Low-temperature meat-derived lactic acid bacteria and application thereof in matured beef production

Abstract

The application belongs to the technical field of food biological processing, and discloses a low-temperature meat-derived lactic acid bacteria and application thereof in mature beef production. The strain is Leuconostoc mesenteroides, which was preserved in the Guangdong Microbial Culture Collection Center on March 23, 2022, and the preservation number is GDMCC No 62311. The strain plays a very good application in the production of mature beef, inhibits the growth of spoilage bacteria and the increase of volatile basic nitrogen in a low-temperature environment, helps to reduce the generation of a spoiled crust of mature beef, and can improve the tenderness of mature beef and give the beef a unique flavor. Moreover, the production cost of the strain is low, the activity maintenance time of the strain is long, the performance of the strain is stable, the application of the strain is simple, the strain is safe for humans and animals, and the strain has a good application prospect in the processing and production of mature beef.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of food biological processing, and particularly relates to a low-temperature meat-derived lactic acid bacteria and application thereof in matured beef production. BACKGROUND

[0002] Matured beef is a kind of beef that is placed in a cold storage room with controlled temperature, humidity and wind speed for 21-45 days for maturation. During this period, the juice is absorbed by the meat, and the muscle fibers and fat are decomposed by enzymes, so that the beef is juicy, tender and has rich flavor.

[0003] In recent years, matured beef has been favored by consumers due to its high tenderness and rich flavor. According to reported data, the matured beef market in the United States reached 15 billion US dollars in 2020, and the export volume reached 9 billion US dollars. With the further introduction of technology, the domestic matured beef market has just begun to take shape. However, during the low-temperature maturation process, the proliferation of low-temperature spoilage bacteria on the surface of beef leads to surface spoilage of the beef skin, and only the "meat core" can be eaten after cutting off the spoiled outer shell. The spoiled outer shell cannot be eaten and accounts for 25-30% of the beef. Therefore, it is difficult to produce matured beef in a factory, and it is usually produced in small batches in a maturation cabinet, resulting in high prices of matured beef in the market, which cannot be widely promoted as "popular food". Therefore, how to prevent spoilage of matured beef in a low-temperature environment is one of the problems that need to be solved at present.

[0004] Currently, chemical preservatives and natural preservatives are mainly used for low-temperature meat preservation. Chemical preservatives (such as sodium benzoate and sodium lactate) have good preservative effect, simple operation and other advantages, but there are certain food safety risks, and sodium benzoate and calcium propionate also have an odor.

[0005] The use of natural preservatives mainly involves the application research of plant extracts, chitosan, Nisin, lactic acid bacteria antibacterial peptides, etc. The use of lactic acid bacteria is mostly limited to the use of lactic acid bacteria metabolites to extend the shelf life of low-temperature meat products, but the production cost is high. SUMMARY

[0006] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present application is to provide a low-temperature meat-derived lactic acid bacteria, which is a probiotic lactic acid bacteria derived from matured beef, Leuconostoc mesenteroides.

[0007] Another purpose of the present application is to provide an application of the above-mentioned low-temperature meat-derived lactic acid bacteria in matured beef production. The strain has obvious preservative function for matured beef.

[0008] The purpose of the present application is achieved by the following technical solutions:

[0009] A low-temperature meat-derived lactic acid bacteria, which is Leuconostoc mesenteroides, named SCB2557, was preserved in the Guangdong Microbial Culture Collection Center on March 23, 2022, and the preservation number is GDMCC No 62311.

[0010] The application of the above-mentioned low-temperature meat-derived lactic acid bacteria in the production of mature beef is as follows: inoculate the low-temperature meat-derived lactic acid bacteria into sterilized soy milk or mixed emulsion to culture for 8-48h to obtain a fermentation broth, spray the obtained fermentation broth as a preservative on the surface of beef, and then mature the beef.

[0011] Preferably, the application specifically follows the following steps: inoculate the low-temperature meat-derived lactic acid bacteria into sterilized MRS liquid medium, culture at 30℃ for 48h to obtain a seed culture solution; then inoculate the seed culture solution into pasteurized soy milk or mixed emulsion medium at an inoculation amount of 1×10 5 cfu / mL~5×10 7 cfu / mL, culture at 30℃ for 8-48h to obtain a fermentation broth, spray the fermentation broth as a preservative on the beef by a spraying method to form a thin film on the surface of the beef, and then place the beef in a maturation refrigerator with a temperature of 2-6℃ and a humidity of 75-85% for maturation. During the maturation period, the temperature and humidity of the refrigerator are controlled by using a four-stage variable temperature and humidity method.

[0012] Preferably, the four-stage variable temperature and humidity method includes the following steps: in the first stage, a relatively high temperature and high humidity environment is used to make the low-temperature meat-derived lactic acid bacteria rapidly grow and reproduce on the surface of the beef; in the second stage, a relatively low temperature environment is used to stabilize the bacterial community structure on the surface of the beef; in the third stage, which is a flavor forming stage, a relatively moderate temperature and humidity environment is used to activate proteases and lipases inside and outside the beef, so as to decompose muscle fibers and fat in the beef, increase the tenderness of the beef, and form rich flavors; in the fourth stage, which is a post-maturation stage, a relatively low temperature and low humidity environment is used to continue the maturation of the beef while inhibiting the growth of spoilage bacteria; the relatively high temperature and high humidity environment has a temperature of 4-6℃ and a humidity of 83-88%; the relatively low temperature environment has a temperature of 2-4℃ and a humidity of 80-85%; the relatively moderate temperature and humidity environment has a temperature of 3-5℃ and a humidity of 77-80%; and the relatively low temperature and low humidity environment has a temperature of 1-3℃ and a humidity of 75-78%.

[0013] Preferably, the formula of the MRS liquid culture medium is: 10.0 g / L of proteose peptone, 5.0 g / L of beef extract powder, 4.0 g / L of yeast extract powder, 20.0 g / L of glucose, 1.0 mL / L of Tween-80, 2.0 g / L of potassium phosphate, 5.0 g / L of sodium acetate, 15.0 g / L of agar, 2.0 g / L of trisodium citrate, 0.2 g / L of magnesium sulfate (MgSO4·7H2O), and 0.05 g / L of manganese sulfate (MnSO4·4H2O), and the sterilization method is 121 DEG C sterilization for 20 min, and the pH is adjusted to 6.2+ / -0.2 when used.

[0014] Preferably, the formula of the soy milk is: 40-110 g / L of sugar-free soybean powder, and 10-80 g / L of white granulated sugar, and the sterilization method is 105 DEG C to 115 DEG C sterilization for 15 min to 20 min.

[0015] Preferably, the formula of the mixed emulsion is: 20-50 g / L of whole milk powder, 20-60 g / L of sugar-free soybean powder, and 10-80 g / L of white granulated sugar, and the sterilization method is 105 DEG C to 115 DEG C sterilization for 15 min to 20 min.

[0016] Preferably, the formula of the mixed emulsion is: 20-50 g / L of whole milk powder, 20-60 g / L of sugar-free soybean powder, and 10-80 g / L of white granulated sugar, and the sterilization method is 105 DEG C to 115 DEG C sterilization for 15 min to 20 min.

[0017] The low-temperature meat-derived lactic acid bacteria strain is separated and screened from the surface of mature beef, the strain can not only grow in a low-temperature environment (4 DEG C), but also has a good antibacterial effect on the dominant spoilage bacteria of mature beef, i.e. Pseudomonas, and the diameter of the inhibition zone of the strain on the indicator bacteria, i.e. Pseudomonas lundensis, is 13.2+ / -1.72 mm, the beef inoculated with the strain can effectively inhibit the production of Pseudomonas on the surface of the beef, and the strain can grow in a low-temperature environment and occupy the ecological niche on the surface of the beef, thereby achieving the purpose of preventing the growth of spoilage bacteria and the spoilage of beef.

[0018] Compared with the prior art, the present application has the following advantages and effects:

[0019] (1) The lactic acid bacteria strain screened from mature beef can grow in a low-temperature environment, and the soy milk (or mixed milk) is used as a culture medium to optimize and promote the growth of lactic acid bacteria; the lactic acid bacteria is sprayed on the surface of beef, and can occupy the ecological niche on the surface of beef through interspecific competition, compress the growth space of spoilage bacteria, and reduce the growth probability of spoilage bacteria on mature beef, thereby regulating the microbial community structure on the surface of mature beef and having a good preservative capacity.

[0020] (2) The application of the strain can inhibit the growth of spoilage bacteria and the increase of volatile basic nitrogen in a low-temperature environment, help to reduce the production of spoilage crust of matured beef, and improve the tenderness and unique flavor of the matured beef. The strain has low production cost, long active retention time, stable performance, simple application, safety to humans and animals, and good application prospect in the processing and production of matured beef. BRIEF DESCRIPTION OF DRAWINGS

[0021] Figure 1 For general matured beef "belong" population structure analysis, beef samples matured for 7d, 14d and 21d are numbered as groups A, B and C respectively;

[0022] Figure 2 For the low-temperature growth curve of the strain SCB2557;

[0023] Figure 3 For the sensory evaluation results of different treatment matured beef;

[0024] Figure 4 Population structure analysis of beef at different matured periods treated with bacteria; DETAILED DESCRIPTION

[0025] The application will be further described in detail below in combination with examples and drawings, but the embodiments of the application are not limited thereto.

[0026] Example 1, isolation and identification of low-temperature meat-derived lactic acid bacterial strain

[0027] I. Isolation

[0028] 1. Sample preparation and lactic acid bacterial isolation

[0029] The sample was taken from the matured beef of Weiran Food (Shenzhen) Co., Ltd., which was obtained by maturing beef at 4℃ and 80% humidity in a cold storage for 21 days. A small amount of white bacterial colonies were found on the surface of the matured beef, which was shiny and moist, and the beef did not stick and had no odor, suspected to be lactic acid bacterial colonies.

[0030] When sampling, the white bacterial colony part of the skin was cut off with scissors, and 25g of the beef sample was dissolved in 225mL of sterile physiological saline to prepare a sample solution. 1mL of the sample solution was taken with a sterile syringe, and was mixed with 9mL of sterile physiological saline to prepare a 10 -1 dilution solution. In succession, the dilution was continued to 10 -4 , 10 -2 , 10 -3 , 10 -4Gradient respectively 1 mL of diluent to the culture dish, and pour 45 ℃ or so sterilization MRS agar medium, mix evenly. 37 ℃ culture 48 h. From the surface of the plate pick up the growth better, individual larger cream-colored colonies, line purification.

[0031] 2. Strain purification preservation

[0032] The inoculation ring is used to pick up the suspected lactic acid bacteria single colony on the surface and bottom of the plate, which is round or shuttle-shaped and cream-colored, and is inoculated on the MRS plate, and is cultured at 37 ℃ for 48 h. After three generations of plate purification, it is stored in a glycerol tube after microscopic examination.

[0033] 3. Strain screening

[0034] 3.1 Preliminary screening

[0035] 3.1.1 Observation of bacterial morphology: after 24 h of culture, single colonies are picked up, gram staining experiment is performed, and phenomena are observed and recorded under an optical microscope.

[0036] 3.1.2 Hydrogen peroxide enzyme test: the colonies on the solid culture medium are picked up with an inoculation ring, 2 mL of 3% hydrogen peroxide solution is added, and the results are observed. If no bubbles are generated, it is negative.

[0037] 3.1.3 Glucose gas production test: a Durham tube is inverted in a test tube containing liquid MRS medium, the strain is inoculated and cultured at 37 ℃ for 24 h, and whether bubbles are generated in the Durham tube is observed.

[0038] 3.1.4 Glucose acid production test: 1.6 g / 100 mL of bromocresol purple is added as an indicator in a test tube containing liquid MRS medium, the addition amount is 1.4 mL / L, the strain is inoculated, and is cultured at 37 ℃ for 24 h. If the culture medium changes from purple to yellow, it proves that acid is produced.

[0039] 3.2 Rescreening

[0040] 3.2.1 Preparation of lactic acid bacteria fermentation broth

[0041] According to the inoculation amount of 1 × 10 6 The activated lactic acid bacteria are inoculated in the MRS liquid medium at an inoculation amount of 1 × 10

[0042] 3.2.2 Preparation of common mature beef surface bacterial population broth

[0043] 3.2.2.1 Analysis of common mature beef surface bacterial population composition

[0044] The samples were taken from the ordinary mature beef (made by traditional process) of Weirun Food (Shenzhen) Co., Ltd. Three sampling points of beef matured for 7d, 14d and 21d were selected respectively and numbered as A, B and C groups. The genus level population composition was analyzed by metagenomics, and the results are shown in Figure 1 Table 1. The dominant spoilage bacteria of mature beef were Pseudomonas, which accounted for 75.3% of the ecological niche of beef surface when matured for 7d. Pseudomonas basically completely occupied the ecological niche of beef surface when matured for 14d and 21d (the proportions were 99.4% and 99.8% respectively).

[0045] 3.2.2.2 Preparation of Pseudomonas liquid

[0046] The indicator strain Pseudomonas lundensis SCB2605 strain (isolated and screened from mature beef and preserved in Fermentation Engineering Laboratory of South China Agricultural University) was cultured on TSB (tryptone soy broth liquid medium) slant medium at 30℃ for 24h, washed with sterile normal saline, and the bacterial concentration was adjusted to 1×10 8 mL.

[0047] 4. Determination of antibacterial activity of strain

[0048] 20mL of TSB medium with a temperature of 45℃ was injected into a culture dish, 20μL of Pseudomonas liquid was added, and after the medium was solidified, a sterile punch with an outer diameter of 7.8mm was used to punch holes on the coated medium, three holes were punched at equal intervals on each plate, 100μL of lactic acid bacteria fermentation liquid was added to the punched holes, and the plate was placed in a 37℃ constant temperature incubator for culture for 24h. The diameter of the inhibition zone was observed and recorded.

[0049] A lactic acid bacteria strain with good antibacterial effect on Pseudomonas lundensis was obtained by screening, which was named SCB2557.

[0050] II. Identification of low-temperature meat-derived lactic acid bacteria strain

[0051] 1. Colony and individual morphology observation

[0052] Bacterial morphology observation: the strain SCB2557 was inoculated in MRS agar medium, cultured for 24h, and single colonies were picked for Gram staining experiment. The phenomenon was observed and recorded under optical microscope. Characteristics: milk white colony, smooth surface, round or bean-shaped, diameter less than 1.0mm. Microscopic observation showed that the cells were spherical, arranged in short chains, non-motile, no spores, Gram-positive and facultative anaerobic.

[0053] 2. 16S rDNA sequence determination

[0054] The template DNA of the strain was extracted by using a genomic extraction kit, and the template solution was added to the PCR system to amplify the 16S rDNA of the strain. The PCR product was sequenced and analyzed by Beijing Genesee Biotechnology Co., Ltd., and the Blast gene sequence comparison was performed on the NCBI website. It is shown that the strain SCB2557 has the highest homology with the mesenteric Candida, and the similarity is 99%. Therefore, according to the results of morphological and 16S rDNA sequence analysis, the strain is mesenteric Candida. The sequence is as follows:

[0055]

[0056] Example 2, Bacteriostatic performance of strain SCB2557

[0057] 20 mL of TSB medium at 45°C was injected into a culture dish, 20 μL of Pseudomonas SCB2605 strain liquid was added, and after the medium was solidified, a sterile punch with an outer diameter of 7.8 mm was used to punch the coated medium, three holes were punched at equal intervals on each plate, 100 μL of strain SCB2557 fermentation broth was inoculated into the punched holes, and the plate was placed in a 37°C constant temperature incubator for 24 h. The diameter of the inhibition zone was observed and recorded. The experiment was repeated 3 times, and the results were averaged.

[0058] The diameter of the inhibition zone was 13.2 ± 1.72 mm.

[0059] Example 3, Growth curve of strain SCB2557 at low temperature and preservative effect on mature beef

[0060] I. Low temperature growth curve

[0061] The activated strain SCB2557 was inoculated in sterile soy milk medium at an inoculation amount of 1 × 10 7 cfu / mL, and cultured at 4°C for 21 days. Samples were taken at 0 (3h), 3d, 7d, 14d, and 21d, and the total number of lactic acid bacteria was determined according to the "GB 4789.2-2016 Food Safety National Standard Method for Determination of Total Number of Colonies". The low temperature curve was determined, and Lactobacillus bulgaricus SCB0693 strain (isolated and screened from Kefir particles (Siberia region, stored in the Fermentation Engineering Laboratory of South China Agricultural University)) was used as a control.

[0062] II. Preservative effect of strain SCB2557 on mature beef

[0063] 1. Preparation of bacterial liquid

[0064] Sterilized soy milk medium preparation: 70 g / L sugar-free soy flour, 30 g / L white sugar, 108°C sterilization for 15 min.

[0065] The activated strain SCB2557 was inoculated in sterile soy milk medium at 30°C for 32h to obtain seed culture liquid; then the seed culture liquid was inoculated in sterile soy milk medium at an inoculation amount of 1 × 10 6 cfu / mL, and cultured at 30°C for 24h to obtain bacterial liquid, which was stored at 4°C for use (within 3 days).

[0066] 2. Preservative experiment of mature beef

[0067] Fresh beef was divided into 6 pieces, each weighing 3 kg, and the size was consistent.

[0068] Table 1 Preservative experiment grouping

[0069]

[0070] The strain SCB2557 group is to spray the above bacteria liquid on the surface of beef, while the control group is not treated as a control. Then put the two groups of meat into the aging refrigerator (1-6℃, humidity 75-85%) for 21 days.

[0071] During the aging period, the refrigerator uses "four-stage" variable temperature and humidity method, the scheme is as follows:

[0072] First stage (0-1d): refrigerator temperature 4-6℃, humidity 83-88%

[0073] Second stage (2-7d): refrigerator temperature 2-4℃, humidity 80-85%

[0074] Second stage (8-11d): refrigerator temperature 3-5℃, humidity 77-80%

[0075] Fourth stage (12-21d): refrigerator temperature 1-3℃, humidity 75-78%

[0076] On the 21st day, the total volatile base nitrogen (TVB-N) content, pH value, sensory evaluation, microbial indicators (total number of colonies, total number of lactic acid bacteria and total number of pseudomonas) were determined, the scheme is as follows.

[0077] pH value: take 10g meat sample, grind thoroughly, then add distilled water without CO2 to 100mL, shake, stand for 30min, then filter the filtrate and measure the pH with pH meter.

[0078] Volatile basic nitrogen content: During the storage of meat samples, basic nitrogen-containing substances such as ammonia, primary amines, secondary amines and tertiary amines generated by protein decomposition can be volatilized in an alkaline solution at 37°C and then collected by an absorbent solution. The volatile basic nitrogen content of the meat sample is determined by referring to the third method for determining volatile basic nitrogen in the GB 5009.228-2016 Food Safety National Standard Food, i.e., the micro-diffusion method. 10 g of the meat sample is ground in a mortar and stirred uniformly, transferred into a 100 mL volumetric flask and diluted with distilled water to 100 mL. The flask is shaken from time to time and immersed for 30 min, and then filtered to obtain a meat sample leachate. A water-based glue is coated on the outer chamber grinding mouth of a Conway dish, 1 mL of boric acid absorbent (20 g / L) and 1 drop of a mixed indicator of methyl red-ethanol indicator (2 g / L) and methylene blue indicator (1 g / L) are added to the inner chamber, 1 mL of the meat sample leachate is added to one side of the outer chamber, a glass cover is placed (the gap is not tightly covered), and then 1 mL of saturated potassium carbonate solution is added to the other side of the outer chamber through the gap in the glass cover. The glass cover is tightly covered, and the outer chamber sample solution and the alkaline solution are mixed uniformly by gently rotating. The Conway dish is placed in a 37°C constant temperature box for 2 hours, taken out, cooled to room temperature, and the cover is removed. The boric acid absorbent in the inner chamber is titrated with a hydrochloric acid standard solution (0.010 mol / L), and the end point is blue-violet. Two independent determination experiments are performed under repeatable conditions, and a blank test is also performed. The volatile basic nitrogen content in the meat sample is calculated by referring to the following formula:

[0079]

[0080] In the formula:

[0081] X: the content of volatile basic nitrogen in the meat sample, in units of milligrams per hundred grams (mg / 100 g);

[0082] V2: the volume of the standard acid solution consumed in titrating the sample, in units of milliliters (mL);

[0083] V3: the volume of the standard acid solution consumed in titrating the blank, in units of milliliters (mL);

[0084] C: the actual concentration of the hydrochloric acid standard solution, in units of moles per liter (mol / L);

[0085] m: the mass of the meat sample, in units of grams (g);

[0086] 14: the mass of nitrogen corresponding to 1.00 mL of the hydrochloric acid standard titration solution [c(HCl) = 1.000 mol / L], in units of milligrams (mg).

[0087] Microbial indicators: 25 g of the beef sample is cut into small pieces, accurately weighed into a sterile homogenization bag, 225 mL of sterilized physiological saline is added, and the sample is shaken for 1 min to obtain a 10 -1Diluted liquid, then 1 mL of sterile gun head to suck 1 mL of diluent, put into 9 mL of sterile saline, shake and mix into 10 -2 Diluted liquid. In turn, the continuous dilution to 10 -4 . According to the "GB 4789.2-2016 food safety national standard colony count method" to determine the total number of colonies; "SN / T 4044-2014 export grade meat products in the counting method of pseudomonas" to determine the total number of pseudomonas; "GB 4789.35-2010 food safety national standard food microbiology test lactic acid bacteria test" to determine the total number of lactic acid bacteria.

[0088] Sensory evaluation: from the refrigerator to take the meat sample, after thawing in warm water for 15 minutes, please 5 food professionals on its cut surface color, texture, smell evaluation. After the sensory identification of meat samples were fried, the taste of beef after cooking, taste evaluation. Scoring criteria are shown in Table 2.

[0089] Table 2 sensory evaluation scoring criteria

[0090]

[0091]

[0092] III. Results

[0093] 3.1 Strain low temperature growth curve

[0094] As Figure 2 shown, strain SCB2557 has good cold tolerance, can grow rapidly to 0.8-1.2 x 10 9 cfu / mL in 4℃ environment, and keep the number of bacteria stable in subsequent time; while the non cold tolerant Lactobacillus bulgaricus SCB0693 does not grow and reproduce in low temperature environment, and the number of bacteria decreases significantly in later period. Therefore, strain SCB2557 has good cold tolerance and can grow in low temperature environment.

[0095] 3.2 Antisepsis effect

[0096] Table 3 changes of physicochemical and microbial indicators of beef aging for 21 days

[0097]

[0098] Note: ** Indicates that the control group p<0.01, extremely significant difference; + indicates increase, - indicates decrease.

[0099] The changes of physicochemical and microbial indicators of beef aging for 21 days at 4℃ are shown in Table 3.

[0100] In terms of pH, the pH of the beef group sprayed with strain SCB2557 was slightly lower than that of the control group during the low-temperature aging of beef, with a decrease of 5.75%, which was due to the acid production of strain SCB2557. The decrease in pH is conducive to the inhibition of the growth of spoilage bacteria, but the decrease is small and is not enough to affect the taste of beef.

[0101] In terms of TVB-N content, the TVB-N content of beef before aging was 10.81 mg / 100 g. After 21 days of treatment, the TVB-N content of the control group was 33.29 mg / 100 g, while the TVB-N content of the beef sprayed with strain SCB2557 was 19.25 mg / 100 g, which was significantly lower than that of the control group by 42.17% (p<0.01). This indicates that the addition of the strain can effectively reduce the production of volatile basic nitrogen.

[0102] In terms of microbial indicators, after 21 days of beef aging, the SCB2557 treatment group and the control group had significant differences in various indicators (p<0.01). Compared with the control group, the total number of colonies and the total number of spoilage bacteria (Pseudomonas) in the treatment group were significantly reduced, with a decrease of 95.31% in the total number of colonies and a decrease of 99.33% in the number of Pseudomonas. The total number of lactic acid bacteria in the treatment group was significantly higher than that in the control group, with an increase of 108.05% compared with the control group. This indicates that strain SCB2557 has good competitiveness on the surface of beef in a low-temperature environment, can quickly occupy the ecological niche on the surface of beef, effectively inhibit the growth of spoilage bacteria, and achieve the effect of effectively preventing beef spoilage.

[0103] The sensory evaluation of beef after 21 days of aging is shown in Table 1. Figure 3 The sensory evaluation of beef after 21 days of aging is shown in Table 1.

[0104] Example 4, Analysis of the Microbial Community Structure of Strain SCB2557 Aged Beef

[0105] I. Preparation of Bacterial Solution

[0106] The activated strain SCB2557 was inoculated into sterilized soy milk medium and cultured at 30°C for 48 h to obtain a seed culture solution. Then, the seed culture solution was inoculated into sterilized soy milk medium at an inoculation amount of 1×10 7 cfu / mL and cultured at 30°C for 16 h to obtain a bacterial solution, which was stored at 4°C for later use (used within 3 days).

[0107] II. Aging beef preservation experiment

[0108] The above bacterial solution was sprayed on the surface of fresh beef, and then placed in an aging refrigerator (1-6°C, humidity 75-85%) for aging for 21 days. During the aging period, the refrigerator used a "four-stage" variable temperature and humidity method, with the following scheme:

[0109] First stage (0-1d): refrigerator temperature 4-6°C, humidity 83-88%

[0110] Second stage (2-7d): refrigerator temperature 2-4°C, humidity 80-85%

[0111] Second stage (8-11d): refrigerator temperature 3-5°C, humidity 77-80%

[0112] Fourth stage (12-21d): refrigerator temperature 1-3°C, humidity 75-78%

[0113] Every 7 days, samples were taken for metagenomic analysis.

[0114] III. Results

[0115] As shown in Figure 4 , after aging beef was sprayed with strain SCB2557, the bacterial community structure of the aged beef was greatly changed compared to the original bacterial community structure Figure 1 . At 7d, there was almost no growth of Pseudomonas, while at 14d and 21d, the relative abundance of Pseudomonas was about 50%. After 21 days of aging, the proportion of surface lactic acid bacteria increased significantly compared to the bacterial community structure on the surface of ordinary aged beef, and the niche of lactic acid bacteria on the surface of the aged beef increased greatly. This indicates that strain SCB2557 has good preservation effect and can inhibit the growth of spoilage bacteria through population competition.

[0116] Example 5, Effect of strain SCB2557 culture time on aging beef preservation

[0117] I. Bacterial solution preparation

[0118] Sterilized soy milk medium preparation: 50g / L sugar-free soy flour, 60g / L white sugar, 112°C sterilization for 15min.

[0119] The activated strain SCB2557 was inoculated into the sterilized soy milk medium and cultured at 30°C for 24h to obtain a seed culture solution; then the seed culture solution was inoculated into the sterilized soy milk medium at an inoculation amount of 1x10 6 cfu / mL and cultured at 30°C for 16h, 24h, and 32h, respectively, to obtain a bacterial solution, which was stored at 4°C for later use (within 3 days).

[0120] II. Aging beef preservation experiment

[0121] Fresh beef 12 pieces, each 3 kg, uniform size, divided into A, B, C, D four groups, each group of three pieces.

[0122] Among them, group A sprays the bacterial liquid cultured for 16h, group B sprays the bacterial liquid cultured for 24h, group C sprays the bacterial liquid cultured for 32h, and group D does not spray the bacterial liquid. And put it into the ripening refrigerator (1-6℃, humidity 75-85%) for 21 days. During the ripening period, the refrigerator adopts "four-stage" variable temperature and humidity method, and the scheme is as follows:

[0123] First stage (0-1d): refrigerator temperature 4-6℃, humidity 83-88%

[0124] Second stage (2-7d): refrigerator temperature 2-4℃, humidity 80-85%

[0125] Second stage (8-11d): refrigerator temperature 3-5℃, humidity 77-80%

[0126] Fourth stage (12-21d): refrigerator temperature 1-3℃, humidity 75-78%

[0127] On the 21st day, the pH value, microbial indicators (total number of colonies, total number of lactic acid bacteria and total number of pseudomonas) were determined.

[0128] III. Results

[0129] Table 4 Effect of strain SCB2557 incubation time on ripening beef

[0130]

[0131] Note: * Significant difference compared with the control group (p<0.05); ** Extremely significant difference compared with the control group (p<0.01)

[0132] As shown in Table 4, compared with the control group, the pH, total number of colonies and total number of pseudomonas of A, B and C groups were relatively small, with extremely significant difference (p<0.01), and the total number of lactic acid bacteria was relatively high, with good preservative effect. For A, B and C groups, the longer the incubation time of strain SCB2557, the lower the pH and the total number of pseudomonas, and the higher the total number of lactic acid bacteria. It shows that within a certain time limit, the longer the incubation time, the more dominant the strain SCB2557 occupies the ecological niche on the surface of beef, thereby inhibiting the growth of spoilage bacteria and improving the preservative effect.

[0133] Example 6, Effect of mixed milk ratio on the preservative effect of ripening beef

[0134] I. Preparation of sterile mixed milk culture medium

[0135] Three sterilized mixed milk culture media were prepared according to the following scheme:

[0136] Group A (3:1): 60 g / L of sugar-free soybean powder, 20 g / L of whole milk powder, 20 g / L of white sugar, sterilized at 105°C for 20 min

[0137] Group B (1:1): 40 g / L of sugar-free soybean powder, 40 g / L of whole milk powder, 20 g / L of white sugar, sterilized at 105°C for 20 min

[0138] Group C (1:3): 20 g / L of sugar-free soybean powder, 60 g / L of whole milk powder, 20 g / L of white sugar, sterilized at 105°C for 20 min

[0139] II. Preparation of bacterial solution

[0140] The activated strain SCB2557 was inoculated into the sterilized mixed milk culture media of groups A, B and C respectively, and incubated at 30°C for 24 h to obtain seed culture solution; then the seed culture solution was inoculated into the corresponding sterilized mixed milk culture media at an inoculation amount of 1 x 10 6 cfu / mL, and incubated at 30°C for 24 h to obtain bacterial solution, which was stored at 4°C for later use (used within 3 days).

[0141] III. Preservation experiment of cooked beef

[0142] Twelve pieces of fresh beef, each weighing 3 kg and having the same size, were divided into four groups, with three pieces in each group.

[0143] The bacterial solution of strain SCB2557 cultured in the sterilized mixed milk culture media of groups A, B and C was sprayed on the beef in the three groups respectively, and the beef in group D was not sprayed with bacterial solution, and then the beef was placed in a cooked cold storage (1-6°C, humidity 75-85%) for 21 days. During the cooking period, the cold storage was subjected to "four-stage" temperature and humidity changes, and the scheme was as follows:

[0144] First stage (0-1d): cold storage temperature 4-6°C, humidity 83-88%

[0145] Second stage (2-7d): cold storage temperature 2-4°C, humidity 80-85%

[0146] Second stage (8-11d): cold storage temperature 3-5°C, humidity 77-80%

[0147] Fourth stage (12-21d): cold storage temperature 1-3°C, humidity 75-78%

[0148] On the 21st day, the microbial indicators (total number of colonies, total number of lactic acid bacteria and total number of Pseudomonas) were determined.

[0149] IV. Results

[0150] Table 5 Effect of mixed milk ratio on cooked beef

[0151]

[0152] Note: There is no same letter between groups, and there is adjacent letter, which means p < 0.05

[0153] From Table 5, A, B, C three groups in four indicators are significantly different (p < 0.05). The ratio of soy milk and cow milk in mixed milk will affect the preservation effect of strain SCB2557 on mature beef. With the increase of the ratio of soy milk in mixed milk, the proliferation rate of strain SCB2557 increases, which makes the population competition advantage of strain SCB2557 on the surface of mature beef stronger, and better inhibits the growth of spoilage bacteria.

[0154] The above examples are the preferred embodiments of the present application, but the embodiments of the present application are not limited to the above examples, and any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present application should be equivalent replacement methods, which are all included in the protection scope of the present application.

Claims

1. A psychrophilic meat-derived lactic acid bacterium, characterized in that: This bacterium is Leuconostoc mesenteriae ( Leuconostoc mesenteroides It was named SCB2557 and deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 23, 2022, with the preservation number GDMCC No: 62311.

2. Use of the psychrophilic meat-derived lactic acid bacterium according to claim 1 in the production of matured beef, characterized in that: The application is carried out according to the following steps: inoculating low-temperature meat-derived lactic acid bacteria into sterilized soy milk or mixed emulsion to culture for 8-48h to obtain a fermentation liquor, spraying the obtained fermentation liquor as a preservative on the surface of beef, and then aging the beef; The formula of the soy milk is: 40-110g / L of sugar-free soybean powder and 10-80g / L of white granulated sugar, and the sterilization method is 105-115℃ sterilization for 15-20min. The formula of the mixed emulsion is: 20-50g / L of full-fat milk powder, 20-60g / L of sugar-free soybean powder and 10-80g / L of white granulated sugar, and the sterilization method is 105-115℃ sterilization for 15-20min; or: mixing pure milk with 40-120g / L of soy milk at a volume ratio of 1:1, and 10-80g / L of white granulated sugar, and the sterilization method is 105-115℃ sterilization for 15-20min.

3. Use according to claim 2, characterized in that: The application specifically follows the following steps: inoculating low-temperature meat-derived lactic acid bacteria into sterilized MRS liquid culture medium, culturing at 30℃ for 48h to obtain seed culture solution; then inoculating the seed culture solution at an inoculation amount of 1×10 5 cfu / mL~5×10 7 cfu / mL into pasteurized soy milk or mixed emulsion culture medium, culturing at 30℃ for 8-48h to obtain fermentation liquor, spraying the fermentation liquor as a preservative onto beef in a spraying manner to form a thin film on the surface of the beef, and then placing the beef in a ripening cold storage with a temperature of 2-6℃ and humidity of 75-85% to ripen, wherein the temperature and humidity of the cold storage are controlled by using a four-stage variable temperature and variable humidity method. The four-stage variable-temperature and variable-humidity method includes the following steps: in the first stage, the low-temperature meat-derived lactic acid bacteria are rapidly grown and reproduced on the surface of beef by using a relatively high-temperature and high-humidity environment; in the second stage, the bacterial community structure on the surface of beef is stabilized in a relatively low-temperature environment; in the third stage, which is a flavor forming stage, the protease and lipase inside and outside the beef are activated in a relatively medium-temperature and medium-humidity environment, so as to decompose the muscle fibers and fat of beef, increase the tenderness of beef, and form rich flavors; in the fourth stage, which is a post-aging stage, the beef continues to age in a relatively low-temperature and low-humidity environment while the growth of spoilage bacteria is inhibited; the relatively high-temperature and high-humidity environment has a temperature of 4-6℃ and a humidity of 83-88%; the relatively low-temperature environment has a temperature of 2-4℃ and a humidity of 80-85%; the relatively medium-temperature and medium-humidity environment has a temperature of 3-5℃ and a humidity of 77-80%; and the relatively low-temperature and low-humidity environment has a temperature of 1-3℃ and a humidity of 75-78%.

4. Use according to claim 3, characterized in that: The formula of the MRS liquid medium is: 10.0g / L of peptone, 5.0g / L of beef extract powder, 4.0g / L of yeast extract powder, 20.0g / L of glucose, 1.0mL / L of Tween-80, 2.0g / L of potassium phosphate dibasic, 5.0g / L of sodium acetate, 15.0g / L of agar, 2.0g / L of trisodium citrate, 0.2g / L of magnesium sulfate and 0.05g / L of manganese sulfate, and the sterilization method is 121℃ sterilization for 20min, and the pH is adjusted to 6.2±0.2 when used.

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