Lactobacillus brevis and application thereof

Abstract

The invention discloses lactobacillus brevis and application thereof, and belongs to the technical field of food microorganisms. The invention provides a lactobacillus brevis CHOL1, which is separated from pickled Chinese cabbages, can be applied to food through safety evaluation, has relatively strong nitrite degradation capability, can react with myoglobin to generate nitrosylmyoglobin, and can be used for degrading nitrite in food. After being inoculated into the fermented meat product, the strain can reduce nitrite and nitrosamine residues in the fermented meat product and improve the color and luster of the meat product at the same time. As a leavening agent, the strain can improve the quality and safety of fermented meat products, and can be used as a natural effective substitute for partially replacing nitrite in the fermented meat products.

Description

Technical Field

[0001] The invention relates to a strain of Lactobacillus brevis CHOL1 and application thereof in fermented meat products, belonging to the technical field of food microorganisms. Background Art

[0002] Nitrite is a substance commonly found in fermented meat products. Its sources are mainly from three aspects: on the one hand, it comes from nitrite food additives added to meat products; on the other hand, it is generated by nitrate impurities in salt during pickling under the reduction of microorganisms; in addition, nitrite can be formed by microbial metabolism and digestion of Nitrosomonas from proteins in meat products. Although nitrite has the functions of fixing color, improving texture, inhibiting the growth of foodborne pathogens and anti-oxidation in meat products, it can react with amines as a precursor to form highly carcinogenic nitrosamine compounds, and the large accumulation of nitrosamines in the body can cause great harm to human health. Therefore, it is very important to seek methods that can replace the function of nitrite and reduce the residual nitrite and nitrosamines in meat products to improve the safety of fermented meat products.

[0003] Studies have confirmed that lactic acid bacteria have the ability to promote the color of myoglobin and reduce nitrite and nitrosamine. Its application in fermented sausages is expected to partially replace the function of nitrite and reduce the amount of nitrite used; its application in fermented fish products can reduce nitrite and nitrosamine residues. At present, studies have screened out lactic acid bacteria that can promote the color of myoglobin or degrade nitrite. For example, Li et al. screened out a fermented lactobacillus that can convert myoglobin into nitrosomyoglobin and promote the color of meat products; Wang et al. found that inoculation of sake lactobacillus can degrade nitrite in meat products. However, a lactic acid bacteria strain has not yet been screened out that not only has a strong nitrite degradation ability, but also can promote the color of myoglobin and improve the color of meat products. In addition, as a strong carcinogen nitrosamine commonly present in meat, reducing its residue in meat products is crucial to human life and health. However, no study has reported that lactic acid bacteria fermentation can achieve the triple effect of degrading nitrite, promoting the color of meat products, and reducing nitrosamine residues in meat products. For fermented fish, especially mandarin fish, no studies have reported that lactic acid bacteria alone can degrade nitrite and nitrosamines at the same time. Therefore, we aimed to screen a lactic acid bacterium with outstanding color development ability and strong nitrite degradation ability, and apply it to fermented meat products such as fermented sausages and fermented fish, which can not only promote the color development of meat products and degrade nitrite residues, but also reduce nitrosamines in meat products, thereby improving the quality and safety of fermented meat products. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a strain of Lactobacillus brevis and its application, aiming to solve the technical problem in the prior art that lactic acid bacteria as a fermentation agent cannot simultaneously degrade nitrite and nitrosamine residues and improve the color of meat products.

[0005] The first technical solution provided by the present invention is a strain of Lactobacillus brevis (Levilactobacillus brevis) CHOL1, which has been deposited in the Guangdong Microbiological Culture Collection Center with a deposit number of GDMCC NO.64656 and a deposit date of May 20, 2024. The deposit address is Guangdong Microbiological Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0006] The Lactobacillus brevis CHOL1 grows well in the MRS solid culture medium and can generate round, smooth-surfaced, milky yellow colonies after being cultured at 37° C. for 24 hours.

[0007] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus brevis CHOL1 described in the first technical solution.

[0008] In certain embodiments, the microbial preparation is a preparation in solid or liquid form, and may be a lyophilized powder, a fermentation broth, or the like.

[0009] In certain embodiments, in the microbial preparation, the amount of Lactobacillus brevis CHOL1 added is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL.

[0010] Furthermore, in the microbial preparation, the amount of Lactobacillus brevis CHOL1 added is not less than 1×10 7 CFU / g or 1×10 7 CFU / mL.

[0011] The third technical solution provided by the present invention is a starter culture, wherein the starter culture contains the Lactobacillus brevis CHOL1 described in the first technical solution or the microbial preparation described in the second technical solution.

[0012] In certain embodiments, other additives or microbial preparations may be added to the fermentation agent.

[0013] In some embodiments, the amount of Lactobacillus brevis CHOL1 added to the starter is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL.

[0014] Furthermore, the amount of Lactobacillus brevis CHOL1 added to the starter is not less than 1×10 7CFU / g or 1×10 7 CFU / mL.

[0015] In certain embodiments, the preparation method of the starter is specifically as follows: a single colony of Lactobacillus brevis CHOL1 is picked and inoculated into 10 mL of MRS liquid culture medium, and cultured at 37° C., 200-250 rpm for 24 hours to obtain a bacterial solution A; then 3 mL of bacterial solution A is inoculated into 150 mL of MRS liquid culture medium, and cultured at 37° C., 200-250 rpm for 24 hours to obtain a bacterial solution B, the bacterial solution B is centrifuged at 8000 rpm / min for 5 minutes, and the bacterial bodies are collected, and the Lactobacillus brevis CHOL1 bacterial bodies are diluted with a sodium chloride aqueous solution with a mass fraction of 0.9% to prepare a bacterial solution.

[0016] The fourth technical solution provided by the present invention is the use of Lactobacillus brevis CHOL1 described in the first technical solution, or the microbial preparation described in the second technical solution, or the starter described in the third technical solution in the preparation of fermented meat products.

[0017] In certain embodiments, the fermented meat products include but are not limited to fermented sausages, fermented fish (especially mandarin fish), etc.

[0018] The fifth technical solution provided by the present invention is the role of Lactobacillus brevis CHOL1 described in the first technical solution, or the microbial preparation described in the second technical solution, or the fermentation agent described in the third technical solution in degrading nitrite and / or nitrosamine.

[0019] The sixth technical solution provided by the present invention is a method for reducing nitrite and / or nitrosamines in fermented meat products, which method comprises introducing the Lactobacillus brevis CHOL1 described in the first technical solution, or the preparation described in the second technical solution, or the starter described in the third technical solution into the fermentation system of the fermented meat product.

[0020] In certain embodiments, in the fermentation system, the concentration of Lactobacillus brevis CHOL1 is 1×10 6 ~1×10 8 CFU / g.

[0021] In certain embodiments, the fermented meat products include but are not limited to fermented sausages, fermented fish (especially mandarin fish), etc.

[0022] In some embodiments, the preparation of the fermented sausage comprises the following steps:

[0023] S1. Freshly slaughtered lean meat and fat meat are minced in a meat grinder with a 1.5 cm aperture sieve plate, and the mixture is in the ratio of lean meat to fat meat = 9:1-7:3;

[0024] S2, taking the total mass of the lean meat in step S1 as 100%, adding 1.5-2.5wt% salt, 0.2%-0.5wt% monosodium glutamate, 0.5%-1.5wt% liquor, 1.0-4.0wt% sugar, 1.0-1.5wt% soy sauce, 0.5-1.5wt% chili powder, 0.01wt% sodium nitrite, and inoculating the Lactobacillus brevis CHOL1 described in the first technical solution, the preparation described in the second technical solution or the starter described in the third technical solution; stirring evenly, marinating at room temperature for 30 minutes, and then enema;

[0025] S3. The sausage obtained in step S2 is fermented at 10-15° C. for 10-12 days to obtain air-dried sausage.

[0026] In certain embodiments, the preparation of the fermented fish meat comprises the following steps:

[0027] S1. Remove the internal organs, scales and gills of fresh mandarin fish;

[0028] S2, weigh drinking water of the same weight as the mandarin fish obtained in step S1, pour it into a container, and add 3wt% salt, 1wt% green onion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili, 0.01wt% Zanthoxylum bungeanum, and inoculate the Lactobacillus brevis CHOL1 described in the first technical solution, the microbial preparation described in the second technical solution, or the starter described in the third technical solution;

[0029] S3, immersing the mandarin fish obtained in step S1 in the fermentation liquid, compacting the fish body with stones, and fermenting at 12° C. for 5 days.

[0030] In certain embodiments, the preparation of the fermented fish meat comprises the following steps:

[0031] S1, remove the viscera, scales, gills, etc. of fresh mandarin fish, drain and weigh;

[0032] S2, preparation of auxiliary materials: according to the weight of fish body, weigh 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% pepper, 0.002% chili powder, 0.6% fresh ginger, 1% green onion, and add Lactobacillus brevis CHOL1 described in the first technical solution, the microbial preparation described in the second technical solution or the starter described in the third technical solution;

[0033] S3, spread the auxiliary materials evenly on the inner and outer surfaces of the mandarin fish, place it in a sealed container, and ferment it at 12°C for 7 days.

[0034] Compared with the existing method, the present invention has the following beneficial effects:

[0035] The present invention provides a Lactobacillus brevis CHOL1 strain, which is isolated from sauerkraut and can be applied to food after safety evaluation. The strain has a strong nitrite degradation ability and can also react with myoglobin to generate nitrosylmyoglobin. It is inoculated into air-dried sausage and found that it can reduce the nitrite and nitrosamine residues in the air-dried sausage and improve the color of meat products. Inoculation into smelly mandarin fish can reduce the nitrite and nitrosamine residues. The strain can be used as a fermentation agent to improve the quality and safety of fermented meat products and can be used as a natural and effective substitute for partially replacing nitrite in fermented meat products.

[0036] Biomaterial Deposit

[0037] The Lactobacillus brevis CHOL1 strain is classified and named Levilactobacillus brevis, and has been deposited in the Guangdong Provincial Microbiological Culture Collection Center with a deposit number of GDMCC NO.64656, a deposit date of May 20, 2024, and a deposit address of 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is a photo of the MRS solid medium culture of Lactobacillus brevis CHOL1;

[0039] Figure 2 This is the color result diagram of the myoglobin simulation system inoculated with Lactobacillus brevis. DETAILED DESCRIPTION

[0040] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0041] The culture media used in the present invention are all prepared by conventional methods. If the specific experimental conditions and methods are not specified for the molecular biology operations involved in the examples, refer to Sambrook J et al., ed., Science Press, 2002, Molecular Cloning Experiment Guide (3rd Edition); or refer to the product instructions.

[0042] The culture medium involved in the following examples is as follows:

[0043] MRS liquid culture medium: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7±0.2.

[0044] MRS solid medium: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5±0.2.

[0045] YPD solid medium: peptone 10.0 g / L, glucose 20.0 g / L, yeast extract powder 5.0 g / L, agar 14.0 g / L.

[0046] YPD liquid medium: peptone 20.0 g / L, glucose 20.0 g / L, yeast extract powder 10.0 g / L, pH 6.5±0.2.

[0047] In the following comparative examples, Lactobacillus plantarum CICC 20242, CICC 22205, CICC 20264, CICC 20038, CICC 20659, CICC 22220, Pediococcus pentosaceus CICC 22227, CICC 21862, Lactobacillus brevis CICC 6139, CICC24450, CICC 20297, CICC 20269, Leuconostoc mesenteroides CICC 21725, CICC 21861, Lactobacillus acidophilus CICC6255, CICC 20248, CICC 20244, Streptococcus thermophilus CICC 20364, CICC 25032, CICC 20367, and Staphylococcus equi CICC 10435 were purchased from China Industrial Microbiological Culture Collection Administration Center; Lactobacillus plantarum ATCC 8014, Pediococcus pentosaceus ATCC 33316, Staphylococcus fish fermentation ATCC 51136, ATCC 51137, Staphylococcus equi ATCC 43958, Staphylococcus xylosus ATCC 29971, 21145 were purchased from Jitai Biotechnology; Saccharomyces cerevisiae CGMCC 2.1364, 2.148 were purchased from Guangdong Microbiological Culture Collection Center.

[0048] Example 1: Isolation and identification of Levilactobacillus brevis CHOL1.

[0049] (1) Collection of strains: isolated from homemade sauerkraut.

[0050] (2) Identification of strain CHOL1:

[0051] The genome of strain CHOL1 was extracted for 16s rDNA identification, and the genome extraction method was carried out according to the glass bead method in the "Concise Molecular Biology Experiment Guide".

[0052] PCR conditions were as follows:

[0053] The amplification system is: 2×Taq Master Mix 25μL, Primer 27F, 2μL, Primer 1492R, 2μL, Sterile Water 19μL, Template 2μL. PCR reaction conditions are: 95℃ pre-denaturation for 3min; 95℃ denaturation for 15s; 55℃ annealing for 15s; 72℃ extension for 1min; 72℃ for 5min; 30 cycles; 4℃ infinity. After the PCR is completed, agarose gel (1.0%) electrophoresis is performed to detect the PCR product. The genome with bright bands is the genome that has been successfully amplified by PCR, and the successful genome can be sent for sequencing.

[0054] The 16s rDNA sequence of the strain CHOL1 is shown in SEQ ID NO:1.

[0055] The sequencing results were subjected to Blast n analysis, and it was found that the bacteria had the highest homology with Levilactobacillus brevis, reaching 99.93%. Therefore, the strain was identified as Levilactobacillus brevis and named as Lactobacillus brevis CHOL1. The Lactobacillus brevis CHOL1 grew well in MRS solid medium and was cultured at 37°C for 24 hours. Figure 1 As shown, the colonies formed by Lactobacillus brevis are round, smooth, and milky yellow in color.

[0056] Based on morphological and 16s rDNA identification, the strain CHOL1 was tentatively identified as Levilactobacillus brevis and was deposited in Guangdong Microbiological Culture Collection Center on May 20, 2024, with the deposit number: GDMCCNO.64656. The deposit address is: Guangdong Microbiological Culture Collection Center, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province.

[0057] Example 2 Safety evaluation of Lactobacillus brevis CHOL1.

[0058] (1) Pathogenicity experiment of Lactobacillus brevis CHOL1

[0059] The pathogenicity experiment of the strain was carried out with reference to the provisions of the "Technical Guidelines for Safety Inspection and Evaluation of Bacterial Species Used in Health Food Ingredients (2020 Edition)", and 10 mice were selected for each group of experiments.

[0060] Table 1 Results of pathogenicity experiment of Lactobacillus brevis CHOL1 (oral administration)

[0061]

[0062]

[0063] Note: Lowercase letters in the table indicate significant differences in male mice, and uppercase letters indicate significant differences in female mice.

[0064] From the experimental results, it can be seen that there is no significant difference between the experimental group and the control group, so it can be determined that the strain is not pathogenic.

[0065] (2) Drug resistance evaluation experiment of Lactobacillus brevis CHOL1:

[0066] The drug resistance evaluation method of Lactobacillus brevis CHOL1 and the critical values ​​of each antibiotic refer to the "Guidance on the assessment of bacterial susceptibility to antimicrobials of human and veterinary importance" issued by the European Food Safety Authority in 2012. The minimum inhibitory concentration (MIC) of each antibiotic was determined by the continuous two-fold dilution method using the activated Lactobacillus brevis CHOL1 as the indicator bacteria. The drug resistance of the strain was evaluated by comparing the MIC with the critical value. When the minimum inhibitory concentration is equal to or less than the established critical value, the strain is considered to be sensitive to the antibiotic; when the minimum inhibitory concentration is greater than the established critical value, the strain is considered to be resistant to the antibiotic.

[0067] Table 2 Results of antibiotic sensitivity test of Lactobacillus brevis CHOL1

[0068]

[0069] The results showed that Lactobacillus brevis CHOL1 was sensitive to ampicillin, vancomycin, gentamicin, kanamycin, streptomycin, erythromycin, tetracycline, chloramphenicol and clindamycin antibiotics, indicating that the strain had good safety.

[0070] (3) Hemolysis experiment of Lactobacillus brevis CHOL1

[0071] Culture medium: Columbia agar, 5% defibrinated sheep blood.

[0072] Take Lactobacillus brevis CHOL1 (order of magnitude about 10 8 CFU / mL) were streaked on Columbia plates containing 5% defibrinated sheep blood, and the hemolysis around the colonies was observed after culturing at 37°C for 24 hours. Staphylococcus aureus ATCC 25923 was used as a positive control.

[0073] The results showed that a hemolytic ring of about 6-8 mm, with clear boundaries and completely transparent, appeared around Staphylococcus aureus ATCC 25923, which was typical β-hemolysis. However, no hemolytic ring appeared around the colony of Lactobacillus brevis CHOL1 provided by the present invention, indicating that the strain was non-hemolytic and was a safe strain.

[0074] Example 3: The method for determining the nitrite degradation ability of Lactobacillus brevis CHOL1 is as follows:

[0075] (1) Draw the sodium nitrite standard curve: Pipette 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL, 1.50mL, 2.00mL, and 2.50mL of sodium nitrite standard solution (5μg / mL) and place them in 15mL centrifuge tubes respectively. Add 2mL of 4g / L p-aminobenzenesulfonic acid solution to the tube, mix well, let stand for 3-5min, then add 1mL of 2g / L naphthaleneethylenediamine hydrochloride solution, add water to 10mL, mix well, and let stand for 15min. Measure the absorbance at a wavelength of 538nm and draw the standard curve. Simultaneously make a reagent blank.

[0076] (2) Determination of nitrite degradation ability of Lactobacillus brevis CHOL1: Take frozen Lactobacillus brevis CHOL1 and culture it on MRS solid medium at 37°C for 1 day, pick a single colony from the plate, inoculate it into MRS liquid medium and culture it for 1 day, then inoculate it into MRS liquid medium containing 100μg / mL sodium nitrite with an initial OD of 1.5 and an inoculum of 1% for culture, and culture it at 37°C for 36h. Take the cultured bacterial liquid and centrifuge it at 10000rpm / min for 5min, take 1mL of the bacterial liquid supernatant in a 15ml centrifuge tube, add 2mL of p-aminobenzenesulfonic acid, let it stand for 5min, then add 1mL of hydrochloric acid solution of naphthylethylenediamine, add 6mL of deionized water, mix well and let it stand for 15min, measure the absorbance value at a wavelength of 538nm, and convert the nitrite content according to the standard curve. The blank control group is MRS liquid medium with 100ug / mL sodium nitrite added. The calculation formula of nitrite degradation rate is as follows:

[0077]

[0078] The units of the initial nitrite content and the nitrite content after cultivation in the formula are both μg / mL.

[0079] It was determined that the degradation rate of nitrite by Lactobacillus brevis in MRS liquid culture medium containing sodium nitrite was 99.03%, indicating that Lactobacillus brevis CHOL1 had a strong nitrite degradation ability.

[0080] Example 4: The method for determining the color development ability of Lactobacillus brevis CHOL1 is as follows:

[0081] The myoglobin stock solution (20 mg / mL) was placed in a 50℃ water bath for 30 minutes, and the myoglobin stock solution after the water bath was centrifuged (10000g, 5min, 4℃), and the supernatant was filtered through a 0.22μm filter membrane and added to the MRS liquid culture medium to prepare a simulation system with a myoglobin concentration of 2.0 mg / mL. 40μL of bacterial solution was inoculated into it and anaerobically cultured at 37℃ for 18h, and the color was measured after centrifugation (10000g, 5min, 4℃). The MRS broth system containing myoglobin with 0.015% sodium nitrite was used as a positive control, and the MRS liquid culture medium system containing myoglobin was used as a blank group.

[0082] The results of the color development ability test of Lactobacillus brevis CHOL1 are shown in the attached Figure 2 As shown, group 1 is CHOL1 in Lactobacillus brevis, group Y is the positive control group, and group C is the blank group. After being cultured in the MRS liquid culture medium system containing myoglobin, the a* / b* value of Lactobacillus brevis was 1.42±0.08, the a* / b* value of the positive control group with sodium nitrite added was 1.20±0.20 after culture, and the a* / b* value of the blank group was 0.41±0.03. This shows that Lactobacillus brevis can convert myoglobin into a red myoglobin derivative, and even achieve a color development ability similar to that of sodium nitrite.

[0083] Example 5: Lactobacillus brevis CHOL1 was isolated at 7×10 6 CFU / g were inoculated into air-dried intestines.

[0084] S1: Freshly slaughtered pork lean meat and fat were minced in a meat grinder with a 1.5 cm aperture sieve plate and mixed at a ratio of lean meat to fat = 9:1.

[0085] S2: Bacterial liquid preparation:

[0086] A single colony of Lactobacillus brevis CHOL1 was selected and inoculated into 10 mL of MRS liquid culture medium, and cultured at 37°C, 200-250 rpm for 24 h to obtain bacterial solution A; then 3 mL of bacterial solution A was inoculated into 150 mL of MRS liquid culture medium, and cultured at 37°C, 200-250 rpm for 24 h to obtain bacterial solution B, and the bacterial solution B was centrifuged at 8000 rpm / min for 5 min to collect the bacterial cells, and the Lactobacillus brevis CHOL1 bacterial cells were diluted to a mass fraction of 10 with a sodium chloride aqueous solution with a mass fraction of 0.9%. 6 CFU / mL bacterial solution.

[0087] S3: Based on the total weight of fat and lean meat as 100%, 1.5wt% salt, 0.5wt% monosodium glutamate, 1.5wt% liquor, 1.0wt% sugar, 1.5wt% soy sauce, 0.5wt% chili powder, 0.01wt% sodium nitrite, and Lactobacillus brevis CHOL1 bacterial solution were added; wherein the Lactobacillus brevis CHOL1 bacterial solution was 7×106 CFU / g were inoculated into the meat, stirred evenly and marinated at room temperature for 30 min before enema.

[0088] S4: Fermentation: The sausages prepared in S3 are fermented at 15° C. for 12 days to obtain air-dried sausages. The product is named Example 5.

[0089] Example 6: Lactobacillus brevis CHOL1 was isolated at 5×10 7 CFU / g were inoculated into air-dried intestines.

[0090] Specific implementation method Except for the step of "diluting the Lactobacillus brevis CHOL1 cells with a sodium chloride aqueous solution having a mass fraction of 0.9% to a value of 10 7 CFU / mL bacterial solution”, S3 “Lactobacillus brevis CHOL1 bacterial solution at 5×10 7 CFU / g inoculated into meat” is different from Example 5, and the rest is carried out according to the implementation method of Example 5.

[0091] Example 7: Lactobacillus brevis CHOL1 was isolated at 3×10 8 CFU / g inoculated into air-dried intestine

[0092] Specific implementation method Except for the step of "diluting the Lactobacillus brevis CHOL1 cells with a sodium chloride aqueous solution having a mass fraction of 0.9% to a value of 10 8 CFU / mL bacterial solution”, S3 “Lactobacillus brevis CHOL1 bacterial solution at 3×10 8 CFU / g inoculated into meat” is different from Example 5, and the rest is carried out according to the implementation method of Example 5.

[0093] Example 8: Lactobacillus brevis CHOL1 was isolated at 7×10 6 CFU / mL inoculated in wet pickled smelly mandarin fish

[0094] S1: Fresh mandarin fish without viscera, scales, gills, etc.

[0095] S2: Bacterial liquid preparation

[0096] A single colony of Lactobacillus brevis CHOL1 was selected and inoculated into 10 mL of MRS liquid culture medium, and cultured at 37°C, 200-250 rpm for 24 h to obtain bacterial solution A; then 3 mL of bacterial solution A was inoculated into 150 mL of MRS liquid culture medium, and cultured at 37°C, 200-250 rpm for 24 h to obtain bacterial solution B, and the bacterial solution B was centrifuged at 8000 rpm / min for 5 min to collect the bacterial cells, and the Lactobacillus brevis CHOL1 bacterial cells were diluted to 10 with a sodium chloride aqueous solution with a mass fraction of 0.9%. 7 CFU / mL bacterial solution.

[0097] S3: Preparation of fermentation broth: Weigh drinking water of the same weight as the mandarin fish obtained in step S1, pour it into a container, and add 3wt% salt, 1wt% green onion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili, 0.01wt% prickly ash, and Lactobacillus brevis CHOL1 bacterial solution, based on the weight of the drinking water as 100%; the concentration of Lactobacillus brevis CHOL1 in the fermentation broth is 7×10 6 CFU / mL.

[0098] S4: Fermentation: The mandarin fish obtained in step S1 is immersed in the fermentation liquid, the fish body is compacted with stones, and fermented at 12° C. for 5 days. The product is named Example 8.

[0099] Example 9: Lactobacillus brevis CHOL1 was isolated at 4×10 7 CFU / mL inoculated in wet pickled smelly mandarin fish

[0100] Specific implementation method Except for the step of "diluting the Lactobacillus brevis CHOL1 cells with a 0.9% sodium chloride aqueous solution to 10 8 CFU / mL bacterial solution” and the concentration of Lactobacillus brevis CHOL1 in the fermentation broth in S3 was 4×10 7 CFU / mL” is different from Example 8, and the rest are carried out according to the implementation method of Example 8.

[0101] Example 10: Lactobacillus brevis CHOL1 was isolated at 7×10 6 CFU / mL inoculated in dry salted smelly mandarin fish

[0102] S1: Remove the viscera, scales, gills, etc. of fresh mandarin fish, drain and weigh.

[0103] S2: Same as Example 8S2.

[0104] S3: Preparation of auxiliary materials: according to the weight of fish, weigh 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% pepper, 0.002% chili powder, 0.6% fresh ginger, 1% onion, and Lactobacillus brevis CHOL1 bacterial solution. The concentration of Lactobacillus brevis CHOL1 is 7×10 6 CFU / g.

[0105] S4: The auxiliary materials were evenly applied to the inner and outer surfaces of the mandarin fish, placed in a sealed container, and fermented at 12° C. for 7 days. The product was named Example 10.

[0106] Example 11: Lactobacillus brevis CHOL1 was isolated at 4×10 7 CFU / mL inoculated in dry salted smelly mandarin fish

[0107] S1: Remove the viscera, scales, gills, etc. of fresh mandarin fish, drain and weigh.

[0108] S2: The bacterial solution was prepared in the same manner as in Example 8S1.

[0109] S3: Preparation of auxiliary materials: According to the weight of fish, weigh 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% pepper, 0.002% chili powder, 0.6% fresh ginger, 1% onion, and Lactobacillus brevis CHOL1 bacterial solution. The concentration of Lactobacillus brevis CHOL1 is 4×10 7 CFU / g.

[0110] S4: The auxiliary materials were evenly spread on the inner and outer surfaces of the mandarin fish, placed in a sealed container, and fermented at 12° C. for 7 days. The product was named Example 11.

[0111] Comparative Example 1: Add 0.01% sodium nitrite to naturally ferment and air-dry sausage.

[0112] S1: Freshly slaughtered pork lean meat and fat were minced in a meat grinder with a 1.5 cm aperture sieve plate and mixed at a ratio of lean meat to fat = 9:1.

[0113] S2: Based on the total weight of fat and lean meat as 100%, add 1.5wt% salt, 0.5wt% monosodium glutamate, 1.5wt% liquor, 1.0wt% sugar, 1.5wt% soy sauce, 0.5wt% chili powder, and 0.01wt% sodium nitrite, stir evenly, marinate at room temperature for 30 minutes, and then perform enema.

[0114] S3: Fermentation: The sausages prepared in S2 are fermented at 15° C. for 12 days to obtain air-dried sausages. The product is named Comparative Example 1.

[0115] Comparative Example 2: Add 0.015% sodium nitrite to naturally ferment and air-dry the sausage.

[0116] Specific implementation method is the same as comparative example 1, wherein the added amount of sodium nitrite is 0.015% of the total mass of fat and lean meat.

[0117] Comparative Example 3: Wet Natural Fermentation of Smelly Mandarin Fish

[0118] S1. Remove the internal organs, scales and gills of fresh mandarin fish.

[0119] S2. Prepare fermentation liquid: weigh drinking water of the same mass as that of the mandarin fish obtained in step S1, and add 3wt% salt, 1wt% green onion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili, and 0.01wt% Sichuan pepper, taking the mass of the drinking water as 100%.

[0120] S3: Fermentation: The mandarin fish obtained in S1 was immersed in the fermentation liquid obtained in S2, the fish body was compacted with stones, and fermented at 12° C. for 5 days. The obtained product was named Comparative Example 3.

[0121] Comparative Example 4: Dry method natural fermentation of smelly mandarin fish

[0122] S1: Remove the viscera, scales, gills, etc. of fresh mandarin fish, drain and weigh.

[0123] S2: Preparation of auxiliary materials: According to the weight of fish body, weigh 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% pepper, 0.002% chili powder, 0.6% fresh ginger, and 1% onion.

[0124] S3: The auxiliary materials were evenly spread on the inner and outer surfaces of the mandarin fish, placed in a sealed container, and fermented at 12° C. for 7 days. The product was named Comparative Example 4.

[0125] Comparative Example 5: A microbial composite preparation of Lactobacillus plantarum CICC 20242, Pediococcus pentosaceus CICC 22227, and Lactobacillus brevis CICC6139 was added and inoculated into air-dried intestines.

[0126] S1: Same as Example 5S1.

[0127] S2: Bacterial liquid preparation:

[0128] Single colonies of Lactobacillus plantarum CICC 20242, Pediococcus pentosaceus CICC 22227, and Lactobacillus brevis CICC 6139 were picked up and inoculated into 10 mL of MRS liquid culture medium, and cultured at 37° C., 200-250 rpm for 24 h to obtain bacterial liquids A1, A2, and A3; then 3 mL of bacterial liquids A1, A2, and A3 were respectively inoculated into 150 mL of MRS liquid culture medium, and cultured at 37° C., 200-250 rpm for 24 h to obtain bacterial liquids B1, B2, and B3, and the bacterial liquids B1, B2, and B3 were centrifuged at 8000 rpm / min for 5 min, and then the bacterial bodies were collected, and the bacterial bodies of B1, B2, and B3 were diluted to 10 with a sodium chloride aqueous solution with a mass fraction of 0.9%. 6 CFU / mL bacterial solution.

[0129] S3: Preparation of fermentation broth: Lactobacillus plantarum CICC 20242, Pediococcus pentosaceus CICC 22227 and Lactobacillus brevis CICC6139 were mixed at a ratio of 1:1:1 to prepare fermentation broth. The final concentration of the fermentation broth was 10 6 CFU / mL.

[0130] S4: Based on the total weight of fat and lean meat as 100%, 1.5wt% salt, 0.5wt% monosodium glutamate, 1.5wt% liquor, 1.0wt% sugar, 1.5wt% soy sauce, 0.5wt% chili powder, and 0.01wt% sodium nitrite were added to mix the fermentation liquid; wherein the mixed fermentation liquid is the fermentation liquid prepared in S3, which is 7×10 6 CFU / g were inoculated into the meat, stirred evenly and marinated at room temperature for 30 min before enema.

[0131] S5: Fermentation: The sausages prepared in S4 are fermented at 15° C. for 12 days to obtain air-dried sausages. The product is named Comparative Example 5.

[0132] Comparative Example 6: Lactobacillus plantarum CICC 22205, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteroides CICC 21725, Lactobacillus acidophilus CICC 6255, and Lactobacillus brevis CICC 24450 were added in a volume ratio of 4:4:2:3:4 to prepare a mixed starter culture, which was then inoculated into air-dried intestines.

[0133] S1: Same as Example 5S1.

[0134] S2: Bacterial liquid preparation:

[0135] Single colonies of Lactobacillus plantarum CICC 22205, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteroides CICC21725, Lactobacillus acidophilus CICC 6255, and Lactobacillus brevis CICC 24450 were picked respectively to prepare bacterial solutions B1, B2, B3, B4, and B5 according to the implementation of S2 in Comparative Example 5.

[0136] S3: Preparation of fermentation broth: Lactobacillus plantarum CICC 22025, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteroides CICC 21725, Lactobacillus acidophilus CICC 6255, and Lactobacillus brevis CICC 24450 were mixed in a ratio of 4:4:2:3:4 to prepare a fermentation broth. The final concentration of the fermentation broth was 10 6 CFU / mL.

[0137] The implementation methods of S4 and S5 are the same as those of Comparative Example 5 S4 、 S5.

[0138] Comparative Example 7: Lactobacillus plantarum CICC 20264, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteroides CICC 21725, Lactobacillus acidophilus CICC 20248, and Lactobacillus brevis CICC20297 were added in a volume ratio of 4:4:2:3:4 to prepare a mixed starter, which was then inoculated into air-dried intestines.

[0139] S1: Same as Example 5S1.

[0140] S2: Bacterial liquid preparation:

[0141] Single colonies of Lactobacillus plantarum CICC 20264, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteroides CICC21725, Lactobacillus acidophilus CICC 20248, and Lactobacillus brevis CICC 20297 were picked respectively to prepare bacterial solutions B1, B2, B3, B4, and B5 according to the implementation of S2 in Comparative Example 5.

[0142] S3: Preparation of fermentation broth: Lactobacillus plantarum CICC 20264, Pediococcus pentosaceus CICC 21862, Leuconostoc mesenteroides CICC 21725, Lactobacillus acidophilus CICC 20248, and Lactobacillus brevis CICC 20297 were mixed in a ratio of 4:4:2:3:4 to prepare a fermentation broth. The final concentration of the fermentation broth was 10 6 CFU / mL.

[0143] The implementation methods of S4 and S5 are the same as those of Comparative Examples 5S4 and S5.

[0144] Comparative Example 8: Lactobacillus plantarum ATCC 8014, Pediococcus pentosaceus ATCC 33316, Leuconostoc mesenteroides CICC21861, Lactobacillus acidophilus CICC 20244, and Lactobacillus brevis CICC 20269 were added in a volume ratio of 4:4:2:3:4 to prepare a mixed starter, which was then inoculated into air-dried intestines.

[0145] S1: Same as Example 5S1

[0146] S2: Bacterial liquid preparation:

[0147] Single colonies of Lactobacillus plantarum ATCC 8014, Pediococcus pentosaceus ATCC 33316, Leuconostoc mesenteroides CICC21861, Lactobacillus acidophilus CICC 20244, and Lactobacillus brevis CICC 20269 were picked respectively to prepare bacterial solutions B1, B2, B3, B4, and B5 according to the implementation of S2 in Comparative Example 5.

[0148] S3: Fermentation broth preparation: Lactobacillus plantarum ATCC 8014, Pediococcus pentosaceus ATCC 33316, Leuconostoc mesenteroides CICC 21861, Lactobacillus acidophilus CICC 20244, and Lactobacillus brevis CICC 20269 were mixed at a ratio of 4:4:2:3:4 to prepare fermentation broth. The final concentration of the fermentation broth was 10 6 CFU / mL.

[0149] The implementation methods of S4 and S5 are the same as those of Comparative Examples 5S4 and S5.

[0150] Comparative Example 9: Streptococcus thermophilus CICC 20364 was added and inoculated into the air-dried intestines.

[0151] Except that the inoculated strain is Streptococcus thermophilus CICC 20364, the rest of the implementation is the same as that of Example 5.

[0152] Comparative Example 10: Streptococcus thermophilus CICC 25032 was added and inoculated into the air-dried intestines.

[0153] Except that the inoculated strain is Streptococcus thermophilus CICC 25032, the rest of the implementation is the same as that of Example 5.

[0154] Comparative Example 11: Streptococcus thermophilus CICC 20367 was added and inoculated into the air-dried intestine.

[0155] Except that the inoculated strain is Streptococcus thermophilus CICC 20367, the rest of the implementation method is the same as that of Example 5.

[0156] Comparative Example 12: Fish fermentation Staphylococcus aureus ATCC 51136 was added and inoculated into air-dried sausage.

[0157] Except that the inoculated strain is Staphylococcus aureus ATCC 51136, the remaining implementation methods are the same as those in Example 5.

[0158] Comparative Example 13: Fish fermentation Staphylococcus aureus ATCC 51137 was added and inoculated into the air-dried sausage.

[0159] Except that the inoculated strain is Staphylococcus aureus ATCC 51137, the remaining implementation methods are the same as those in Example 5.

[0160] Comparative Example 14: Staphylococcus equi ATCC 43958 was added and inoculated into the air-dried intestines.

[0161] Except that the inoculated strain is Staphylococcus equi ATCC 43958, the rest of the implementation is the same as that of Example 5.

[0162] Comparative Example 15: Staphylococcus equi CICC 10435 was added and inoculated into the air-dried intestines.

[0163] Except that the inoculated strain is Staphylococcus equi CICC 10435, the rest of the implementation is the same as that of Example 5.

[0164] Comparative Example 16: Lactobacillus plantarum CICC 20038 was added and inoculated into the air-dried sausage.

[0165] Except that the inoculated strain is Lactobacillus plantarum CICC 20038, the rest of the implementation is the same as that of Example 5.

[0166] Comparative Example 17: Lactobacillus plantarum CICC 20242 was added and inoculated into the air-dried sausage.

[0167] Except that the inoculated strain is Lactobacillus plantarum CICC 20242, the rest of the implementation is the same as that of Example 5.

[0168] Comparative Example 18: Lactobacillus plantarum CICC 20659 was added and inoculated into the air-dried sausage.

[0169] Except that the inoculated strain is Lactobacillus plantarum CICC 20659, the rest of the implementation is the same as that of Example 5.

[0170] Comparative Example 19: Inoculate Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylosus ATCC29971 in equal proportions into wet-pickled smelly mandarin fish

[0171] S1: Fresh mandarin fish without viscera, scales, gills, etc.

[0172] S2: Bacterial liquid preparation

[0173] First, bacterial cells are prepared, and the bacterial cell preparation method is as follows: single colonies of Lactobacillus plantarum CICC 22220 and Staphylococcus xylosus ATCC 29971 are respectively picked up and inoculated into 10 mL of MRS liquid culture medium, and cultured at 37° C. and 200-250 rpm for 24 hours to obtain bacterial liquids A1 and A2; then 3 mL of bacterial liquids A1 and A2 are inoculated into 150 mL of MRS liquid culture medium, and cultured at 37° C. and 200-250 rpm for 24 hours to obtain bacterial liquids B1 and B2, the bacterial liquids B1 and B2 are centrifuged at 8000 rpm / min for 5 minutes, and then the bacterial cells are collected, and the bacterial cells of Lactobacillus plantarum CICC 22220 and Staphylococcus xylosus ATCC 29971 are diluted to 10% by mass with a sodium chloride aqueous solution with a mass fraction of 0.9%; 7 CFU / mL bacterial solution.

[0174] The preparation method of the yeast CGMCC 2.1364 cell is as follows: a single colony of the yeast CGMCC 2.1364 is picked up and inoculated into 10 mL of YPD solid culture medium, and cultured at 30° C. and 200-250 rpm for 24 h to obtain a bacterial solution A3; then 3 mL of the bacterial solution A3 is inoculated into 150 mL of YPD liquid culture medium, and cultured at 30° C. and 200-250 rpm for 24 h to obtain a bacterial solution B3, the bacterial solution B3 is centrifuged at 8000 rpm / min for 5 min, and the bacterial cells are collected, and the yeast CGMCC 2.1364 cell is diluted to 10 with a sodium chloride aqueous solution having a mass fraction of 0.9% 7 CFU / mL bacterial solution.

[0175] S3: Preparation of fermentation liquid: Weigh drinking water of the same weight as the mandarin fish obtained in step S1, pour it into a container, and add 3wt% salt, 1wt% green onion, 0.6wt% ginger, 0.1wt% star anise, 0.05wt% fennel, 0.05wt% cumin, 0.01wt% chili, 0.01wt% prickly ash, Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC2.1364, Staphylococcus xylosus ATCC 29971 bacterial liquid in an equal volume ratio of 7×10 6 CFU / mL.

[0176] S4: Fermentation: The mandarin fish obtained in step S1 is immersed in the fermentation liquid, the fish body is compacted with stones, and fermented at 12° C. for 5 days. The product is named Comparative Example 19.

[0177] Comparative Example 20: Inoculate Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylosus CICC21145 in equal proportions into wet-pickled smelly mandarin fish

[0178] S1: Fresh mandarin fish without viscera, scales, gills, etc.

[0179] S2: Bacterial liquid preparation

[0180] The preparation method of the bacterial solution of Lactobacillus plantarum CICC 20242 and Staphylococcus xylosus CICC 21145 refers to the preparation method of the bacterial solution of Lactobacillus plantarum CICC 22220 and Staphylococcus xylosus ATCC 29971 in Comparative Example 19S2.

[0181] The preparation method of the yeast culture medium of Saccharomyces cerevisiae CGMCC 2.1364 refers to the preparation method of the yeast culture medium of Saccharomyces cerevisiae CGMCC 2.1364 in Comparative Example 19S2.

[0182] S3: Preparation of fermentation broth: The preparation method of fermentation broth of Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylosus CICC 21145 refers to the preparation method of fermentation broth of Comparative Example 19 S3.

[0183] S4 is the same as the implementation method of comparison example 19S4.

[0184] Comparative Example 21: Inoculate Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylosus ATCC29971 in equal proportions into dry-salted smelly mandarin fish

[0185] S1: Fresh mandarin fish without viscera, scales, gills, etc.

[0186] S2: Preparation of composite bacterial solution

[0187] First, bacterial cells are prepared, and the bacterial cell preparation method is as follows: single colonies of Lactobacillus plantarum CICC 22220 and Staphylococcus xylosus ATCC 29971 are respectively picked up and inoculated into 10 mL of MRS liquid culture medium, and cultured at 37° C. and 200-250 rpm for 24 hours to obtain bacterial liquids A1 and A2; then 3 mL of bacterial liquids A1 and A2 are inoculated into 150 mL of MRS liquid culture medium, and cultured at 37° C. and 200-250 rpm for 24 hours to obtain bacterial liquids B1 and B2, the bacterial liquids B1 and B2 are centrifuged at 8000 rpm / min for 5 minutes, and then the bacterial cells are collected, and the bacterial cells of Lactobacillus plantarum CICC 22220 and Staphylococcus xylosus ATCC 29971 are diluted to 10% by mass with a sodium chloride aqueous solution with a mass fraction of 0.9%; 7 CFU / mL bacterial solution.

[0188] The preparation method of the yeast CGMCC 2.1364 cell is as follows: a single colony of the yeast CGMCC 2.1364 is picked up and inoculated into 10 mL of YPD solid culture medium, and cultured at 30° C. and 200-250 rpm for 24 h to obtain a bacterial solution A3; then 3 mL of the bacterial solution A3 is inoculated into 150 mL of YPD liquid culture medium, and cultured at 30° C. and 200-250 rpm for 24 h to obtain a bacterial solution B3, the bacterial solution B3 is centrifuged at 8000 rpm / min for 5 min, and the bacterial cells are collected, and the yeast CGMCC 2.1364 cell is diluted to 10 with a sodium chloride aqueous solution having a mass fraction of 0.9% 7 CFU / mL bacterial solution.

[0189] The bacterial liquids of Lactobacillus plantarum CICC 22220, Saccharomyces cerevisiae CGMCC 2.1364, and Staphylococcus xylosus ATCC 29971 were mixed in equal volume ratio to prepare 7×10 6 CFU / mL of composite bacterial solution.

[0190] S3: Preparation of auxiliary materials: according to the weight of fish, weigh 6% salt, 0.02% fennel, 0.04% cumin, 0.06% star anise, 0.03% pepper, 0.002% chili powder, 0.6% fresh ginger, 1% onion, and composite bacterial solution; the amount of bacterial solution added is 7×10 6 CFU / g.

[0191] S4: The auxiliary materials were evenly spread on the inner and outer surfaces of the mandarin fish, placed in a sealed container, and fermented at 12° C. for 7 days. The product was named Comparative Example 21.

[0192] Comparative Example 22: Inoculate Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylosus CICC21145 in equal proportions into dry-salted smelly mandarin fish

[0193] S1: Fresh mandarin fish without viscera, scales, gills, etc.

[0194] S2: Bacterial liquid preparation

[0195] The preparation method of the bacterial solution of Lactobacillus plantarum CICC 20242 and Staphylococcus xylosus CICC 21145 refers to the preparation method of the bacterial solution of Lactobacillus plantarum CICC 22220 and Staphylococcus xylosus ATCC 29971 in Comparative Example 21S2.

[0196] The preparation method of the yeast culture medium of Saccharomyces cerevisiae CGMCC 2.1364 refers to the preparation method of the yeast culture medium of Saccharomyces cerevisiae CGMCC 2.1364 in Comparative Example 21S2.

[0197] The bacterial liquids of Lactobacillus plantarum CICC 20242, Saccharomyces cerevisiae CGMCC 2.148, and Staphylococcus xylosus ATCC 29971 were mixed in equal volume ratio to prepare 7×10 6 CFU / mL of composite bacterial solution.

[0198] S3, S4: Same as Comparative Example 21S3, S4.

[0199] Test Case

[0200] The embodiments of the present invention and the comparative examples were tested:

[0201] 1. Determination of nitrite residue

[0202] To determine the residual nitrite in the examples and comparative examples, the spectrophotometric method was used according to GB 5009.33-2016 "National Food Safety Standard Determination of Nitrite and Nitrate in Food". The specific method is as follows:

[0203] (1) Nitrite extraction: Weigh 5 g (accurate to 0.001 g) of homogenized sample (if water is added during the preparation process, it should be converted according to the amount of water added), place it in a 250 mL stoppered conical flask, add 12.5 mL of 50 g / L saturated borax solution, add about 150 mL of water at about 70°C, mix well, heat in a boiling water bath for 15 min, take out and cool in a cold water bath, and let it stand at room temperature. Quantitatively transfer the above extract to a 200 mL volumetric flask, add 5 mL of 106 g / L potassium ferrocyanide solution, shake well, and then add 5 mL of 220 g / L zinc acetate solution to precipitate protein. Add water to the scale, shake well, let it stand for 30 min, remove the upper fat layer, filter the supernatant with filter paper, discard 30 mL of the initial filtrate, and set aside the filtrate.

[0204] (2) Determination of nitrite: Pipette 40.0mL of the above filtrate into a 50mL stoppered colorimetric tube, and plute 0.00mL, 0.20mL, 0.40mL, 0.60mL, 0.80mL, 1.00mL, 1.50mL, 2.00mL, and 2.50mL of sodium nitrite standard solution (5μg / mL) into 50mL stoppered colorimetric tubes, respectively. Add 2mL of 4g / L p-aminobenzenesulfonic acid solution into the standard tube and sample tube, mix well, let stand for 3-5min, then add 1mL of 2g / L naphthaleneethylenediamine hydrochloride solution into each tube, add water to the scale, mix well, let stand for 15min, use a 1cm colorimetric cup, adjust the zero point with a zero tube, measure the absorbance at a wavelength of 538nm, and draw a standard curve for comparison. Simultaneously make a reagent blank.

[0205] The residual amounts of nitrite in the examples and comparative examples are shown in Table 3. From the results, it can be seen that for air-dried sausages, the residual amounts of nitrite in Examples 5, 6, and 7 are lower, with the lowest being 1.78 mg / kg, and the nitrite in the stinky mandarin fish inoculated with Lactobacillus brevis is reduced to undetectable. This is attributed to the high nitrite degradation ability of Lactobacillus brevis CHOL1, and its inoculation in fermented meat products can effectively reduce the residual nitrite.

[0206] Table 3 Sodium nitrite residue in air-dried intestines (mg / kg)

[0207]

[0208]

[0209] 2. Determination of Nitrosamine Residues

[0210] According to the results of the determination of nitrite residue, the experimental group that can degrade nitrite was screened to further determine its nitrosamine residue. The specific determination method is as follows:

[0211] Sample pretreatment: weigh 10g of homogenized sample, add 10uL NDPA-d14 internal standard (2μg / L), add 15mL acetonitrile, vortex mix, freeze in a -20℃ refrigerator for 30min, add 4g magnesium sulfate and 1g sodium chloride, oscillate for 30s, and centrifuge at 8000rpm / min at 4℃ for 10min; take 6mL of supernatant and add it to a 15mL centrifuge tube containing 50mg PSA, 150mg C18E and 900mg anhydrous sodium sulfate, vortex thoroughly for 1min, centrifuge at 4℃ 8000rpm / min for 10min, take 5mL of supernatant, concentrate to 1mL by nitrogen blowing, pass through 0.22um organic filter membrane, and wait for detection.

[0212] GC parameters: carrier gas is helium; constant flow rate: 1.0 mL / min; temperature program: hold at 40 °C for 0.5 min, increase to 115 °C at a rate of 6 °C / min, hold for 1 min, then increase to 150 °C at a rate of 15 °C / min, hold for 0.5 min, then increase to 180 °C at a rate of 2 °C / min, hold for 2 min; solvent delay: 8.5 min;

[0213] MS parameters: ionization mode: EI; ion source temperature: 240°C; injection port temperature: 240°C; quadrupole temperature: 150°C; interface temperature: 240°C; detection mode: MRM; gain factor: 1.

[0214] The results of the determination of the residual nitrosamines in the embodiment and the comparative example are shown in Table 4. It can be seen from the table that the residual amount of each nitrosamine and the total amount of nitrosamines in the air-dried intestines after adding Lactobacillus brevis CHOL1 are lower than those in the comparative example, indicating that Lactobacillus brevis CHOL1 can effectively reduce the residual nitrosamines in the air-dried intestines. Compared with the comparative example, the inoculation of Lactobacillus brevis significantly reduced the residual amount of nitrosamines in the smelly mandarin fish. Therefore, the use of Lactobacillus brevis CHOL1 to inoculate fermented meat products can better reduce the residual nitrosamines in the fermented meat.

[0215] Table 4 Residual nitrosamines in air-dried intestines (μg / kg)

[0216]

[0217]

[0218] 3. Chroma Measurement

[0219] According to the results of the determination of nitrosamine residues, the group that can reduce nitrosamine residues was screened and its color was further determined. The specific determination method is as follows:

[0220] The color of the air-dried sausage and smelly mandarin fish was measured using an UltraScan Pro colorimeter. The instrument was set to reflectance mode and calibrated using a white standard plate. The a* / b* value was used as the color index for evaluating the examples and comparative examples. Each sample was measured 6 times in parallel to calculate the average value.

[0221] Table 5 shows the chromaticity values ​​of the embodiments and comparative examples. In practical applications, the a* / b* value can more accurately reflect the results of human naked eye observation. The larger the a* / b* value, the brighter the product color. From the chromaticity results, it can be seen that Lactobacillus brevis significantly improves the a* / b* value of air-dried sausage, which is attributed to the excellent color development ability of Lactobacillus brevis and can be used as a natural bacterial agent to partially replace nitrite. Inoculation with Lactobacillus brevis has no significant effect on the color of smelly mandarin fish, which may be because its myoglobin content is low and Lactobacillus brevis has no obvious effect on it.

[0222] Table 5 Chromaticity value (a* / b*)

[0223]

[0224]

[0225] Combined with the above results, compared with existing studies, the Lactobacillus brevis CHOL1 provided by the present invention can significantly reduce the residual sodium nitrite and nitrosamines in the fermented meat, and improve the safety of the fermented meat products, as a fermentation agent for air-dried sausages, smelly mandarin fish, etc. In addition, the excellent color development ability of Lactobacillus brevis CHOL1 helps to improve the color of fermented sausages and improve product quality.

[0226] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Lactobacillus brevis CHOL1, characterized in that: It was deposited in Guangdong Provincial Microbiological Culture Collection Center on May 20, 2024, with the deposit number GDMCC NO.64656.

2. A microbial preparation containing the Lactobacillus brevis CHOL1 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that In the microbial preparation, the amount of Lactobacillus brevis CHOL1 added is not less than 1×10 6 CFU / g or 1×10 6 CFU / mL.

4. A leavening agent, characterized in that The fermentation agent contains the Lactobacillus brevis CHOL1 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. Use of the Lactobacillus brevis CHOL1 according to claim 1, or the microbial preparation according to claim 2 or 3, or the starter according to claim 4 in the preparation of fermented meat products.

6. The use according to claim 5, characterized in that: The fermented meat products include fermented sausages and fermented fish.

7. The effect of Lactobacillus brevis CHOL1 according to claim 1, or the microbial preparation according to claim 2 or 3, or the fermentation agent according to claim 4 in degrading nitrite and / or nitrosamine.

8. A method for reducing nitrite and / or nitrosamine in fermented meat products, characterized in that: The method comprises introducing the Lactobacillus brevis CHOL1 described in claim 1, or the microbial preparation described in claim 2 or 3, or the starter described in claim 4 into a fermentation system of a fermented meat product.

9. The method according to claim 8, characterized in that In the fermentation system, the concentration of Lactobacillus brevis CHOL1 is 1×10 6 ~1×10 8 CFU / g.

10. The method according to claim 8, characterized in that The fermented meat products include fermented sausages and fermented fish.

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