CN111424001A

CN111424001A - Lactobacillus sake B2-4 and application thereof in aquatic product preservation

Info

Publication number: CN111424001A
Application number: CN202010323120.0A
Authority: CN
Inventors: 妥彦峰; 牟光庆; 刘佳秀; 时逸馨; 钱方
Original assignee: Dalian Polytechnic University
Current assignee: Dalian Polytechnic University
Priority date: 2020-04-22
Filing date: 2020-04-22
Publication date: 2020-07-17
Anticipated expiration: 2040-04-22
Also published as: CN111424001B

Abstract

The invention discloses lactobacillus sake B2-4 (L actinobacillus sakei B2-4) and application thereof in aquatic product preservation, belonging to the technical field of biological application.

Description

Lactobacillus sake B2-4 and application thereof in aquatic product preservation

Technical Field

The invention belongs to the technical field of biological application, and particularly relates to lactobacillus sake B2-4 (L actinobacillus sakei B2-4) and application thereof in aquatic product preservation.

Background

Lactic acid bacteria are gram-positive bacteria. Can release antibacterial substances such as organic acid, bacteriocin, diacetyl and the like in the growth and metabolism process of the microbial agent, can inhibit the growth of pathogenic bacteria and putrefying bacteria, and prevent the product from being putrefy caused by microorganisms.

Cyprinus carpio, a freshwater fish, Chinese alias Cyprinus carpio, fructus eriocarpi, and Rubus ocephalus, belongs to brown fish in Cyprinidae, native Asia, and later introduced into Europe, North America and other regions, and belongs to a fish with omnivory. 96% of the protein of the carp can be digested by the human body, and the necessary nutrient substances of the human body such as amino acid, mineral substances, vitamin A, vitamin D and the like can be provided. The carp has large volume and high meat yield, can be steamed and fried in clear in the cooking history of China for many years, can also be used as stuffing of dumplings, soup for cooking and the like, and has good color and taste. The Chinese carps are preserved by introducing oxygen through flowing water and are killed, so that the preservation cost is greatly increased, the consumers are required to perform self-treatment, the processing utilization rate is low, and the added value is reduced.

Fresh carps can be preserved after being sliced, the most common preservation method at present comprises chemical and physical preservation, the chemical preservation is generally to utilize chemical preservative to spray or soak on the surface of fish meat to prolong the preservation period of the fish meat, the chemical preservative is not beneficial to the health of human bodies in consideration of health, the low-temperature preservation technology commonly used in China can cause damage to fish meat cells, so that the water loss of the fish meat cells is caused, the taste of the fish meat is relatively poor, and the sensory score is sharply reduced.

In order to improve the commercial utilization level of carp fish meat, prolong the preservation time of carp fish meat, prevent potential food safety problems caused by a chemical preservation method and prevent the reduction of the sensory quality of food per se caused by a physical method, lactobacillus bacterium lactobacillus sake B2-4 which has long edible safety history and inhibits the growth of harmful microorganisms is utilized to biologically preserve the fish meat, so that the quality guarantee period of the fish meat can be prolonged, the safety of the fish meat can be ensured, and the economic value of a product can be improved.

Disclosure of Invention

The invention aims to provide lactobacillus sake B2-4 and application thereof in aquatic product preservation.

The invention provides lactobacillus sake (L actinobacillus sakei) B2-4, wherein the preservation number of the strain is CCTCC NO: M2018555.

The invention also provides application of the lactobacillus sake B2-4 in aquatic product preservation.

Further, the aquatic product includes carp meat.

Further, the aquatic product preservation method comprises the following steps:

(1) inoculating lactobacillus sake B2-4 to MRS culture medium for culture to obtain lactobacillus sake B2-4 culture solution;

(2) aquatic product treatment: cutting the edible meat of the aquatic product into slices or blocks for later use;

(3) centrifuging the culture solution of the lactobacillus sake B2-4 obtained in the step (1), removing the supernatant, washing thalli, and suspending the washed thalli in sterilized physiological saline to prepare lactobacillus sake B2-4 bacterial suspension;

(4) soaking the aquatic product obtained in the step (2) in the lactobacillus sake B2-4 bacterial suspension obtained in the step (3);

(5) and (4) taking out the aquatic product obtained in the step (4), draining and preserving.

Further, the inoculation amount of the lactobacillus sake B2-4 in the step (1) is 2-6% by volume, and the culture time is 18-24 h. Preferably, the inoculation amount of the lactobacillus sakei B2-4 in the step (1) is 2% by volume, and the culture time is 18 h.

Further, centrifuging for 10 minutes at 8000rmp/min under the centrifugation condition of 4-10 ℃ in the step (3); the solvent for washing the cells was sterilized normal saline with a concentration of 0.85%. Preferably, the centrifugation condition in the step (3) is 4 ℃, and the centrifugation is carried out for 10 minutes at 8000 rmp/min;

further, the concentration of the lactobacillus sake B2-4 bacterial suspension in the step (3) is 10⁵～10⁸CFU/m L, preferably, the concentration of the lactobacillus sakei B2-4 bacterial suspension is 10⁶CFU/mL。

Further, the soaking time in the step (4) is 20-60 minutes, and preferably, the soaking time is 30 minutes.

Further, the preservation temperature in the step (5) is 0-8 ℃, and preferably, the preservation temperature is 4 ℃.

Advantageous effects of the invention

The method provided by the invention provides the lactobacillus sake B2-4, and the lactobacillus sake B2-4 is applied to the preservation of the carp meat of aquatic products, so that the generation of biogenic amine, thiobarbituric acid and volatile basic nitrogen in the carp meat can be effectively reduced, the sensory quality of the carp meat is stabilized, and the shelf life of the carp meat is prolonged.

Drawings

FIG. 1 is a microscope photograph of Lactobacillus sake B2-4 (100 × 10).

FIG. 2 shows the pH change of surimi of carp under Sakei B2-4 stored at 4 deg.C, which is accessed L.

FIG. 3 shows the change of volatile basic nitrogen during storage of surimi of sakei B2-4 carp at 4 ℃ under L.

FIG. 4 shows the change of thiobarbituric acid during storage of surimi of Sakei B2-4 carp at 4 ℃ under L.

FIG. 5 shows the change of putrescine in the process of storing the minced carp with sakei B2-4 at 4 ℃ under L.

FIG. 6 shows the change of cadaverine during storage of surimi of Sakei B2-4 carp at 4 ℃ under L.

FIG. 7 shows the histamine change of minced carp inoculated with L sakei B2-4 during storage at 4 ℃.

Fig. 8 is a venn chart showing the similarity and overlapping of the flora composition in carps at the OUT level.

Fig. 9 is a carp surimi partial least squares regression model based on OUT levels.

FIG. 10 is the strain abundance of carp flesh as determined by genus level 16S rRNA after 12 days of storage at 4 ℃.

Detailed Description

The following non-limiting examples will allow one of ordinary skill in the art to more fully understand the present invention, but are not intended to limit the invention in any way.

Example 1

The lactobacillus sake B2-4 is screened in the intestinal tract of the turbot, and the method comprises the following steps:

a. strain separation method

(1) MRS Medium preparation

Weighing and uniformly mixing 20 g/L of glucose, 10 g/L of peptone, 5 g/L of yeast extract, 10 g/L of beef extract, 1m L of Tween 80, 2 g/L of dipotassium hydrogen phosphate, 2 g/L of diammonium citrate, 5 g/L of sodium acetate, 0.58 g/L of magnesium sulfate heptahydrate and 0.25 g/L of manganese sulfate tetrahydrate, dissolving the obtained mixture in deionized water, and sterilizing the obtained solution at 121 ℃ for 20min to obtain the MRS culture medium.

Weighing 1g of the intestinal tissue of the turbot, putting the weighed sample into 4ml of sterilized physiological saline with the concentration of 0.85 percent by weight, performing gradient dilution on the sample by adopting a ten-fold dilution method, then coating the sample in the culture medium containing 2 percent agar in the step (1), performing anaerobic culture for 48 to 72 hours at the temperature of 37 ℃, picking out a single colony, inoculating the single colony in an MRS solid culture medium containing 2 percent agar again, and performing culture for 24 to 48 hours at the temperature of 37 ℃.

b. Stain of the Strain

Staining the strain by gram staining method, morphologically observing by microscope, simultaneously performing catalase test, and strictly performing aseptic operation in aseptic environment.

The gram stain visualization procedure was as follows:

the gram staining procedure is smear fixing, staining, washing, air drying and microscopic examination, and specifically comprises the following steps:

(1) smearing and fixing: taking a clean glass slide (without oil stain), adding a drop of distilled water in the center of the glass slide, taking a bacterium liquid smear according to an aseptic technique, uniformly mixing, coating into a film, and drying: the smear was allowed to air dry naturally.

(2) Crystal violet dyeing: placing the glass slide on a glass slide shelf of a waste liquid tank, adding a proper amount of crystal violet staining solution (Biyuntian) to stain for 1-2 min; washing with water: the staining solution was decanted and carefully rinsed with water.

(3) Mordant dyeing: carrying out mordant dyeing for 1min by using iodine solution; washing with water: the iodine solution was washed with water.

(4) And (3) decoloring: inclining the slide, continuously dropwise adding 95% ethanol for decolorizing for 20-25s until the effluent is colorless, and immediately washing with water.

(5) Counterdyeing: dripping yellow-reddish staining solution for counterstaining for 2-3 min; washing with water: flushing staining solution for removing red stain on smear

(6) Drying: the dyed smear was blotted dry with absorbent paper.

(7) Microscopic examination: the gram staining reactivity of the bacteria is judged by observing the bacteria with an oil lens firstly with low power and then with high power and finally with an oil lens. Gram-positive bacteria are stained with bluish purple bacteria, and gram-negative bacteria are stained with red bacteria.

c. Strain catalase test

The gram-positive strain is picked up by an inoculating loop and placed on a glass slide containing 3% of hydrogen peroxide solution drops, if a large amount of bubbles are generated, the gram-positive strain is positive, otherwise, the gram-positive strain is negative.

Inoculating the strain into an MRS liquid culture medium, culturing at 37 ℃ for 24-48 h, entrusting Dalianbao bio-company to perform 16S rDNA sequencing identification, wherein the sequence is shown as SEQ ID NO.1, and the identification result is lactobacillus sake (L actabacillus sakei).

The strain is preserved in China center for type culture collection (CCTCC for short, address: Wuchang Lojia mountain, China academy of sciences, postal code 430072.) and the strain preservation number of lactobacillus sake (L actinobacillus sakei) B2-4 is M2018555, the preservation date is 8 months and 21 days in 2018, the strain is classified and named as lactobacillus sake (L actinobacillus sakei), and the strain name is B2-4.

Example 2

The invention relates to application of lactobacillus sake B2-4 in biological preservation of fresh carp meat, which comprises the following steps:

(1) preparing MRS culture medium, namely weighing the raw materials according to the proportion of 20 g/L glucose, 10 g/L peptone, 5 g/L yeast extract, 10 g/L beef extract, 1m L Tween 80, 2 g/L dipotassium hydrogen phosphate, 2 g/L diammonium citrate, 5 g/L sodium acetate, 0.58 g/L magnesium sulfate heptahydrate and 0.25 g/L manganese sulfate tetrahydrate, uniformly mixing, mixing the obtained mixture in deionized water, and sterilizing the obtained solution at 121 ℃ for 20min to obtain the MRS culture medium.

(2) Activating the strain: and (3) streaking and inoculating the freeze-preserved lactobacillus sake B2-4 into the MRS culture medium obtained in the step (1) containing 2% agar, culturing at the constant temperature of 37 ℃ for 24 hours, and picking a single colony to culture in the MRS culture medium obtained in the step (1) for 18 hours.

(3) Strain culture: adding the activated strain obtained in the step (2) into the MRS culture solution obtained in the step (1) according to the inoculation amount of 2% of the weight of the MRS culture medium, culturing for 18 hours in a constant-temperature incubator at the temperature of 37 ℃, and continuously carrying out passage twice to obtain the culture solution of the lactobacillus sake B2-4.

(4) Carp treatment: removing scales, heads and viscera of carps at room temperature, cleaning, and cutting into fish slices with fish meat thickness of 2cm along the direction perpendicular to the spine;

(5) preparing fresh-keeping bacterial liquid: centrifuging the culture solution of the lactobacillus sake B2-4 obtained in the step (3) at 4 ℃ and 8000rmp/min for 10 minutes, washing with sterilized 0.85% physiological saline for 2-3 times, and suspending the washed thallus in the sterilized physiological saline to prepare 10⁶CFU/M L Lactobacillus sake B2-4 bacterial suspension (group M6);

(6) soaking the carp fish meat obtained in the step (4) in the lactobacillus sake B2-4 bacterial suspension obtained in the step (5) for 30 minutes;

(7) taking out the carp flesh obtained in the step (6), draining, mincing the carp flesh into carp surimi by using a tissue mincing machine, and preserving at 4 ℃.

Example 3

(2) Activating the strain: and (3) streaking and inoculating the freeze-preserved lactobacillus sake B2-4 into the MRS solid culture medium obtained in the step (1) containing 2% agar, culturing at the constant temperature of 37 ℃ for 48h, and picking out a single colony to culture in the MRS culture medium obtained in the step (1) for 18 h.

(3) Strain culture: adding the activated strain obtained in the step (2) into the MRS culture solution obtained in the step (1) according to the inoculation amount of 2% of the weight of the MRS culture medium, culturing for 18 hours in a constant-temperature incubator at the temperature of 37 ℃, and continuously carrying out passage twice to obtain a culture solution of lactobacillus sake B2-4;

(5) preparing fresh-keeping bacterial liquid: centrifuging the lactobacillus sake B2-4 obtained in the step (3) at 4 ℃ at 8000rmp/min for 10 minutes, washing with sterilized PBS or 0.85% normal saline for 2-3 times, preparing bacterial suspension of the washed thallus in sterilized normal saline, and preparing the bacterial suspension into 10⁸CFU/M L Lactobacillus sake B2-4 bacterial suspension (group M8);

(6) soaking the carp fish meat obtained in the step (4) in the lactic acid bacteria fresh-keeping solution obtained in the step (5) for 30 minutes;

Comparative example 1

In this comparative example, only the step (4) of example 2 was carried out, and the fish meat was immersed in sterilized 0.85% physiological saline for 30min to set as a blank control group (CK group), minced into carp minced fillet using a tissue mincing machine, and stored in a refrigerator at 4 ℃ for 12 days.

Example 4

The fish meat samples of example 2, example 3 and comparative example 1 were subjected to sensory evaluation tests as follows:

in the present test, the sensory evaluation was measured according to sensory evaluation criteria. The 6 sensory persons performed sensory evaluation of the preserved surimi at 0d and 12d, respectively. Before the experiment is started, all carp minced fillet samples are preserved in a sterilized culture dish to ensure the original flavor substances. Randomly assigning 6 sensory persons for sensory evaluation; the evaluation indices and descriptive criteria are shown in table 1:

TABLE 1 sensory evaluation criteria for carp surimi

Note: the appearance, texture and smell were scored separately and then scored together to obtain an average

The sensory evaluation results of carp surimi on day 0 and day 12 are shown in table 2, and the experimental results in table 2 show that the color of carp surimi of M6 group still maintains the original pink color of fish meat, the sticky condition of surimi is light, the sensory score is higher than that of CK and M8, and the sensory evaluation personnel can accept the sensory evaluation results.

TABLE 2 sensory evaluation results of carp surimi on

days

0 and 12

Example 5

The fish meat samples of example 2, example 3 and comparative example 1 were subjected to the pH determination test as follows:

in the test, the pH of the sample is measured according to a standard method of GB5009237-2016 national food safety standard pH value measurement, 2.00g (accurate to 0.01g) of minced fillet is weighed and placed in a 50m L centrifuge tube, 10m L distilled water is added, a vortex oscillator is used for uniformly mixing, the mixture is placed at room temperature for 30min, and the pH value of the supernatant is measured.

Example 6

The fish meat samples of example 2, example 3 and comparative example 1 were subjected to the volatile basic nitrogen TVBN determination test as follows:

in the test, 2.00g of sample TVBN (accurate to 0.01g) is weighed and placed in a 50m L centrifugal tube, 10m L distilled water is added, a vortex oscillator is used for uniformly mixing, standing and extracting are carried out for 30min, 8000r/min is carried out at 4 ℃ for 10min, TVBN is carried out according to a third method of national standard GB 5009.228-2016 plate diffusion method, the TVB-N value of the fresh marine fish is within 15mg/100g and is the first-order freshness according to the national standard, and the fresh marine fish cannot be sold on a shelf when the TVB-N value is more than 30mg/100g, and the effect of two groups of carp fish meat inoculated with lactobacillus sake B2-4 on the 12 th day is better than that of the CK group and is more close to the TVBN value specified by the national standard, as shown by the experimental result in figure 3.

Example 7

Fish meat samples of example 2, example 3 and comparative example 1 were subjected to thiobarbituric acid TBA determination test as follows:

in the test, sample TBA is measured according to GB 5009.181-2016 determination of malonaldehyde in national food safety standard, 1g (accurate to 0.01g) of sample is weighed and placed in a 50m L test tube, 10m L7.5.5% trichloroacetic acid solution is absorbed and added into a centrifuge tube, the centrifuge tube is shaken up, a cover is sealed, the centrifuge tube is placed on a constant temperature oscillator, the centrifuge tube is shaken up for 30min at 50 ℃, the sample is taken out and cooled to room temperature, residue filtration is carried out by using double-layer quantitative slow speed filter paper, initial primary filtrate is discarded, the residual filtrate is used for sample determination, 1m L filtrate and 1ml of standard are respectively added into 1m L TBA, the mixed solution is mixed evenly, the mixture is placed in a water bath kettle at 90 ℃ for reaction for 30min, the sample is taken out and cooled to room temperature, the sample is adjusted to zero point at blank, the absorbance values of the sample solution and the standard solution are determined at 532nm, the abscissa is the mass concentration of the standard solution, and the ordinate is the absorbance value of the determination, and a standard curve and.

The results are shown in FIG. 4, with the addition of 10⁶CFU/g and 10⁸The TBA of carp minced fillet of the CFU/g of the lactobacillus sake B2-4 is always lower than that of the CK group before 12 days, and the lactobacillus sake B2-4 is shown to be capable of delaying the oxidation of fat in fish meat. Lactobacillus sake B2-4The alleviation of fat oxidation may be due to the ability of Lactobacillus sake B2-4 to produce antioxidant substances such as catalase, superoxide dismutase and glutathione peroxidase, etc.

Example 8

The fish meat samples of example 2, example 3 and comparative example 1 were subjected to the biogenic amine BAs assay as follows:

in this test, the BAs sample is measured by measuring biogenic amine in GB5009.208-2016 food safety national standard food, weighing 2.00g (accurate to 0.01g) minced fillet, placing in a 15m L centrifuge tube, adding 4m L.5% trichloroacetic acid solution, ultrasonic vibration extracting for 30min, centrifuging (8000r/min,4 ℃,10min) to obtain supernatant, centrifuging the residue once with 4m L.5% trichloroacetic acid, combining the two supernatants, taking a 0.75m L supernatant sample or standard solution in a test tube, adding 150 μm saturated sodium carbonate, mixing, adding 0.75m L derived reagent dansyl chloride, mixing, incubating in 45 ℃ water bath for 30min, adding 150 μ L% ammonia water in the test tube to remove residual dansyl chloride, incubating at 45 ℃ for 15min, adding 0.2m L acetonitrile to dissolve organic residue, adding 254.22 min before the supernatant, filtering with acetonitrile, filtering with 254.22 min, eluting with a flow rate of acetonitrile under 3640 nm wavelength of 20.40 mm of ultraviolet absorption chromatography, and detecting the sample flow rate of the sample by ultraviolet absorption chromatography (L nm).

TABLE 3 HP L C mobile phase ratio and flow Rate information

FIGS. 5 to 7 are respectively the changes of putrescine, cadaverine and histamine during the storage of carp surimi inoculated with the Lactobacillus sake B2-4 of the present invention at 4 ℃.

As shown in the experimental result of FIG. 5, on the 8 th day, the putrescine content of the untreated surimi is as high as 3.56 +/-0.48 mg/kg, which is far higher than that of the added 10⁶CFU/g and 10⁸1.49 +/-0.76 mg/kg and 1.84 +/-0.28 mg/kg (p) of carp surimi of CFU/g lactobacillus sake B2-4<0.05) putrescine content, which indicates that the increase of the concentration of lactobacillus sake B2-4 can inhibit the production of putrescine.

As shown in the experimental results of FIG. 6, on the 8 th day, the cadaverine content of the untreated surimi is as high as 8.45 +/-01.41 mg/kg, which is far higher than that of the minced fillet added with 10 percent of cadaverine⁶CFU/g and 10⁸2.36 +/-1.49 mg/kg and 2.50 +/-0.17 mg/kg (p) of carp surimi of CFU/g lactobacillus sake B2-4<0.05) putrescine content, which indicates that the increase of the concentration of lactobacillus sake B2-4 can inhibit the production of putrescine.

As shown in the experimental results of FIG. 7, there was no significant difference in the histamine content (p >0.05) during the 12-day storage period, and the addition of Lactobacillus sake B2-4 had no inhibitory effect on histamine.

Example 9

The fish meat samples of example 2, example 3 and comparative example 1 were subjected to the volatile component determination test as follows:

measuring volatile components by gas mass spectrometry, accurately weighing 3.5g of prepared carp surimi and 1m L saturated NaCl solution, mixing the two solutions, placing the mixture in a 20m L gas sample bottle, placing the mixture in a 50 ℃ constant-temperature magnetic stirrer for water bath, wherein the water bath time is 15min, inserting an aged extraction head for adsorption, wherein the adsorption time is 40min, rapidly inserting the mixture into a gas chromatography sample inlet, the resolution time of the extraction head is 5min, adopting an HP-5MS capillary column (30m × 0.25mm × 0.25 mu m), the sample inlet temperature is 250 ℃, the column temperature is 35 ℃, keeping the temperature for 3min, heating to 60 ℃ by a 5 ℃/min program, heating to 140 ℃ by a 6 ℃/min program, heating to 230 ℃ by 8 ℃/min, keeping for 5min, taking helium (He) as carrier gas, the flow rate is 1.0m L/min, the resolution temperature is 250 ℃, an undivided flow mode is adopted, the mass spectrometry conditions are that the ion source temperature is 280 ℃, the scanning range is 550 m/z, the voltage is 4670 He, the volatile components in a gas chromatography, and the volatile components are subjected to a four-stage chromatography ionization analysis, and the volatile components are analyzed by a four-stage chromatography, and the volatile component analysis is performed by a four-stage chromatography^-6And (4) showing.

Table 4 the results of the experiments show that Lactobacillus sake B2-4 was added to fish meat to a concentration of 10⁸CFU/g and 10⁶CFU/g can inhibit the generation of putrefactive volatile components in fish meat, which indicates that Lactobacillus sake B2-4 can delay the putrefaction process.

TABLE 4 change of volatile components of carp surimi during storage at 4 deg.C

Example 10

The fish meat sample of example 2, example 3 and comparative example 1 was tested for bacterial composition by a third party testing company.

A. Collecting samples: the minced fillet is picked up under a clean bench for the first day and the last day, and stored in a sterilized centrifuge tube at-80 ℃ for sample delivery.

B. DNA extraction and PCR amplification: according to

The total DNA extraction was performed using the soil kit (Omega Bio-tek, Norcross, GA, U.S.) instructions, DNA concentration and purity were determined using NanoDrop2000, and DNA extraction quality was determined using 1% agarose gel electrophoresis; V3-V4 variable region was PCR amplified with primers 338F (5'-ACTCCTACGGGAGGCAGCAG-3') and 806R (5 '-GGACTACHVGGGTWTCTAAT-3'), using the following protocol: pre-denaturation at 95 ℃ for 3min, 27 cycles (denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 30s), and final extension at 72 ℃ for 10min (PCR instrument:

9700 type) amplification system was 20. mu. L, 4. mu. L5 × FastPfu buffer, 2. mu. L2.5.5 mM dNTPs, 0.8. mu. L primer (5. mu.M), 0.4. mu. L FastPfu polymerase, 10ng DNA template.

C. IlluminaMiseq sequencing: the PCR product was recovered using a 2% agarose gel, purified using AxyPrepDNaGelExtractionkit (Axygen Biosciences, UnionCity, Calif., USA), eluted with Tris-HCl, and detected by 2% agarose electrophoresis. Utilizing QuantiFluor^TM-ST(Promega,USA) was measured. The purified amplified fragments were used to construct a library of PE2 x 300 according to the standard protocol of the Illumina miseq platform (Illumina, san diego, USA).

D. The library construction steps comprise ① connection of a Y-shaped joint, ② removal of joint self-connection fragments by magnetic bead screening, ③ enrichment of library templates by PCR amplification, ④ sodium hydroxide denaturation to generate single-stranded DNA fragments, sequencing by a MiseqPE300 platform of Illumina, and uploading raw data to an NCBI database.

E. And (3) data processing, namely performing quality control on the original sequencing sequence by using Trimmomatic software and splicing by using F L ASH software:

① setting a 50bp window, if the average quality value in the window is lower than 20, cutting all the sequences at the back end of the base from the front end of the window, then removing the sequences with the length lower than 50bp after quality control, ② splicing the sequences at the two ends according to the overlapped base overlap, the maximum mismatching rate between the overlap is 0.2 during splicing, the length is more than 10bp, ③ splitting the sequence to each sample according to the barcodes and the primers at the head and the tail of the sequence, the barcodes need to be matched accurately, the primer allows the mismatching of 2 bases, and removes the sequences with fuzzy bases.

F. Using UPARSE software (version7.1http:// drive5.com/UPARSE /), OTU clustering was performed on the sequences according to 97% similarity, and single sequences and chimeras were removed during clustering. Species class annotation was performed on each sequence using rdpclasifier (http:// rdp. cme. msu. edu /), and the Silva data (SSU123) were aligned, setting the alignment threshold at 70%.

As shown in fig. 8, fig. 9 and fig. 10, the lactobacillus sakei B2-4 has an effect on the abundance of the flora in the carp meat preservation process, and lactobacillus sakei B2-4 inhibits the growth and propagation of some bacteria in the fish meat, such as putrefying bacteria such as B. Has good fresh-keeping effect.

The foregoing examples are provided for illustration and description of the invention only and are not intended to limit the invention to the scope of the described examples. Furthermore, it will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that many variations and modifications may be made in accordance with the teaching of the present invention, which variations and modifications fall within the scope of the invention as claimed.

SEQUENCE LISTING

<110> university of Dalian Industrial university

<120> lactobacillus sake B2-4 and application thereof in aquatic product preservation

<130>2020

<160>1

<170>PatentIn version 3.5

<210>1

<211>1413

<212>DNA

<213> Lactobacillus sake (L actabacillus sakei)

<400>1

aacgcactct cgtttagatt gaaggagctt gctcctgatt gataaacatt tgagtgagtg 60

gcggacgggt gagtaacacg tgggtaacct gccctaaagt gggggataac atttggaaac 120

agatgctaat accgcataaa acctaacacc gcatggtgta gggttgaaag atggtttcgg 180

ctatcacttt aggatggacc cgcggtgcat tagttagttg gtgaggtaaa ggctcaccaa 240

gaccgtgatg catagccgac ctgagagggt aatcggccac actgggactg agacacggcc 300

cagactccta cgggaggcag cagtagggaa tcttccacaa tggacgaaag tctgatggag 360

caacgccgcg tgagtgaaga aggttttcgg atcgtaaaac tctgttgttg gagaagaatg 420

tatctgatag taactgatca ggtagtgacg gtatccaacc agaaagccac ggctaactac 480

gtgccagcag ccgcggtaat acgtaggtgg caagcgttgt ccggatttat tgggcgtaaa 540

gcgagcgcag gcggtttctt aagtctgatg tgaaagcctt cggctcaacc gaagaagtgc 600

atcggaaact gggaaacttg agtgcagaag aggacagtgg aactccatgt gtagcggtga 660

aatgcgtaga tatatggaag aacaccagtg gcgaaggcgg ctgtctggtc tgtaactgac 720

gctgaggctc gaaagcatgg gtagcaaaca ggattagata ccctggtagt ccatgccgta 780

aacgatgagt gctaggtgtt ggagggtttc cgcccttcag tgccgcagct aacgcattaa 840

gcactccgcc tggggagtac gaccgcaagg ttgaaactca aaggaattga cgggggcccg 900

cacaagcggt ggagcatgtg gtttaattcg aagcaacgcg aagaacctta ccaggtcttg 960

acatcctttg accactctag agatagagct ttcccttcgg ggacaaagtg acaggtggtg 1020

catggttgtc gtcagctcgt gtcgtgagat gttgggttaa gtcccgcaac gagcgcaacc 1080

cttattacta gttgccagca tttagttggg cactctagtg agactgccgg tgacaaaccg 1140

gaggaaggtg gggacgacgt caaatcatca tgccccttat gacctgggct acacacgtgc 1200

tacaatggat ggtacaacga gttgcgagac cgcgaggttt agctaatctc ttaaaaccat 1260

tctcagttcg gattgtaggc tgcaactcgc ctacatgaag ccggaatcgc tagtaatcgc 1320

ggatcagcat gccgcggtga atacgttccc gggccttgta cacaccgccc gtcacaccat 1380

gagagtttgt aacacccaaa gccggtgagg taa 1413

Claims

1. Lactobacillus sake (L actinobacillus sakei) B2-4 is characterized in that the preservation number of the strain is CCTCC NO: M2018555.

2. The use of lactobacillus sake B2-4 according to claim 1 for the preservation of aquatic products.

3. The use according to claim 2, wherein preservation of aquatic products comprises the steps of:

4. The use of claim 3, wherein the lactobacillus sake B2-4 is inoculated in an amount of 2-6% by volume in step (1), and the culture time is 18-24 h.

5. The use according to claim 3, wherein the centrifugation conditions in step (3) are 4-10 ℃ at 8000rmp/min for 10 minutes; the solvent for washing the cells was sterilized normal saline with a concentration of 0.85%.

6. The use according to claim 3, wherein the concentration of the Lactobacillus sake B2-4 bacterial suspension in the step (3) is 10⁵～10⁸CFU/mL。

7. The use according to claim 3, wherein the soaking time in the step (4) is 20 to 60 minutes.

8. Use according to claim 3, wherein the preservation temperature in step (5) is 0-8 ℃.