Lactobacillus buchneri CZ23, microbial inoculum, microbial inoculum preparation method and application thereof

Abstract

The invention relates to the technical field of microorganisms, and particularly discloses lactobacillus buchneri CZ23, a fungicide, a fungicide preparation method and application, the lactobacillus buchneri CZ23 is preserved in China General Microbiological Culture Collection Center (CGMCC) on September 18, 2024, and the preservation number is CGMCC NO.31986. The lactobacillus buchneri CZ23 has a DNA sequence of SEQ ID NO.1 in a sequence table; the lactobacillus buchneri CZ23 fermentation liquor has the characteristic of inhibiting the growth of harmful microorganisms and can regulate and control fence factors, the pH value of the solution is 3.24-3.25, and the lactobacillus buchneri CZ23 can survive and breed in an environment with a relatively low pH value and is good in acid resistance and high in survival amount.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly relates to a Lactobacillus brenis-like strain CZ23, a bacterial agent, a method for preparing the bacterial agent, and its application. Background Art

[0002] In the processes of food processing and preservation, sterilization and bacteriostasis are key steps to ensure food safety and extend the shelf life of food. Currently, the commonly used sterilization and bacteriostasis methods in the food industry mainly include high-temperature sterilization, pasteurization, room-temperature sterilization, addition of preservatives, etc. High-temperature sterilization usually refers to the sterilization treatment at a temperature of 121°C, which is sufficient to kill most bacteria, molds, and yeasts, including spores. To further enhance the preservation effect of food, a small amount of preservatives, antioxidants, and other additives are usually added during or after the sterilization process; the sterilization conditions for pasteurization are 61 - 63°C for 30 min, or 72 - 75°C for 10 - 15 min. Such conditions can kill some pathogenic bacteria but cannot completely eliminate them, and preservatives (such as potassium sorbate, sodium benzoate, etc.) are usually added; room-temperature sterilization usually refers to the sterilization treatment under room-temperature conditions, mainly relying on physical methods (such as ultraviolet rays, filtration) or chemical methods (such as preservatives).

[0003] However, high-temperature sterilization can lead to a decline in the taste, color, and nutritional value of food, especially for heat-sensitive vitamins and proteins. Pasteurization and room-temperature sterilization cannot kill most bacteria, molds, and yeasts, presenting certain food safety risks and requiring a large amount of preservatives, and long-term intake may have an adverse impact on health.

[0004] Therefore, how to achieve food safety and food quality while carrying out sterilization and bacteriostasis is an important problem commonly faced by the food industry. At present, no perfect solution to the above problems has been found. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide a Lactobacillus brenis-like strain CZ23, a bacterial agent, a method for preparing the bacterial agent, and its application, and to verify the application and effect of this strain in food production.

[0006] To achieve the above object, the present invention provides the following technical solution: A Lactobacillus brenis-like strain CZ23, the Lactobacillus brenis-like strain ( Lentilactobacillus parabuchneri ) CZ23 is deposited in the China General Microbiological Culture Collection Center on September 18, 2024, and the deposit number is: CGMCC NO. 31986; The Lactobacillus brenis-like strain ( Lentilactobacillus parabuchneri ) CZ23 has the DNA sequence of SEQ ID NO.1 in the sequence listing.

[0007] A bacterial agent, the bacterial agent contains the Lactobacillus brantae - like Lentilactobacillus parabuchneri CZ23).

[0008] Furthermore, the viable count of Lactobacillus brantae - like CZ23 in the bacterial agent is (1.04 ± 0.43)×10 Lentilactobacillus parabuchneri 9 .

[0009] Furthermore, the acid - resistant pH is 3.24 - 3.25.

[0010] The preparation method of the above - mentioned bacterial agent is as follows: Inoculate Lactobacillus brantae - like CZ23 into MRS medium with a tomato juice content of 50 mL / L, and culture at 37°C for 24 h; inoculate the obtained bacterial liquid into the fermentation medium according to an inoculation amount of 3 - 5% (volume ratio), and place it in a 37°C incubator for 24 h to obtain the Lactobacillus brantae - like Lentilactobacillus parabuchneri CZ23 bacterial agent; MRS medium with a tomato juice content of 50 mL / L: 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 ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 20 g calcium carbonate, 1000 mL distilled water, 50 mL tomato juice; Fermentation medium: 15.00 g / L glucose, 10.00 g / L lactose, 25.00 g / L soy peptone, 2.29 g / L K2HPO4, 0.80 g / L MgSO4·7H2O.

[0011] Furthermore, the application of Lactobacillus brantae - like CZ23 or the bacterial agent in food production.

[0012] Compared with the prior art, the beneficial effects of the present invention are: The fermentation broth of Lactobacillus brantae - like CZ23 has the characteristic of inhibiting the growth of harmful microorganisms, can regulate the hurdle factors, and the pH value of the solution is 3.24 - 3.25. Lactobacillus brantae - like CZ23 can survive and reproduce in an environment with a relatively low pH value, has good acid resistance and a high survival amount. Description of the Drawings

[0013] Figure 1 It is the plate colony morphology diagram of Lactobacillus brantae - like CZ23; Figure 2 It is the Gram - staining microscope diagram of Lactobacillus brantae - like CZ23; Figure 3 It is the phylogenetic tree constructed from the 16S rDNA sequence of Lactobacillus brantae - like CZ23;​ Figure 4 It is the bacteriostatic circle effect diagram produced by Lactobacillus brantae - like CZ23 in Escherichia coli; Figure 5 It is the bacteriostatic circle effect diagram produced by Lactobacillus brantae - like CZ23 in Bacillus subtilis; Figure 6 It is the bacteriostatic circle effect diagram produced by Lactobacillus brantae - like CZ23 in Staphylococcus aureus; Figure 7 It is the comparison diagram of the bacteriostatic effect in pre - packaged spicy diced chicken with lactic acid solution added with Lactobacillus brantae - like CZ23. Specific implementation mode

[0014] Next, in combination with the accompanying drawings in the embodiments of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments in the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0015] Please refer to Figures 1 - 7 , the present invention provides a Lactobacillus brantae - like CZ23, which is isolated from pickled vegetables. The collection time is May 2024, and the collection place is Zunyi City, Guizhou Province, China. Lactobacillus brantae - like ( Lentilactobacillus parabuchneri ) CZ23 was deposited at the General Microbiological Center of the China National Culture Collection of Microorganisms on September 18, 2024. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and its deposit number is: CGMCC NO.31986.

[0016] Example 1: Isolation, purification and identification of Lactobacillus brantae - like CZ23 1. Isolation and purification of strains Lactobacillus brantae - like CZ23 is isolated from pickled vegetables. The isolation and purification method is: take 0.1 mL of pickled vegetable juice diluted to 10 -4 and evenly coat it on the 2% calcium carbonate MRS liquid medium containing nystatin, and culture it at 37 °C for 48 h.

[0017] MRS liquid medium: 10 g of peptone, 5 g of beef powder, 4 g of yeast powder, 2 g of glucose, 1 mL of Tween 80, 2 g of dipotassium hydrogen phosphate, 5 g of sodium acetate, 2 g of ammonium citrate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of calcium carbonate, 15 g of agar powder, 1000 mL of distilled water.

[0018] Preparation method of MRS liquid medium: Add the above components to distilled water, heat and dissolve, adjust the pH to 6.2, dispense and sterilize at 121 °C for 15 minutes.

[0019] Specific operation: Inoculate the dilution into 2% calcium carbonate MRS liquid medium containing nystatin, shake while cooling to mix the calcium carbonate evenly, taking care not to generate bubbles. After mixing, mix the MRS liquid medium containing 2% calcium carbonate with an appropriate amount of agar, heat until the agar is completely dissolved, and then immediately pour it into a petri dish and let it stand to solidify to obtain the MRS solid medium. Under sterile conditions, pipette 100 μL of the sample and spread it on the surface of the solidified MRS solid medium plate, and incubate it upside down at 37 °C for 48 h.

[0020] After culturing for 48 h, observe the colony characteristics, pick a single colony with a large calcium dissolution zone, streak-culture it on the MRS solid medium to obtain a single colony strain, streak-separate it on the plate, and purify it repeatedly to obtain a purified single bacterium. The colony morphology is as shown in the appendix Figure 1 as follows.

[0021] 2. Preliminary identification of colonies Hydrogen peroxide catalase test: Use an inoculation loop to pick 1 loop of the isolated and purified colony onto a clean glass slide, add 1 drop of 30% hydrogen peroxide. If there are bubbles, it is catalase positive; otherwise, it is negative.

[0022] Gram staining: Pick the isolated and purified colony for Gram staining and observe it under a microscope. The results of the hydrogen peroxide catalase test and Gram staining of the lactic acid bacteria after isolation and purification are as shown in the following table and Figure 2 as follows: Number Catalase (±) Gram stain (G±) Microscopic examination result CZ23 - G+ Rod-shaped, existing singly or in pairs Conclusion: As can be seen from the above table and the appendix Figure 2 it can be seen that the strain is negative for hydrogen peroxide catalase, positive for Gram staining, and rod-shaped. This strain is labeled as CZ23.

[0023] 3. Physiological and biochemical tests Based on "Bergey's Manual of Systematic Bacteriology" and "Classification, Identification and Experimental Methods of Lactic Acid Bacteria", conduct physiological and biochemical identification of the strain. A positive reaction result is indicated by (+), and a negative result is indicated by (-), so as to make a preliminary identification of the genus and species of the target strain. The results are shown in the following table: Test item CZ23 Sorbitol - Mannitol + Arabinose + Lactose - Cellobiose - Raffinose - Trehalose + Ribose + Melezitose + Conclusion: As can be seen from the above table, CZ23 belongs to the genus Lactobacillus buchneri.

[0024] 4. 16S rDNA sequence analysis The genomic DNA of the target strain was extracted using a bacterial genomic DNA extraction kit. Using the genomic DNA of the target strain as a template, 27F and 1492R were selected as universal primers for PCR amplification of 16S rDNA. PCR reaction system (25 μL): 1 μL each of the 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 min, denaturation at 94°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, for a total of 30 cycles, and finally extension at 72°C for 7 min to end the reaction. The PCR product was sent to BGI Tech Solutions Co., Ltd. for purification and sequencing. The obtained PCR product sequence and the 16S rDNA sequence of closely related lactic acid bacteria were used to construct a phylogenetic tree through MEGA 5.05 software. The phylogenetic tree is shown in Figure 3 the appendix.

[0025] The sequencing results were as follows: GTGGGGGACTGACGTACTGAGCTGAGGCTCGAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAAGAGGTGGAGGGTTTCCGCCCTTCATTGCTGAAGTTAACGCATTAAGCACTCCGCCTGGGAAGAACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCGCCCAAGCGGGGAAGCTTGGGGTTTAATTCAATGCTACGCGAAAAACCTTACCGGGTCTTGACTTCTTCTGCCAACCTAAAAAATTAGGGGTTCCCTTCGGAAACAGAATGACAGGGGTGGCATGGTTGTCGCCAGCTCGGGTCGGGAGAGGTTGGGTTAAGCCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCAGGATTCGGTTGGGCACTCTAACAAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACGGTACAACGAGACGCGAAACCGCGAGAGCAAGCTAATCTCTTAAAGCCGGTCTCAGTTCGGATTGTAGGCTGCAACTCGACTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTGTGTAACACCCAAAGCCGGTGAGGTAACCTTCGGGGGACCAGCCGTCTAATTTGGGACAGATGACTGGGGGTGAAGCGCAA Based on the sequencing results, the target strain CZ23 was identified as Lactobacillus delbrueckii subsp. Lentilactobacillus parabuchneri .

[0026] Example 2. Changes in pH during the fermentation process of Lactobacillus delbrueckii subsp. CZ23 Inoculate Lactobacillus brenneri CZ23 into MRS medium with 50 mL / L tomato juice content and culture at 37°C for 24 h; inoculate the cultured bacterial liquid into the fermentation medium at an inoculation amount of 3 - 5% (volume ratio) and culture in a 37°C incubator for 24 h. Use a pH meter to measure the pH after inoculation until the pH remains stable.

[0027] MRS medium with 50 mL / L tomato juice content: 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 ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 20 g calcium carbonate, 1000 mL distilled water, 50 mL tomato juice.

[0028] Fermentation medium: 15.00 g / L glucose, 10.00 g / L lactose, 25.00 g / L soy peptone, 2.29 g / L K2HPO4, 0.80 g / L MgSO4·7H2O.

[0029] Preparation method of MRS medium with 50 mL / L tomato juice content and fermentation medium: Add the formula components to distilled water according to the ratio, heat and dissolve, and autoclave at 121°C for 15 minutes.

[0030] The pH change after inoculating the medium is shown in the following table: Time (h) pH (with 50 ml / L tomato juice added) 0 6.14 12 3.65 18 3.25 24 3.25 30 3.24 Conclusion: Measuring the pH change during the culture process can reflect the fermentation status of lactic acid bacteria. Lactobacillus brenneri CZ23 can lower the pH value of the fermentation broth during growth, and the pH value continuously decreases with the prolongation of fermentation time. The pH drops rapidly at the beginning and then slowly drops and tends to level off after 18 h. It can also be seen from this experiment that Lactobacillus brenneri CZ23 can survive and reproduce in an environment with a relatively low pH value, reflecting the acid resistance of Lactobacillus brenneri CZ23 of the present invention.

[0031] Example 3. Determination of the viable count in the fermentation broth of Lactobacillus brenneri CZ23 at different culture times Take three batches of fermentation broth fermented for 18 h, 24 h, and 30 h respectively, and use the plate spreading method or the pouring method to determine the viable count. Prepare MRS agar medium, autoclave at 121°C for 15 min, and after the sterilization is completed, wait for the medium to cool to 48°C and then pour the plates. Pour about 15 - 20 mL / plate of MRS medium into the petri dishes. Take samples with a tomato juice addition of 50 mL / L for gradient dilution. Use a pipette to aspirate 1 mL of the bacterial liquid into a test tube containing 9 mL of sterile normal saline, and dilute the sample to 10 -1 , and repeat the operation to dilute to 10 -7 . Use a pipette to aspirate 200 μl of 10-5 , 10 -6 , 10 -7 Dilute the bacterial suspension onto the solidified MRS agar medium, spread it evenly with a sterile spreading stick, and incubate it upside down in a constant temperature incubator at 37°C for 48 hours before counting.

[0032] The number of viable bacteria in the fermentation broth of Lactobacillus lentus CZ23 at different culture times is shown in the following table:

[0033] Conclusion: As shown in the table above, the number of live bacteria of Lactobacillus lentus CZ23 changes little between 24h and 30h of fermentation, and 24h is selected as the optimal fermentation time of Lactobacillus lentus CZ23.

[0034] Example 4: Antibacterial effect of Lactobacillus lentus CZ23 The Oxford cup double-layer agar diffusion method was used to determine bacteriocins: the fermentation liquid of the tested Lactobacillus lentus was centrifuged at 4000r / min for 3min, and the supernatant was taken. Escherichia coli, Bacillus subtilis and Staphylococcus aureus were used as indicator bacteria, and 100μL of the supernatant was placed in an Oxford cup with a diameter of 8.0mm. The cup was cultured in a 37℃ incubator for 24-48h, the inhibition zone was observed, and the diameter of the inhibition zone was measured and recorded. The results are shown in the following table and attached. Figures 4 - 6 As shown: Indicator bacteria Inhibition zone diameter (mm) Escherichia coli 12.45±0.35 Bacillus subtilis 13.55±0.45 Staphylococcus aureus 13.27±0.27 Conclusion: Based on the above table and attached Figures 4 - 6 As shown in the figure, Lactobacillus lentus CZ23 inhibits the growth of Escherichia coli, Bacillus subtilis and Staphylococcus aureus, among which the best inhibitory effect is on Bacillus subtilis, with the diameter of the inhibition zone reaching 13.55mm; the second best inhibitory effect is on Staphylococcus aureus, with an average inhibition zone diameter of 13.27mm; the average inhibition zone diameter is 12.45mm when Escherichia coli is used as an indicator bacteria. Lactobacillus lentus CZ23 has different degrees of inhibitory effect on common pathogens.

[0035] Example 5: Effect of adding different amounts of fermentation liquid on the quality of pre-packaged spicy chicken The activated Lactobacillus brenneri - like CZ23 was inoculated at an inoculation amount of 3% by mass ratio and fermented at 37 °C for 24 h to obtain a fermentation broth. The comprehensive sensory quality evaluation was used to test the effect of the fermentation broth with different amounts of Lactobacillus brenneri - like CZ23 added on the quality of soft pre - packaged spicy diced chicken. The groups were divided into: blank group (without adding any preservatives or fresh - keeping agents), experimental group 1 (the fermentation broth and the sample were added in a ratio of 2:100), experimental group 2 (the fermentation broth and the sample were added in a ratio of 4:100), experimental group 3 (the fermentation broth and the sample were added in a ratio of 8:100), experimental group 4 (the fermentation broth and the sample were added in a ratio of 16:100), and the above ratios were all by mass ratio. Among them, the comprehensive sensory quality evaluation included 4 evaluation indexes: taste, flavor, aroma, and color. Each index was evaluated on a scale of "1 - 10", and the higher the score, the better the quality. The specific evaluation results are shown in the following table.

[0036] Group Taste (score) Flavor (score) Aroma (score) Color (score) Average score (score) Blank group 8 9 8 8 8.25±0.50 Test group 1 8 7 8 8 7.75±0.50 Test group 2 8 7 8 8 7.75±0.50 Test group 3 8 7 7 8 7.50±0.58 Test group 4 4 5 5 4 4.50±0.58

[0037] Conclusion: As can be seen from the above table, with the increase in the addition amount of the fermentation broth, the sensory quality of the pre - packaged spicy diced chicken decreases. To ensure the quality of the pre - packaged spicy diced chicken to the greatest extent, the addition ratio of the fermentation broth to the sample does not exceed 8:100.

[0038] Example 6. Effect of adding fermentation broth on the shelf life of pre - packaged spicy diced chicken The activated Lactobacillus brenneri - like CZ23 was inoculated at an inoculation amount of 3% by mass ratio and fermented at 37 °C for 24 h to obtain a fermentation broth. The antibacterial, bactericidal, antiseptic, and fresh - keeping effects of the fermentation broth with Lactobacillus brenneri - like CZ23 added in soft pre - packaged spicy diced chicken were tested.

[0039] Experimental conditions: From August 15, 2024 to October 17, 2024 in Weifang, Shandong, at a normal temperature of 18 °C - 38 °C throughout the day.

[0040] Experimental groups: conventional preservative group (adding nisin, with an addition amount of 0.5 g / kg), experimental group (the fermentation broth and the sample were added in a ratio of 8:100).

[0041] The conventional preservative group and the experimental group were each subjected to 5 parallel tests. The appearance of the soft pre - packaged spicy diced chicken was detected every 7 days. Staphylococcus aureus was detected according to GB 4789.10, Salmonella was detected according to GB 4789.4, and Escherichia coli was detected according to GB 4789.3. The two samples were compared, as shown in the following table and Figure 7 as follows.

[0042] Group Day 7 Day 14 Day 21 Day 28 Day 35 Day 42 Conventional preservative group Normal Only Staphylococcus aureus detected Only Staphylococcus aureus detected, with browning and swelling Only Staphylococcus aureus detected, with browning and juice leakage Only Staphylococcus aureus detected, with browning and juice leakage Only Staphylococcus aureus detected, with browning and black water leakage Test group Normal Normal Normal Normal Normal Staphylococcus aureus detected

[0043] In summary, the experimental group adding the fermentation broth of Lactobacillus brenis CZ23 has significantly better effect in inhibiting Staphylococcus aureus than the conventional preservative group, and significantly extends the shelf life of spicy diced chicken by up to 5 times.

[0044] The above are only the preferred embodiments of the present invention, and are not limitations on the present invention in other forms. Any person skilled in the art may use the technical content disclosed above to make changes or modifications into equivalent embodiments with equivalent changes. However, any simple modifications, equivalent changes and modifications made to the above examples based on the technical essence of the present invention without departing from the technical solution content of the present invention still belong to the protection scope of the technical solution of the present invention.

Claims

1. Lactobacillus delbrueckii subsp. lactis CZ23, characterized in that: The Lactobacillus brevis - like bacterium ( Lentilactobacillus parabuchneri ) CZ23 was deposited at the China General Microbiological Culture Collection Center on September 18, 2024, with the deposit number: CGMCC NO. 31986; The Lactobacillus delbrueckii subsp. bulgaricus-like Lentilactobacillus parabuchneri CZ23 has the DNA sequence of Sequence Listing SEQ ID NO.

1.

2. A bacterial agent, characterized in that: The bacterial agent contains Lactobacillus brenisoides Lentilactobacillus parabuchneri CZ23 as described in claim 1.

3. A microbial agent according to claim 2, characterized in that: The bacterial agent contains Lactobacillus brantae-like Lentilactobacillus parabuchneri) The viable count of CZ23 is (1.04±0.43)×10 9 .

4. The microbial agent according to claim 2, wherein: The acid tolerance pH is 3.24 - 3.

25.

5. The microbial agent according to claim 2, wherein: The bacterial agent is prepared according to the following method: Lactobacillus brevis - like strain CZ23 was inoculated into MRS medium containing 50 mL / L of tomato juice and cultured at 37°C for 24 h; the cultured bacterial solution was inoculated into the fermentation medium at an inoculum size of 3 - 5% (volume ratio) and cultured in an incubator at 37°C for 24 h to obtain the Lactobacillus brevis - like strain ( Lentilactobacillus parabuchneri ) CZ23 bacterial agent; MRS medium with 50 mL / L tomato juice: 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 ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 20 g calcium carbonate, 1000 mL distilled water, 50 mL tomato juice; Fermentation medium: 15.00 g / L glucose, 10.00 g / L lactose, 25.00 g / L soy peptone, 2.29 g / L K2HPO4, 0.80 g / L MgSO4·7H2O.

6. Use of Lentilactobacillus parabuchneri CZ23 according to claim 1 or the bacterial agent according to claim 2 in food production.

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