medium plate and incubated inverted at 37°C. After 24-48 hours of incubation, the colony characteristics were observed, and single colonies with large calcium-dissolving rings were selected and streaked onto MRS solid medium to obtain single colonies. These strains were then streaked onto the plate and purified repeatedly.

[0015] MRS liquid medium: peptone 10.0 g, beef extract 10.0 g, yeast extract 5.0 g, glucose 20.0 g, diammonium citrate 2.0 g, sodium acetate 5.0 g, MgSO4 2.0 g, MnSO4 0.05 g, K2HPO4 2.0 g, Tween 80 1.0 mL; distilled water 1000 mL; adjust pH 6.2-6.4, culture temperature 37°C, culture time 16 h.

[0016] 2. Preliminary identification of colonies Hydrogen peroxide catalase test: Use an inoculating loop to take 1 loop of isolated and purified colonies and place it on a clean glass slide. Add 1 drop of 30% hydrogen peroxide. If bubbles are present, the test is catalase positive, otherwise it is negative.

[0017] Gram staining: Pick the isolated and purified colonies for Gram staining and observe them under a microscope. The hydrogen peroxide catalase test and Gram staining of the isolated and purified lactic acid bacteria are shown in the following table: serial number Hydrogen peroxide catalase (±) Gram stain (G±) Microscopic examination results CZ24 - G+ Rod-shaped, present in pairs or singly Conclusion: From the above table, we can see that the strain is negative for hydrogen peroxide catalase, positive for Gram staining, and rod-shaped. The strain is labeled CZ24.

[0018] 3. Physiological and biochemical tests Different types of sugars were selected to replace glucose as the carbon source to prepare MRS liquid culture medium. The liquid culture medium was aspirated and its OD600 was measured in a 96-well plate. CZ24 was inoculated into MRS liquid culture medium with different carbon sources at a 1% inoculation volume and cultured at 37°C for 24 hours. The bacterial liquid was aspirated and its OD600 was measured in a 96-well plate. Based on the "Bergey's Manual of Systematic Bacteriology" and "Classification, Identification and Experimental Methods of Lactic Acid Bacteria", the strain was subjected to physiological and biochemical identification. Positive reaction results were indicated by (+) and negative by (-), thereby making a preliminary identification of the species of the target strain. The results are shown in the following table: pilot projects CZ24 sorbitol - Mannitol + arabinose + lactose - Cellobiose + Raffinose - Trehalose - Ribose + melezitose - Conclusion: According to Bergey's Manual of Systematic Bacteriology and Lactic Acid Bacteria Classification, Identification and Experimental Methods, the target strain CZ24 was preliminarily identified as Lactobacillus genus.

[0019] 4. 16S rDNA sequence analysis The genomic DNA of the target strain was extracted using a bacterial genome extraction kit. The genomic DNA of the target strain was used as a template, and 27F and 1492R were selected as universal primers to perform PCR amplification of 16S rDNA. PCR reaction system (25 μL): 1 μL each of upstream and downstream primers and genomic template, 12.5 μL of Pre-mix Ex Taq, and 9.5 μL of sterilized ddH2O. PCR amplification conditions: pre-denaturation at 95°C for 4 minutes, denaturation at 94°C for 1 minute, annealing at 58°C for 1 minute, and extension at 72°C for 2 minutes, for a total of 30 cycles. The reaction was terminated after the final extension at 72°C for 7 minutes. The PCR product was then sent to BGI for purification and sequencing. The obtained PCR product sequences and the 16S rDNA sequences of closely related lactic acid bacteria were used to construct a phylogenetic tree using MEGA 5.05 software. The phylogenetic tree is shown in the attached figure. Figure 3 shown.

[0020] The sequencing results are: GGGGGGGCCGTGTCGAACGAACGCTGAGATCGAAGCATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGAGGTGGAGGCCTTCCGCCCTTCATTGCTGCGGTTAACGCATTAAGCACTCCGCCTGGGGAGTACGATCGCAAGATTGAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGGGGAGCTTGTGGTTTAATTCAAAGCAACGCGAAAAACCTTACCAGGTCTTGACATACCATGAAAAGCTAAGAGATTATTCTTTCCCTTCGGGGACATGGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTA TTATCAGTTGCCAGCATTCAGTTGGGCACTCTGGTGAGACTGCCGGTGATAAACCGGAGGAAGGGGGGGACGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTCGGTACAACGTGTTGCGAACTCGCGAGGGCAAGCAAATCACTTAAAACCGATCTCAGTTCGGATTGC AGGCTGCAACTCGCCTGCATGAAGCTGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTGTGTAACACCCAAAAGTCGGTGGATTAACCCTTCGGAGAACTATTCCCCCTAATGTGATACAAGAGAGTAGGGAGAAGCAAC According to the sequencing results, the target strain CZ24 was identified as a digestive-associated Lactobacillus ( Companilactobacillus alimentarius ).

[0021] Example 2: Effect of peanut bud juice addition on the growth of digestive-associated Lactobacillus CZ24 Lactobacillus pneumophila CZ24 was cultured in MRS medium at 37°C for 24 hours. The resulting culture was inoculated into a fermentation medium at a 3% inoculum rate and incubated in a 37°C incubator. Starting at 12 hours of fermentation, samples were taken and OD600 values ​​were measured every 4 hours. The experiment was repeated three times.

[0022] MRS medium: 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 mL Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g triammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 20 g calcium carbonate, and 1000 mL distilled water.

[0023] Fermentation medium containing peanut bud juice digesting Lactobacillus CZ24: peanut bud juice 50 ml / L, glucose 10.00 g / L, lactose 10.00 g / L, soy peptone 25.00 g / L, K2HPO4 2.29 g / L, MgSO4·7H2O 0.80 g / L.

[0024] Fermentation medium without peanut sprout juice digesting associated Lactobacillus CZ24: glucose 10.00 g / L, lactose 10.00 g / L, soy peptone 25.00 g / L, K2HPO4 2.29 g / L, MgSO4·7H2O 0.80 g / L.

[0025] Preparation of MRS medium and fermentation medium: Add the above ingredients into distilled water, heat to dissolve, and sterilize by high pressure at 121°C for 15 minutes.

[0026] The effect of adding peanut sprout juice on the growth of digestive-associated Lactobacillus CZ24 is shown in the following table:

[0027] Conclusion: Adding peanut bud juice can effectively promote the growth of Lactobacillus cinereus CZ24, and after fermentation time exceeds 24 hours, the growth rate of Lactobacillus cinereus CZ24 is slowed down. Therefore, 24 hours of fermentation is the optimal fermentation time.

[0028] Example 3: Antibacterial effect of digestive-associated Lactobacillus CZ24 Take the three batches of fermentation broth mentioned above, centrifuge at 4000r / min for 3 minutes, and set aside the supernatant. The bacteriocin in the supernatant was determined by the Oxford cup double-layer agar diffusion method. Escherichia coli, Bacillus subtilis, and Staphylococcus aureus were used as indicator bacteria, and 200μL of fermentation broth was placed in an Oxford cup. The diameter of the Oxford cup is 8.0mm. Incubate in a 37℃ incubator for 24-48 hours, observe the inhibition zone, measure and record the diameter of the inhibition zone, and the results are shown in the following table and attached. Figure 4-6 As shown: indicator bacteria Diameter of inhibition zone (mm) Escherichia coli 13.95±0.15 Bacillus subtilis 14.00±0.55 Staphylococcus aureus 10.1±0.05 Conclusion: Lactobacillus pneumophila CZ24 inhibited the growth of Escherichia coli, Bacillus subtilis, and Staphylococcus aureus. The strain showed the greatest inhibitory effect against Bacillus subtilis, with an inhibition zone diameter of 14.00 mm. Escherichia coli showed the second-best inhibitory effect, with an average inhibition zone diameter of 13.95 mm. When Staphylococcus aureus was used as an indicator bacteria, the average inhibition zone diameter was 10.1 mm. Lactobacillus pneumophila CZ24 exhibited varying degrees of inhibitory activity against common pathogens.

[0029] Example 4: Effect of adding different amounts of digestive-associated Lactobacillus CZ24 on the quality of pre-packaged crucian carp Activated Lactobacillus pneumophila CZ24 was inoculated at a 3% inoculum by mass and fermented at 37°C for 24 hours to obtain a fermentation broth. A comprehensive sensory quality evaluation was conducted to test the effects of adding different amounts of Lactobacillus pneumophila CZ24 to fermentation broth on the quality of prepackaged crucian carp. The groups were divided into: blank group (no preservatives or antistaling agents added), test group 1 (fermentation broth and sample added at a ratio of 1:100), test group 2 (fermentation broth and sample added at a ratio of 2:100), test group 3 (fermentation broth and sample added at a ratio of 4:100), and test group 4 (fermentation broth and sample added at a ratio of 8:100). All proportions are by mass. The comprehensive sensory quality evaluation includes four evaluation indicators: taste, flavor, aroma, and color. Each indicator is rated on a scale of 1-10, with higher scores indicating better quality. The specific evaluation results are shown in the table below.

[0030] Group Taste (points) Taste (points) Aroma (points) Color (points) Average score (points) Blank group 8 9 8 8 8.25±0.50 Experimental group 1 8 8 8 8 8.00±0.00 Experimental group 2 8 7 8 8 7.75±0.50 Experimental group 3 8 7 7 7 7.25±0.50 Experimental group 4 4 5 5 4 4.50±0.58 Conclusion: The table above shows that the sensory quality of pre-packaged crucian carp decreases with increasing fermentation broth addition. To maximize the quality of pre-packaged crucian carp, the fermentation broth to sample addition ratio should not exceed 4:100.

[0031] Example 5: Effect of Adding Lactobacillus CZ24 Fermentation Broth on the Shelf Life of Prepackaged Crucian Carp The activated Lactobacillus pneumophila CZ24 was inoculated at a mass ratio of 3% and fermented at 37°C for 24 hours to obtain a fermentation broth. The antibacterial and antiseptic effects of the fermentation broth supplemented with Lactobacillus pneumophila CZ24 on soft pre-packaged crucian carp were tested.

[0032] Experimental conditions: Weifang, Shandong, August 15, 2024 - October 17, 2024, normal temperature 18°C-38°C all day.

[0033] Experimental groups: conventional preservative group (nisin added at a dosage of 0.5 g / kg), experimental group 1 (fermentation broth and sample added at a ratio of 4:100), and experimental group 2 (fermentation supernatant and sample added at a ratio of 4:100).

[0034] Five parallel tests were conducted on the conventional preservative group, test group 1, and test group 2, respectively. The appearance of the pre-packaged crucian carp was tested every seven days. Staphylococcus aureus was tested according to GB 4789.10, Salmonella was tested according to GB 4789.4, and Escherichia coli was tested according to GB 4789.3. The three samples were compared, as shown in the table below.

[0035]

[0036] In summary, the effects of experimental groups 1 and 2 in inhibiting Staphylococcus aureus were significantly better than those of the conventional preservative group. The fermentation broth with the addition of Lactobacillus pneumophila CZ24 had a better antibacterial effect than the fermentation supernatant. In addition, the fermentation broth with the addition of Lactobacillus pneumophila CZ24 significantly extended the shelf life of pre-packaged crucian carp by up to 5 times.

[0037] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any other manner. Any person skilled in the art may utilize the above-disclosed technical content to modify or modify the present invention into equivalent embodiments. However, any simple modifications, equivalent variations, and modifications made to the above examples that do not depart from the technical content of the present invention and are based on the technical essence of the present invention remain within the scope of protection of the present invention.

Claims

1. A digestive-associated lactobacillus CZ24, characterized in that: The digestive associated lactobacillus ( Companilactobacillus alimentarius )CZ24 was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 18, 2024, with the deposit number: CGMCC NO.31987; The digestive associated lactobacillus ( Companilactobacillus alimentarius ) CZ24 has the DNA sequence of SEQID NO.1 in the sequence listing.

2. A bacterial agent, characterized in that: The bacterial agent comprises the digestive-associated lactobacillus ( Companilactobacillus alimentarius )CZ24.

3. A bacterial agent according to claim 2, characterized in that: The bacterial agent is prepared according to the following method: The digestive associated lactobacillus CZ24 was inoculated into MRS medium and cultured at 37°C for 24 hours. The obtained bacterial liquid was inoculated into the fermentation medium at an inoculum rate of 3%, and cultured in a 37°C incubator to obtain the digestive associated lactobacillus ( Companilactobacillus alimentarius )CZ24 bacterial agent; Fermentation medium: peanut sprout juice 50 ml / L, glucose 10.00 g / L, lactose 10.00 g / L, soy peptone 25.00 g / L, K2HPO4 2.29 g / L and MgSO4·7H2O 0.80 g / L.

4. Use of the digestive-associated Lactobacillus CZ24 as claimed in claim 1 or the bacterial agent as claimed in claim 2 in food production.

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