CN109097297B - Leuconostoc mesenteroides strain and application thereof - Google Patents

Abstract

The invention relates to the technical field of microorganisms, and provides a lactobacillus plantarum strain and an application thereof, wherein the Leuconostoc mesenteroides is adopted to ferment pickled beef to obtain a fermented beef product with a regular shape, and the surface muscle is rose red and fat white; the elasticity is better, and the section is solid and regular; has mild fragrance and moderate sweet and sour taste.

Description

Leuconostoc mesenteroides strain and application thereof

Technical Field

The invention relates to the technical field of microorganisms, in particular to a leuconostoc mesenteroides strain and application thereof.

Background

The fermented meat product is produced by utilizing the microbial fermentation under natural or artificial control conditions, has special flavor, color and texture and has long shelf life. Through microbial fermentation, the quality of the raw materials is improved, the flavor is increased, and the nutritional value is improved. In the whole processing process of the fermented meat product, endogenous enzymes and microbial enzymes in the meat decompose protein to generate a large amount of amino acids, so that the digestibility of the fermented meat product by a human body is improved. Many of the free amino acids are essential amino acids required by human bodies, and have obvious biological activity, so that the health-care property and the nutrition of the meat product are greatly improved. The amino acid reacts with other substances to generate flavor substances, thereby promoting the fragrance and taste of meat products. Meanwhile, some amino acids in the meat product are compounded with other substances to play a role in resisting oxidation, and the amino acids are used as color development additives to improve the sense of the fermented meat product and influence the quality of the meat product.

Meat products in China are divided into western meat products and medium meat products. The western-style meat products mainly comprise three products of sausage, ham and bacon, and the Chinese-style meat products comprise three products of cured meat products, dried meat products and sauce products. The industrialized production of the western-style fermented meat products is mature, but most products do not accord with the dietary habits of Chinese people.

Although the Chinese meat products have a long history of eating fermented meat products in the minority nationality residential area, the Chinese meat products are mostly local products and are limited by local environment and climate. The traditional salted meat product is generally prepared by taking pork as a raw material and performing anaerobic fermentation by utilizing microorganisms in a natural state. There are the following problems: on one hand, the pork has high fat content and is easy to cause obesity and cardiovascular diseases, on the other hand, the natural fermentation period is long, the control is not good, and the mixed bacteria are easy to pollute.

The beef is rich in mineral substances, B vitamins, potassium, iron, zinc and other nutrient elements, and provides high-quality protein. Compared with pork, the pork has high protein content of 5-10% and low fat content of 20-30%. At present, the beef fermented product in the market mainly takes air-dried beef and belongs to a slightly fermented product. Lack of deep-fermented beef products.

Various commercial fermented meat strains in the market at present have rich production companies and product types, mainly direct-vat-set trial fermentation and are simple and convenient to operate. However, most of the strains belong to western-style fermented meat strains, and the Chinese-style fermented meat strains are rare.

The pickled meat belongs to Chinese-style fermented meat products, and is generally fermented naturally, so that the production period is long, and the mixed bacteria are easily polluted.

The existing commercial leaven is used for western-style fermented meat products, and the produced products are easy to have the same flavor and have no special problems. In addition, few leavening agents are used for special Chinese-style fermented meat products in the market, and the development of corresponding leavening agents is needed to commercialize the Chinese-style fermented meat.

The beef can cause the protein in the beef to be denatured and degraded after fermentation, and the protein is converted into matters such as peptone, peptide, amino acid and the like, so that the product quality is improved, the nutrition is richer, the protein absorption rate is also improved, and the beef is easier to absorb and utilize by a human body. But the market has few deep fermentation products about beef.

Disclosure of Invention

It is necessary to provide a Leuconostoc mesenteroides strain which is preserved in China general microbiological culture Collection center (CGMCC) with the preservation number of CGMCC No.15924 at 6, 11 and 11 days of 2018 at No. 3 of Beijing, facing Yang area, north Chen Xilu No.1 of China.

The application of Leuconostoc mesenteroides in the fermentation of Chinese-style pickled beef is also necessarily provided.

The pickled beef is fermented by using the strain provided by the invention, and the obtained fermented beef product has a regular shape, and surface muscles are rose red and are fat white; the elasticity is better, and the cut surface is solid and regular; has mild fragrance and moderate sweet and sour taste.

The fermentation strain is different from the fermentation strain for fermenting western-style meat in the prior art, and is a special fermentation strain for Chinese-style pickled beef.

Drawings

FIG. 1 shows the individual form of the selected fermentation lactic acid bacteria P6 under 100-fold oil immersion.

FIG. 2 shows the individual forms of the selected cultured lactic acid bacterium K12 under a 100-fold oil mirror.

FIGS. 3-6 are bar graphs of sensory evaluation of the effect of different strains and different fermentation times on the quality of fermented pickled beef. L3, K12, P6 and ZC4 in sequence.

FIGS. 7-10 are bar graphs of sensory evaluation and pH changes of different bacterial species at different temperatures.

FIGS. 11-14 are bar graphs of sensory evaluation and pH changes of different bacterial species at different humidities.

FIGS. 15-18 are histograms of sensory evaluation and pH change for different species at different inoculum sizes.

FIGS. 19 to 24 are graphs showing the results of the lactobacillus viscogenesis experiments.

FIGS. 25-30 are graphs showing the results of the lactic acid bacteria gassing experiments.

FIGS. 31 and 32 show the production of H by lactic acid bacteria ₂ And S, displaying an experimental result.

FIG. 33 is a graph showing the growth of 4 strains of bacteria.

Detailed Description

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following description will be made in conjunction with the embodiments and experimental solutions.

1. The source of the strain

P6: the pickled vegetable is obtained by separating and screening natural fermented pickled vegetables of citizens in Ningxia Yinchuan area.

K12: the natural fermented milk is obtained by separating and screening commercial Tibetan mushroom strains produced in Tibetan glossy ganoderma areas.

2. Strain separation and screening method

1. Casting a flat plate: sterilized MRS +0.5% calcium carbonate agar medium and MSA agar medium in triangular flasks were melted by heating, cooled to about 45℃, and plates were cast, approximately 20mL each.

2. Sample dilution: adding 9mL of sterile water into 1mL of the above natural fermented milk and 1mL of natural fermented pickle juice, respectively, to obtain a dilution of 10 ^-1 Respectively taking 1mL of diluent, putting the diluent in an aseptic test tube, adding 9mL of sterile water, mixing uniformly, sequentially carrying out 10-time serial dilution, and respectively taking a small amount of diluted zymocyte liquid and pickle juice bacteria liquid to separate zymocyte. 3. Plate separation: A. plating and streak inoculation: respectively picking fermentation bacteria liquid and pickle juice bacteria liquid by using an inoculating loop, and carrying out streak inoculation on a flat plate in an aseptic operation; after the streaking is finished, the cover is covered, and the mixture is inverted and cultured in a constant temperature incubator at 40 ℃ for 48 hours. B. Plating and inoculating: taking two sterile plates, respectively adding 0.1mL of zymogen liquid and pickle juice bacterial liquid on the two sterile plates, uniformly coating the bacterial liquid on the plates by using a sterile glass coating rod, flatly placing the plates on an experiment table for 20min, and then inversely placing the plates in a constant-temperature incubator at 25 ℃ for culturing for 48h.

4. And (4) observing colonies: selecting a plate with better colony distribution, observing the colony morphology of the plate, and preliminarily finding out the lactobacillus fermentum colony. (MRS +0.5% calcium carbonate solid medium screening standard: good growth on the surface or inside of the culture medium, white and glossy, neat edges, easy picking up and non-sticking, and a transparent ring.) 5, microscopic examination: colonies are picked from the plate by using an inoculating loop, a bacterium smear is made, gram staining and oil-lens observation are carried out to obtain two forms of strains P6 and K12, and individual shape pictures of the two forms of strains. As shown in fig. 1 and 2.

3. Identification and conclusion of strains

The obtained two individual strains are sampled and labeled with K2, K3, K5, K10, K12, K13, K17, K18, K19, K20, K21, K22, L1, L2, L5, FX3, ZC4, ZC5, ZC7, FXH11, FH2, FH6, P7, Y4, Y6, Y9, Y10, L3 and L7.

Seven comparison experiments in the following (four and strain characteristic comparison experiments) are carried out on the strains in sequence, and 4 strains after screening are respectively carried out (five and single strain fermentation experiments), so that the obtained strains P6 and K12 have various characteristics which are obviously superior to those of other strains, and the P6 and K12 have the best effect on the fermentation of the pickled beef.

Performing molecular biological identification on the strains P6 and K12, performing sequencing after 16S rDNA amplification, and comparing a sequencing result with a sequence in a BLAST database to finally obtain identification results of the two strains: strain P6 was identified as Leuconostoc mesenteroides (Leuconostoc mesenteroides) strain; k12 was identified as Lactobacillus plantarum (Lactobacillus plantarum). See 16S rDNA sequence of strain P6 and 16S rDNA sequence of strain K12.

4. Comparison experiment and conclusion of strain characteristics

4.1 Strain characteristics

(1) Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 were both tolerant to 6% sodium chloride solution, i.e.were cultured in MRS broth containing 6% NaCl at 37 ℃ and reached OD at 600nm of 2.360 and 2.351, respectively.

(2) Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 are tolerant to sodium nitrite solution, i.e.OD 2.517 and 2.611 were measured at 600nm in 150mg/kg MRS broth at 37 ℃ for 24h.

(3) After the Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 were cultured in MRS liquid medium at 37 ℃ for 28 hours, the pH values were reduced to 3.78 and 3.71, respectively.

(4) The two strains are cultured for 48 hours at 37 ℃ on an MRS solid plate culture medium, and no sticky condition of bacterial colonies appears.

(5) The two strains are inoculated in a glucose gas production culture medium with a Duchenne tubule and cultured for 24 hours at 37 ℃, and no gas production phenomenon occurs.

(6) Two strains are punctured and inoculated in the H product ₂ In S medium, no black precipitate line appears and no hydrogen sulfide is produced after 24 hours of culture at 37 ℃.

(7) Punching holes in an Oxford cup on an MRS solid plate culture medium containing 10% skim milk, respectively adding 100 mu L of the two strains of lactobacillus liquid into the holes, and observing that the casein has strong hydrolysis capacity after 24 hours.

Namely: the above test conclusion is that the following seven groups of comparative tests verify that Leuconostoc mesenteroides P6 and Lactobacillus plantarum K12 have the excellent characteristics of high salt resistance, high sodium nitrite resistance, high acid production, no mucus production, no gas production, no hydrogen sulfide gas production and good protein activity degradation.

(1) The salt tolerance experimental scheme and the experimental data of the lactobacillus are shown in the table 1.

Lactic acid bacteria were inoculated into MRS liquid medium containing 2.0%, 4.0%, 6.0% NaCl, cultured at 37 ℃ for 24h, and OD was measured at 600nm, while growth was compared with that of non-inoculated MRS liquid medium as a blank.

TABLE 1 OD value of 30 strains of bacteria at 2.0%, 4.0%, 6.0% salt

By comparing the OD values measured for the strains, it was found that the NaCl resistance was high for strains K2, K3, K5, K10, K12, K13, K18, K20, K21, P6, P7, Y4, Y10, ZC4, ZC5, ZC7, FX3, FXH11, FH2, and L3, and these 20 strains were selected for the subsequent tests.

(2) The sodium nitrite resistance experiment and test data of the lactobacillus are shown in table 2.

Inoculating lactobacillus in the mixture containing NaN0 at 50mg/kg, 100mg/kg, and 150mg/kg ₂ In the MRS liquid culture medium, the OD value is measured under 600nm, and meanwhile, the growth condition of the MRS liquid culture medium without inoculation is compared by taking the MRS liquid culture medium without inoculation as a blank control.

TABLE 2 strains of 20 strains of NaNO at 50mg/kg, 100mg/kg, 150mg/kg ₂ OD value of

By comparing the OD values measured for the strains, it was found that 15 strains K2, K3, K5, K10, K12, K13, K18, K20, L3, P6, Y4, FXH11, ZC4, ZC5, and ZC7 were NaN 0-resistant ₂ The performance is high.

(3) The acid production experiments and test data of lactic acid bacteria are shown in table 3.

Inoculating lactobacillus in MRS liquid culture medium, culturing at 37 deg.C, measuring pH value 24 hr later, and measuring pH value of non-inoculated MRS liquid culture medium each time as blank control to determine the acid production condition of lactobacillus.

TABLE 3 pH of 20 strains

The comparison of the pH values measured 24h after the culture of the strains revealed that the pH values of 15 strains K2, K3, K5, K10, K12, K13, K18, K20, L3, P6, Y4, FXH11, ZC4, ZC5 and ZC7 decreased rapidly, indicating that the acid-producing effect was good.

(4) The mucus production experiments and test results of lactic acid bacteria are shown in FIGS. 19-24.

Inoculating lactobacillus on MRS solid plate culture medium, culturing at 37 deg.C for 48 hr, and picking colony for direct observation.

The bacterial colony is picked up by using the inoculating loop, whether the bacterial colony has wiredrawing or adhesion is observed, and according to the state of the bacterial colony on the observation test flat plate after being picked up by the inoculating loop, the bacterial colony of 7 strains of ZC4, K3, K12, L3, P6, Y4 and K18 can be determined to have no wiredrawing, adhesion and mucus production.

(5) The results of the gassing experiment and test with lactic acid bacteria are shown in FIGS. 25-30.

Inoculating lactobacillus into glucose gas production culture medium with Duchenne tubule, and culturing at 37 deg.C for 24 hr. And observing whether bubbles are generated in the Duchenne tubule or not.

By observing the Du's tubule, it was confirmed that no bubble was generated in the tubules of 15 strains of K2, K3, K5, K10, K12, K13, K18, K20, L3, P6, Y4, FXH11, ZC4, ZC5 and ZC7, and no gas was produced.

(6) Production of H by lactic acid bacteria ₂ S experiment and test results are shown in figures 31 and 32.

Inoculating lactobacillus in the egg H by puncture ₂ Culturing in S medium at 37 deg.C for 24 hr, and if black precipitate line appears, there is H ₂ S is generated, otherwise H is not generated ₂ And S. Protein degradation Activity test

(7) The test method comprises the following steps: a hole is punched in an MRS solid plate culture medium containing 10% skim milk by using an oxford cup, 100 mu l of lactic acid bacteria liquid is added into the hole, and after 24h, a casein hydrolysis ring is observed. And judging the hydrolysis capability of the strain to the protease according to the existence and the size of a hydrolysis ring.

The protein degradation activity test and results are shown in Table 4 below

TABLE 4 results of protein degradation assay of different strains

Name of Strain	Hydrolytic power	Name of Strain	Hydrolytic power	Name of Strain	Hydrolytic power
						K2	--	K13	--	Y4	+
K3	--	K18	+	FXH11	++
						K5	--	K20	--	ZC4	++
K10	--	L3	+	ZC5	+
						K12	++	P6	++	ZC7	--

As is clear from Table 4, it was confirmed that 8 strains of ZC4, ZC5, K12, K18, Y4, FXH11, P6 and L3 had high hydrolysis ability.

4.2 drawing growth curve

According to the seven groups of tolerance experiment results, 6 strains of ZC4, K12, P6, L3, Y4 and K18 which have high salt tolerance, high sodium nitrite resistance, high acid production, no mucus production, no gas production, no hydrogen sulfide gas production and good protein activity degradation are screened out through overall analysis to draw a growth curve. As shown in fig. 33.

As can be seen from the figure, the 4 strains of K12, P6, L3 and ZC4 can reach a stationary phase and grow vigorously within about 20 hours, and the 2 strains of K12 and P6 have high growth speed, can reach stationary phases within about 9 hours and 20 hours respectively and grow vigorously, so the strain can be used as an application strain for subsequent production.

5. Fermentation experiments with a Single Strain

4 strains with excellent performance are selected and inoculated into pickled beef to carry out fermentation test, so as to prove the application and effect of K12 and P6 in the fermentation of the pickled beef.

And 5.1, screening the experimental strains to obtain 4 strains with good performance through the fourth strain characteristic comparison experiment and conclusion.

Lactobacillus plantarum K12、Leuconostoc mesenteroides P6，

Lactobacillus alimentarius L3，Leuconostoc mesenteroides ZC4。

5.2 test methods

(1) Investigation of the inoculum sizes of four different strains: the inoculum size was 0.2%, 1.0%, 2.0% three different inoculum sizes to produce fermented cured meat, and the pH was measured every 5 hours until the pH reached below 5.2 and the fermentation was stopped.

(2) Fermentation temperatures of four different strains: fermenting the pickled meat with lactobacillus at 28 deg.C, 37 deg.C, and 41 deg.C, measuring pH every 4 hr, and stopping fermentation until pH is below 5.2.

(3) Fermenting humidity of four different strains: fermenting the cured meat with lactobacillus at 85%, 90%, 95%, and measuring pH every 5 hr until pH is below 5.2.

(4) Fermentation time of four different strains: fermenting the cured meat with lactobacillus at 6h, 12h, 18h, 24h, and 30h, and performing sensory evaluation of the cured meat product at each time point.

5.3 sensory evaluation

Designing BIB by adopting balanced incomplete partitioning, and carrying out sensory evaluation on the salted meat reaching the fermentation end point, wherein the method specifically comprises the following steps: professionals who study food processing form a 5-person assessment group, and the assessment group is required to have no smoking, no drinking, no spicy food and other stimulating foods within 12 hours before the assessment group. The assessment personnel keep a certain distance, the assessment process does not talk with each other to assess the sensory quality of the fermented pickled beef, the assessment is mainly carried out according to the attributes of the appearance, the tissue state and the flavor of the fermented pickled beef, the assessment standard refers to (table 4) to assess the finished product, the product is rinsed with clear water, the next sample is assessed at the interval of 10min, finally, the assessment table is filled and signed, and the assessment results of the assessment personnel are collected for analysis. And 5 evaluators respectively carry out comprehensive sensory evaluation on each product, and the evaluation range is 0-100.

TABLE 4 sensory evaluation criteria for fermented pickled beef

5.4 results of the experiment

(1) The influence of different strains and different fermentation times on the quality of the fermented pickled beef is shown in figures 3-6.

Fermenting the K12, the P6, the L3, the ZC4 and the four strains for 6h, 12h, 18h, 24h and 30h at the inoculation amount of 0.2 percent and the temperature of 28 ℃ and the humidity of 85 percent. The sensory evaluation of the cured meat products is shown in FIGS. 3-6.

The experimental results show that: the pH value of the four strains generally shows a descending trend along with the prolonging of the fermentation time; the tissue structure aspect and the flavor score are higher and higher; the appearance becomes dark and the fat becomes slightly yellowish, which may be related to the oxidation of the fat and proteins in the meat by the lactic acid bacteria. The lactobacillus K12 is digested to the fastest fermentation end point, the pH value can reach 5.1 within 24 hours, and the lactobacillus K12 is a strain with high acid production potential. Although the fermentation capacity is weaker than that of other three strains, the leuconostoc mesenteroides P6 has the highest sensory evaluation value, the comprehensive sensory evaluation score is 81 points, and is higher than L3 by 2.1 points, K12 by 0.2 point and ZC4 by 0.5 point, the appearance shape of the product is slightly irregular, the surface part of the muscle is brown, and the fat is yellow; in the aspect of tissue state, the elasticity is better, and the section is firm and neat; has soft and fragrant flavor and moderate sour taste.

(2) Different strains were evaluated organoleptically and pH changes at different temperatures, as shown in FIGS. 7-10.

The four strains were fermented at 28 deg.C, 37 deg.C, and 41 deg.C, respectively, with the inoculum size of 0.2% and humidity of 85% unchanged, and the results were observed after 15h of fermentation. The sensory evaluation and pH of the cured meat products at different fermentation temperatures for different bacterial fermentations are shown in FIGS. 7-10.

Experimental results show that the four strains are fermented simultaneously for 15 hours, the pH value is reduced along with the temperature rise, the overall appearance and shape of the produced fermented meat are regular, the surface muscles are dark, the fat is yellow, the tissue state elasticity is good, the cut surfaces are uniform, and the fermented meat has the fermented fragrance but the lactic acid flavor is insufficient. According to overall analysis, sensory evaluation of four strains at 41 ℃ is higher than that of other two fermentation temperatures, the average score is 81.9 points, 7.6 points higher than that of 28 ℃ fermentation, and 3.5 points higher than that of 37 ℃ fermentation. The sensory evaluation score of P6 at 41 ℃ is up to 83.0 points. The acid production effect of lactobacillus plantarum K12 at 41 ℃ is still the best by combining the pH curve.

(3) Different strains were evaluated organoleptically and pH changes at different humidities, as shown in FIGS. 11-14.

The four strains were fermented at 85%, 90%, 95% humidity, respectively, with the inoculum size being maintained at 0.2%, the temperature being 28 ℃, and the results were observed after 15h of fermentation. Sensory evaluation of the cured meat products by fermentation with different strains at different fermentation humidities is shown in FIGS. 11-14.

The experimental result shows that the pH of the four strains integrally shows the trend of firstly decreasing and then increasing along with different humidity. The overall sensory expression of the fermented meat is that the surface of the fermented meat has dark muscle, the fat is yellow, the tissue state is elastic, the cut surface is uniform, the meat has cracks but is not obvious, and the fermented flavor is sufficient. Overall analysis shows that when the humidity is 90% for fermentation, the sensory evaluation of the four strains is higher than that of the other two strains, the average score is 83.8, 8.0 and 3.1 respectively higher than that of 85% and 95% for fermentation. P6 scored a maximum of 85.6 points at 90% sensory. The best acid production performance of lactobacillus plantarum L3 at 41 ℃ can be known by combining a pH curve.

(4) Different species were evaluated organoleptically and pH changes at different inoculum sizes, as shown in FIGS. 15-18.

Decomposing the four strains, inoculating the strains in the inoculum sizes of 0.2%, 1.0% and 2.0%, maintaining the temperature at 41 ℃ and the humidity at 85%, fermenting for 15h, and observing the fermentation result. Different fermentation temperatures different strains were fermented, and the sensory evaluation and pH of the cured meat products are shown in FIGS. 15-18.

The experimental result shows that the pH of the four strains of bacteria integrally shows a descending trend along with the increase of the addition amount, the muscle on the appearance surface becomes dark, the fat becomes yellow, the tissue state and the shape are more regular, the fermentation fragrance is insufficient, and the fragrance of lactic acid fermentation is provided. According to the overall analysis body, the sensory evaluation of the four strains at 2.0% is higher than that of the other two strains, the average score is 76.2, 8 and 2.5 respectively higher than that of 0.2% and 1.0% in fermentation. The sensory evaluation score of P6 at 2.0% was 77.8 points at the highest. The acid production effect of 2.0% lactobacillus plantarum K12 is still the best by combining with the pH curve.

In conclusion, according to sensory evaluation and pH curve charts, the optimal fermentation time at 28 ℃ is 24 hours, the temperature is 41 ℃, the humidity is 90 percent, and the addition amount is 2.0 percent. Although the two strains which are optimal to obtain the P6 and the L3 from the humidity chart, the time chart, the temperature chart and the adding amount chart obtain the optimal strains which are P6 and K12, and the optimal strains which are P6 and K12 are finally selected by combining sensory evaluation.

6. Preparation method of fermented pickled beef

6.1 Strain activation

The activation of the Lactobacillus plantarum P6 according to the invention must be carried out in a sterile operating station. Inoculating a single strain to be inoculated at a position close to flame, burning an inoculating loop until the inoculating loop turns red, and inoculating, but when the inoculating loop is placed into a test tube, firstly cooling the wall of the test tube, picking, inoculating the picked strain into a culture medium test tube before inoculating, wherein the whole process is carried out around the flame. Culturing in the later period; the cells were incubated at 37 ℃ for 24 hours in an incubator. Subculturing for 3 times according to the above method to keep the strain active. And (4) putting the cultured strain in a centrifugal machine for centrifugation at 3000 revolutions for 5 minutes to obtain wet bacterial sludge sediment.

6.2 beef early-stage process treatment

(1) Cutting into blocks: cutting fresh beef into cuboid with length of 2cm, width of 1cm and thickness of 1cm,

(2) mixing seasonings: mixing 3% of salt and 1.25% of fructus Zanthoxyli uniformly to obtain mixed flavoring.

(3) Stir-frying the seasoning with small fire; the frying process is carried out continuously to prevent local scorching. Until the color turned yellow.

(4) Salt bath: the mixed seasonings stir-fried by small fire are uniformly stirred to prevent some meat blocks from being soaked in salt bath.

(5) Pickling and sealing: the meat curing vessel must be checked for tightness before sealing in order to prevent possible air leakage.

6.3 Strain inoculation and fermentation

The inoculation amount of the leuconostoc mesenteroides strain is 2.0 percent of that of the pickled beef after the pickling, and the inoculation is carried out for fermentation. Controlling the fermentation humidity at 90-95%, controlling the temperature at 41 ℃, fermenting for 15-24 hours, and detecting that the pH value of the fermented beef is not more than 5.2 to prepare the fermented pickled beef.

6.4 fermentation pickled beef product characteristics

The fermented and pickled beef product prepared by the process method and the strain has the water content of 72 percent, the pH value of 5.1, the POV value of 2.0mg/100g and the TVB-N value of 6.1 multiplied by 10 ^-6 mg/100g. The fermented product under the process condition has a regular shape, the surface muscle is rose red, and the fat is white; the elasticity is better, and the section is solid and neat; has mild fragrance and moderate sweet and sour taste.

The fermentation strain is different from a fermentation strain for fermenting western-style meat in the prior art, and is a special fermentation strain for Chinese-style pickled beef.

7. The 16S rDNA sequence of strain P6 is:

CAGTCGAACGCACAGCGAAAGGTGCTTGCACCTTTCAAGTGAGTGGCGA ACGGGTGAGTAACACGTGGACAACCTGCCTCAAGGCTGGGGATAACATTT GGAAACAGATGCTAATACCGAATAAAACTTAGTGTCGCATGACACAAAGTT AAAAGGCGCTTCGGCGTCACCTAGAGATGGATCCGCGGTGCATTAGTTAGT TGGTGGGGTAAAGGCCTACCAAGACAATGATGCATAGCCGAGTTGAGAGA CTGATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGC TGCAGTAGGGAATCTTCCACAATGGGCGAAAGCCTGATGGAGCAACGCCG CGTGTGTGATGAAGGCTTTCGGGTCGTAAAGCACTGTTGTATGGGAAGAA CAGCTAGAATAGGAAATGATTTTAGTTTGACGGTACCATACCAGAAAGGGA CGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGTCCCGAGCGTTAT CCGGATTTATTGGGCGTAAAGCGAGCGCAGACGGTTTATTAAGTCTGATGT GAAAGCCCGGAGCTCAACTCCGGAATGGCATTGGAAACTGGTTAACTTGA GTGCAGTAGAGGTAAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATA TATGGAAGAACACCAGTGGCGAAGGCGGCTTACTGGACTGCAACTGACGT TGAGGCTCGAAAGTGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCC ACACCGTAAACGATGAACACTAGGTGTTAGGAGGTTTCCGCCTCTTAGTGC CGAAGCTAACGCATTAAGTGTTCCGCCTGGGGAGTACGACCGCAAGGTTG AAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGT TTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGAAG CTTTTAGAGATAGAAGTGTTCTCTTCGGAGACAAAGTGACAGGTGGTGCAT GGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAG CGCAACCCTTATTGTTAGTTGCCAGCATTCAGATGGGCACTCTAGCGAGAC TGCCGGTGACAAACCGGAGGAAGGCGGGGACGACGTCAGATCATCATGCC CCTTATGACCTGGGCTACACACGTGCTACAATGGCGTATACAACGAGTTGC CAACCCGCGAGGGTGAGCTAATCTCTTAAAGTACGTCTCAGTTCGGATTGT AGTCTGCAACTCGACTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAG CACGCCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACC ATGGGAGTTTGTAATGCCCAAAGCCGGTGGCCTAACCTT。

8. the 16S rDNA sequence of strain K12 is:

CGAACGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTG AGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGG ATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGG TCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCG TATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCG ACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCC TACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGG AGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGT TAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAAC CAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTG GCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTT AAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACT GGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTG AAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGT CTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGAT ACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTC CGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACG GCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGA CATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATAC AGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACT CTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAA ATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTAC AACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAG TTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAAT CGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC CCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTT AGGAACCAGCC。

while the invention has been described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention.

SEQUENCE LISTING

<110> Ningxia university

<120> Leuconostoc mesenteroides strain and application thereof

<130> 2018

<160> 1

<170> PatentIn version 3.3

<210> 1

<211> 1401

<212> DNA

<213> Leuconostoc mesenteroides

<400> 1

cagtcgaacg cacagcgaaa ggtgcttgca cctttcaagt gagtggcgaa cgggtgagta 60

acacgtggac aacctgcctc aaggctgggg ataacatttg gaaacagatg ctaataccga 120

ataaaactta gtgtcgcatg acacaaagtt aaaaggcgct tcggcgtcac ctagagatgg 180

atccgcggtg cattagttag ttggtggggt aaaggcctac caagacaatg atgcatagcc 240

gagttgagag actgatcggc cacattggga ctgagacacg gcccaaactc ctacgggagg 300

ctgcagtagg gaatcttcca caatgggcga aagcctgatg gagcaacgcc gcgtgtgtga 360

tgaaggcttt cgggtcgtaa agcactgttg tatgggaaga acagctagaa taggaaatga 420

ttttagtttg acggtaccat accagaaagg gacggctaaa tacgtgccag cagccgcggt 480

aatacgtatg tcccgagcgt tatccggatt tattgggcgt aaagcgagcg cagacggttt 540

attaagtctg atgtgaaagc ccggagctca actccggaat ggcattggaa actggttaac 600

ttgagtgcag tagaggtaag tggaactcca tgtgtagcgg tggaatgcgt agatatatgg 660

aagaacacca gtggcgaagg cggcttactg gactgcaact gacgttgagg ctcgaaagtg 720

tgggtagcaa acaggattag ataccctggt agtccacacc gtaaacgatg aacactaggt 780

gttaggaggt ttccgcctct tagtgccgaa gctaacgcat taagtgttcc gcctggggag 840

tacgaccgca aggttgaaac tcaaaggaat tgacggggac ccgcacaagc ggtggagcat 900

gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc ttgacatcct ttgaagcttt 960

tagagataga agtgttctct tcggagacaa agtgacaggt ggtgcatggt cgtcgtcagc 1020

tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc aacccttatt gttagttgcc 1080

agcattcaga tgggcactct agcgagactg ccggtgacaa accggaggaa ggcggggacg 1140

acgtcagatc atcatgcccc ttatgacctg ggctacacac gtgctacaat ggcgtataca 1200

acgagttgcc aacccgcgag ggtgagctaa tctcttaaag tacgtctcag ttcggattgt 1260

agtctgcaac tcgactacat gaagtcggaa tcgctagtaa tcgcggatca gcacgccgcg 1320

gtgaatacgt tcccgggtct tgtacacacc gcccgtcaca ccatgggagt ttgtaatgcc 1380

caaagccggt ggcctaacct t 1401

Claims (2)

1. A Leuconostoc mesenteroides strain is preserved in China general microbiological culture Collection center (CGMCC) at 6 months and 11 days in 2018, and the preservation number is CGMCC No.15924.

2. Use of Leuconostoc mesenteroides (Leuconostoc mesenteroides) as defined in claim 1 for curing beef in fermentation.

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