CN116042435A

CN116042435A - Lactobacillus sake subspecies and application thereof

Info

Publication number: CN116042435A
Application number: CN202210936626.8A
Authority: CN
Inventors: 吴永祥; 董心雨; 汪婷婷; 蒋小旋; 刘刚; 李晨; 代琪琪; 杨波; 佘新松; 胡长玉; 金声琅
Original assignee: Huangshan University
Current assignee: Huangshan University
Priority date: 2022-08-05
Filing date: 2022-08-05
Publication date: 2023-05-02

Abstract

The invention discloses a lactobacillus sake subspecies and application thereof. The lactobacillus sake subspecies are named CMC1-8 and are preserved in China Center for Type Culture Collection (CCTCC), and addresses: the preservation number is CCTCC M2022621, and the preservation time is 2022, 5 and 13 days. Researches show that the strain has better salt resistance and low temperature resistance and has remarkable antioxidation and bacteriostasis effects; the method can be well applied to low-temperature fermentation of the mandarin fish, improves sensory evaluation of the mandarin fish, effectively reduces the generation of nitrite, thiobarbituric acid reactant and histamine in the fermentation process of the mandarin fish, improves edible safety of the mandarin fish, and can be widely applied to the field of curing of the mandarin fish.

Description

Lactobacillus sake subspecies and application thereof

Technical Field

The invention relates to the field of bioengineering, in particular to a lactobacillus sake subspecies and application thereof.

Background

Protease is a kind of catalytic enzyme capable of hydrolyzing protein peptide chain, and plays a very important role in industrial fields such as food, washing, leather and the like as commercial enzyme with the largest production and sales in the world. Microorganisms are an important source of proteases, which are produced by plants and animals more easily isolated and purified than proteases produced by them. At present, the microbial protease is mainly middle-temperature protease in industrial application, and has certain limitation because the optimal enzyme activity is about 50 ℃. Proteases can be classified into three categories of acidic (pH value of 2.0-6.0), neutral (pH value of 6.0-9.0) and alkaline proteases (pH value of more than 9.0) according to the optimal reaction pH value, wherein the neutral proteases are the earliest and clear to study; however, there are few reports in the literature that neutral proteases can withstand high temperatures of 60℃and above, and that screening for proteases that are both high and low temperature from lactic acid bacteria is not relevant.

The stink mandarin fish is a traditional flavor fermented food with delicious taste and unique flavor, which is prepared by taking fresh mandarin fish as a raw material and adding a certain spice under the condition of low temperature and low salt and fermenting by means of self microorganisms. The fish meat is degraded into flavor substances such as micromolecular peptide, free amino acid and the like under the low temperature condition by the action of microbial protease in the fermentation process, and the substances are core components contributing to the flavor, so that the delicate flavor and the quality of the stinky mandarin fish are directly influenced. From this, it can be seen that the protease-producing microbial strain of mandarin fish is closely related to the formation of fermentation flavor and quality. However, at present, the traditional mandarin fish is prepared by natural fermentation, which has the problems of long fermentation period, poor product quality, different flavor and the like, so that the standardized and industrialized production of the mandarin fish is seriously hindered, and the market supply requirement cannot be met. Therefore, the dominant lactobacillus for producing the protease is separated and identified from the mandarin fish, and is applied to the curing process of the mandarin fish, so that the method is an effective way for improving the quality of the mandarin fish products, is a key problem to be solved in the mandarin fish industry of the yellow mountain, and has a wide application prospect.

Disclosure of Invention

The invention aims to provide a lactobacillus sake subspecies and application thereof in curing and fermenting stinky mandarin fish.

In order to achieve the above purpose, the invention adopts the following technical means:

the invention discloses a method for preparing a strain which can produce protease with high temperature resistance and low temperature resistance, which comprises the steps of separating lactobacillus sake subspecies from fermented mandarin fish bodies, separating and screening the fermented mandarin fish with the characteristics of CMC1-8, identifying the morphological and physiological chemical characteristics of the strain, determining the 16S rRNA sequence of the strain, comparing and analyzing, and finally identifying the strain as lactobacillus sake subspecies (Lactobacillus sakei subsp.sakei). The strain is preserved in China Center for Type Culture Collection (CCTCC) at 5 and 13 days 2022, and has a preservation number of CCTCC M2022621 at the university of Wuhan in China. The strain has good salt resistance and low temperature resistance, has remarkable antioxidation and bacteriostasis effects, and can be well applied to low-temperature low-salt fermentation of the stink mandarin fish; the optimal reaction temperature of the protease produced by CMC1-8 is 70 ℃, but the protease can keep the enzyme activity of more than 67.65% at 10 ℃, has low-temperature adaptability, can still keep the enzyme activity of 91.95% at 80 ℃, and has better thermal stability; the optimal pH of CMC1-8 protease is about 9, belongs to neutral protease, has higher enzyme activity when the NaCl mass concentration is 3-11%, and has certain salt tolerance.

The application of the lactobacillus sake subspecies in curing and fermenting the stinky mandarin fish comprises the following steps:

1) Removing scales and gills of fresh mandarin fish, removing viscera by cutting, cleaning, and draining;

2) Placing the drained mandarin fish in order into a fermentation tank, adding NaCl solution with the NaCl mass concentration of 3-9% until the mandarin fish is submerged, adding pickling auxiliary materials, uniformly stirring, inoculating lactobacillus sake subspecies suspension with the mass percentage of 0.05-0.8% into the fermentation tank, and uniformly stirring;

3) The sectional fermentation is adopted: the first stage is to set the fermentation temperature at 10-16 ℃ and the fermentation time at 1-2 d; then compacting the stinky mandarin fish by using a heavy object to ferment in the second stage, wherein the weight of the heavy object pressing the stinky mandarin fish is 1.5-3 times of the mass of the mandarin fish in the fermentation tank; the second stage fermentation temperature is 6-10 deg.c and fermentation time is 4-7 d.

The specific preparation method of the lactobacillus sake subspecies bacterial suspension in the step 2) comprises the following steps:

s1: inoculating the lactobacillus sake subspecies CMC1-8 to MRS culture medium for culture, and culturing at 10-16 ℃ for 24-48 h to obtain CMC1-8 culture solution;

s2: centrifuging the culture solution of the lactobacillus sake subspecies CMC1-8 obtained in the step S1 at the temperature of 10-15 ℃ for 5-8 min, setting the rotation speed of a centrifuge to 6000-10000 rpm, and standing for layering;

s3: removing supernatant, washing thallus with sterilized physiological saline, suspending the washed thallus in 0.85% sterilized physiological saline to obtain lactobacillus sake subspecies suspension with a concentration of 10 ⁷ ～10 ⁹ CFU/mL。

The invention has the beneficial effects that: the lactobacillus sake subspecies obtained by screening have better salt resistance and low temperature resistance, and have obvious antioxidation and bacteriostasis effects; can be well applied to low-temperature fermentation of the mandarin fish, improves sensory evaluation of the mandarin fish, effectively reduces the generation of nitrite, thiobarbituric acid reactant and histamine in the fermentation process of the mandarin fish, and improves the edible safety of the mandarin fish. Meanwhile, the optimal reaction temperature of the protease produced by the lactobacillus sake subspecies is 70 ℃, can keep the enzyme activity of more than 67.65% at 10 ℃, has low-temperature adaptability, can still keep the enzyme activity of 91.95% at 80 ℃, and has better thermal stability; the optimal pH of the CMC1-8 protease is about 9, belongs to neutral protease, has higher enzyme activity when the NaCl mass concentration is 3% -11%, has better salt tolerance, can improve the pickling speed of the stink mandarin fish when being applied to pickling the stink mandarin fish, can ensure the consistency of the taste of the stink mandarin fish, and provides favorable conditions for standardized and large-scale production of the stink mandarin fish.

Drawings

FIG. 1A is a colony chart of Lactobacillus sake subspecies CMC1-8 in the present invention.

FIG. 1B is a gram stain of Lactobacillus sake subspecies CMC1-8 according to the present invention.

FIG. 1C is an electron microscope scan of lactobacillus sake subspecies CMC1-8 in accordance with the present invention.

FIG. 2 is a phylogenetic tree of the 16S rRNA sequences of the Lactobacillus sake subspecies CMC1-8 strain of the present invention.

FIG. 3 is a graph showing the growth curve and pH change of the strain CMC1-8 of Lactobacillus sake subspecies according to the present invention.

FIG. 4 is a graph showing the salt tolerance curve and the temperature change resistance curve of the strain CMC1-8 of the lactobacillus sake subspecies of the present invention.

FIG. 5 is a standard graph of casein in the present invention

FIG. 6 is a schematic diagram showing the enzyme activities of proteases produced by the strain CMC1-8 of the Lactobacillus sake subspecies according to the present invention at different temperatures.

FIG. 7 is a schematic diagram showing the enzyme activities of proteases produced by the strain CMC1-8 of the Lactobacillus sake subspecies according to the present invention at different pH values.

FIG. 8 is a schematic diagram showing the enzyme activity of protease produced by the strain CMC1-8 of Lactobacillus sake subspecies at different concentrations of NaCl in the present invention.

FIG. 9 is a graph showing the evaluation of the effect of CMC1-8 strain on the sensory quality of stinky mandarin fish in the present invention.

Detailed Description

The invention will be further described with reference to specific embodiments, and advantages and innovations of the invention will be apparent from the description.

The raw materials, the auxiliary materials, the culture medium, the reagents and the instrument and equipment related to the invention are all general products sold in the market and are not described one by one.

Example 1: screening and identification of the strains of the invention

The protease-producing lactobacillus sake subspecies are derived from fermentation products of siniperca chuatsi, are obtained after multiple screening, are named as CMC1-8, and are identified as lactobacillus sake subspecies (Lactobacillus sakei subsp.sakei). The strain is preserved in China Center for Type Culture Collection (CCTCC) at 5 and 13 days 2022, and has a preservation number of CCTCC M2022621 at the university of Wuhan in China.

The acquisition step of the lactobacillus sake subspecies CMC1-8 comprises the steps of sample acquisition, primary screening of strains, re-screening, identification of protease-producing strains and fermentation characteristic research.

(1) Sample collection: selecting Huang Shanshi Hu Xingtang peach blossom running water food limited company pickled stinky mandarin fish;

(2) Primary screening of strains: taking a certain amount of fish meat from the cured stinky mandarin fish backs, fish maws and fish tails, adding into a conical flask (containing a certain amount of small glass beads) filled with 180mL of sterile physiological saline after shearing, and rapidly vibrating and shaking uniformly to obtain 10 parts ^-1 Sample homogenate with concentration is diluted by gradient with 10 times by taking sterilized normal saline as solvent, and is absorbed 10 times ^-4 ～10 ^-7 100 mu L of diluted sample homogenate is coated on the surface of a Gibbons agar plate containing 1% of skim milk powder, and the mixture is cultured for 3-5 d at the temperature of 12 ℃ in an incubator to observe whether the mixture grows and generate transparent rings.

(3) And (3) re-screening strains: according to the ratio of the radius of the generated transparent ring to the radius of the bacterial colony, selecting the bacterial strain with larger ratio as the protease producing strain of the re-screening. Inoculating to a liquid Gibbons culture medium, culturing for 2-4d under the environmental condition of 12 ℃, centrifuging for 5-8 min by adopting a high-speed centrifuge, taking supernatant after standing at 5000-8000 rpm, measuring the protease activity of the supernatant, and selecting a strain with the maximum protease activity, namely CMC1-8, as the next research.

(4) Identification of protease-producing strains:

(1) gram staining: marking the activated bacterial liquid on an MRS solid culture medium, culturing for 24 hours at 37 ℃, and recording the characteristics of colony color, shape, size and the like; and (5) picking single colonies for gram staining, and observing the characteristics of the strain such as color, shape and the like under a microscope.

(2) Microscopic electron microscope observation: selecting a culture dish with better single colony distribution by using an S-3400N type scanning electron microscope, freezing for 1d at the temperature of minus 20 ℃, selecting a colony by using an inoculating loop, fixing the colony on a sample holder, spraying gold on the cross section of the sample by using an ion sputtering instrument, observing by using a scanning electron microscope (5000 times), and photographing.

(3) Molecular biological identification and construction of phylogenetic tree: and (3) extracting, separating and purifying the obtained strain genome DNA by adopting a bacterial genome extraction kit, and carrying out PCR amplification on the total DNA of the extracted strain to obtain 3 specific bands with the size of about 1500 bp. And then, the obtained specific fragment is purified and sequenced, and BLAST homology comparison is carried out on the sequencing result in NCBI website, so as to construct a phylogenetic tree. Specifically, the sequences were amplified using bacterial universal primer 16S rRNA. PCR amplificates: forward primer 27F:5'-AGAGTTTGATCCTGG CTCAG-3'; reverse primer 1942R: 5'-CTACGGCTACCTTGTTACGA-3'. PCR amplification reaction system: amplification conditions: pre-denaturation at 95 ℃ for 5min; denaturation at 95℃for 30s; annealing at 58 ℃ for 30s; extending at 72℃for 1min 30s; after 35 cycles, the extension was carried out at 72℃for 7min. Primer synthesis and sequencing of PCR products were carried out by Shanghai Sanny Biotechnology Co. The 16S rRNA gene sequences thus obtained were aligned and analyzed for homology in NCBI BLAST system, and a suitable reference strain sequence was selected, and a phylogenetic tree was constructed by the Neighbor-Joing (NJ) method using MEGA11.0 software, and the 16S rRNA sequence phylogenetic tree of CMC1-8 strain was constructed as shown in FIG. 2.

(5) Identification of fermentation characteristics: CMC1-8 strain was inoculated in MRS broth medium for 2 times of activation at 37℃and the OD value of the bacterial suspension was adjusted to 1.0 at 600nm for use.

(1) Growth curve and acid production capacity: the bacterial suspension is inoculated into MRS broth culture medium in a proportion of 1% by volume, and a non-inoculated liquid culture medium is set as a blank control. The incubation was carried out at 37℃for 24 hours, with OD and pH values measured 1 time every 4 hours. The enzyme-labeled instrument was set at 600nm to measure absorbance and pH was measured with a pH meter. The growth curve was plotted by OD change and the pH analysis of the culture process was determined for its acid generating capacity as shown in fig. 3. As shown in FIG. 3, CMC1-8 grows fast in 3-9 hr, and the strain enters logarithmic phase to reach the maximum thallus concentration, and the acidity is lowered obviously. And 9-24 hours starts to enter a stable period, the strain grows and metabolizes slowly, the acid production capacity is also slowed down, and the growth condition of the strain is consistent with the pH change condition. At 24 hours, the pH reaches 5.1, which is consistent with the certain acidity required in the fermentation process of the stinky mandarin fish. By combining the growth curve and the acid production capacity of the strain and combining the fermentation process of the mandarin fish, analysis shows that the strain can be rapidly propagated to produce lactic acid in the initial stage, so that the strain not only can help to shorten the fermentation period, but also can provide a good growth environment for the growth of other fermentation lactic acid bacteria while inhibiting the mixed bacteria, and has important influence on the safety and flavor formation of the fermented mandarin fish.

(2) Temperature resistance: the bacterial suspension is inoculated into MRS broth culture medium in a proportion of 1% by volume, and a non-inoculated liquid culture medium is set as a blank control. OD was measured at 600nm every 4h of incubation at 12℃for 24 h.

(3) Salt tolerance: the bacterial suspension was inoculated into an MRS broth medium containing 3% NaCl at a volume fraction of 1%, while a liquid medium containing NaCl was set as a blank. OD was measured at 600nm every 4h of incubation at 37℃for 24 h.

The results of the temperature resistance and the salt resistance are shown in figure 4, and compared with the high-temperature pickling of the stinky mandarin fish, the low-temperature wet method is beneficial to improving the color and luster of the stinky mandarin fish, enhancing the texture and improving the sensory quality. Therefore, the strain is explored in growth condition under the condition of 12 ℃, and is very beneficial to low-temperature fermentation of the stinky mandarin fish. The graph shows that the growth activity of CMC1-8 at 12 deg.c is very considerable, OD value at 24 hr reaching 1.5, and the growth condition is superior to that at 37 deg.c, and the strain is excellent for low temperature fermentation of mandarin fish.

The salt concentration is critical in the fermentation of the stinky mandarin fish, and the salt addition amount in the pickling of the traditional stinky mandarin fish is about 3-4% (mass fraction), so that the salt plays roles of increasing the flavor, improving the color and inhibiting microorganisms. The tolerance of the strain to salt is explored to judge whether the strain can play a role of dominant bacteria in the fermentation process of the mandarin fish. As can be seen from the graph, the reproductive capacity of CMC1-8 was similar to that in MRS liquid medium for 0-3 h. Along with the extension of the culture time, the growth is obviously inhibited, but the reproductive capacity is stable, the OD value is 0.9-1.0 when the concentration of the thalli is maximum, and the OD value (0.798-2.724) of salt-tolerant strains separated from fermented food fermented soybeans by crowing is reached, so that the strain has good adaptability under the concentration of 3% of salt, and can play a role better.

EXAMPLE 2 determination of the protease-producing Activity of Strain CMC1-8

(1) Preparation of protease: CMC1-8 strain is inoculated into MRS broth culture medium and cultured for 2-4d at 12-20 ℃. Centrifuging the fermentation liquor at the temperature of 4 ℃ and the rotating speed of 8000-10000 rpm for 5-10 min, and taking supernatant; slowly adding ammonium sulfate into the supernatant liquid under stirring at the temperature of 4 ℃ to ensure that the saturation of the ammonium sulfate in the supernatant liquid reaches 60% -80%, and salting out for 8-12 h at the temperature of 4 ℃ after full dissolution to obtain salting-out liquid; centrifuging the salting-out solution at the temperature of 4 ℃ and the rotating speed of 8000-10000 rpm for 5-15 min, taking precipitate, and dissolving the precipitate by using a phosphate buffer solution with the mass concentration of 10-30 mmol/L and the pH value of 7.2 to obtain a protease crude enzyme solution; transferring the protease crude enzyme solution into a 3500-5000D dialysis bag with the molecular weight cutoff, placing the dialysis bag into 3L of phosphoric acid buffer with the mass concentration of 15-30 mmol/L, dialyzing for 16-18 h for desalting, changing the buffer for 2-3 times, and freeze-drying the dialysis solution to obtain CMC1-8 products: a protease.

(2) Preparation of a standard curve: accurately weighing 0.100g of L-tyrosine standard substance which is dried at 105 ℃ in advance, dissolving with 20mL of 1mol/L hydrochloric acid solution, and then fixing the volume to 100mL by distilled water to obtain the product100. Mu.g/mL L-tyrosine standard stock solution. Accurately sucking 0.00, 1.00, 2.00, 3.00, 4.00, 5.00 and 6.00mL of L-tyrosine standard solution into a 10mL volumetric flask respectively, fixing the volume of distilled water to 10mL, and shaking uniformly to prepare standard working solutions respectively containing 0.0, 10.0, 20.0, 30.0, 40.0, 50.0 and 60.0 mug of L-tyrosine. 0.2mL of standard working solution is respectively sucked, 1.0mL of 0.4mol/L sodium carbonate solution and 0.2mL of furin reagent are added, and the mixture is uniformly mixed. The standard tube is simultaneously placed in a water bath at 40 ℃ for reaction for 20min, taken out and placed in cold water for rapid cooling to room temperature, the blank tube is used for zeroing, and the absorbance value is measured at 680nm of a spectrophotometer. Drawing a standard curve with L-tyrosine content as abscissa and absorbance as ordinate to obtain y=0.0386x+0.0458 (R ² = 0.9954) linear regression equation.

(3) Measurement of protease Activity: the Folin method is adopted for measurement, namely, a substrate and an enzyme solution are respectively preheated at 40 ℃ for 2min, 0.1mL of diluted enzyme solution is taken, 0.1mL of substrate is added, the reaction is carried out for 10min at 40 ℃, and then 0.2mL of 10% trichloroacetic acid is added for stopping the reaction. After 10min incubation at 40℃the solution was centrifuged at 12 000r/min for 2min, 0.2mL of supernatant was taken, 1mL of 0.4mol/L sodium carbonate was added, after mixing, 0.2mL of Fulin reagent (Solarbio) was added, incubation was performed at 40℃for 20min, and then the OD at 680nm was determined by spectrophotometry. The assay was run against buffer and substrate reactions. Protease activity is represented by X, calculated as follows:

wherein: c is the tyrosine concentration of the sample tube, g/mL; v is total volume of enzyme reaction, mL; n is the dilution of the sample; v (V) ₀ mL for the amount of enzyme involved in the reaction; 10 is the reaction time, min.

The enzyme activity is defined as: the amount of enzyme that hydrolyzes casein to produce 1. Mu. Mol tyrosine per ml of enzyme solution at a certain temperature and pH is one activity unit (U).

(4) Influence of different temperature conditions on protease activity: the protease activity was measured by performing an enzymatic reaction at 10℃at 20℃at 30℃at 40℃at 50℃at 60℃at 70℃and at 80℃for 10min, respectively. The results are shown in FIG. 6. As is clear from FIG. 6, CMC1-8 produces proteinase with an optimal enzyme activity temperature of 70 deg.C, and belongs to high temperature resistant proteinase. The enzyme produced by CMC1-8 can maintain the enzyme activity of more than 67.65% at 10 ℃. Along with the increase of the temperature, the enzyme activity is gradually enhanced to be the highest at 70 ℃, the enzyme activity is reduced after the temperature exceeds 70 ℃, and the enzyme activity can still be kept at 91.95% at 80 ℃. The results show that CMC1-8 produces proteinase with high activity at 10-80 deg.c, and the proteinase has high heat resistance and low temperature resistance and high heat stability.

(5) Effect of different concentration pH conditions on protease activity: buffers of pH 3.0, 5.0, 7.0, 9.0 and 11.0 were prepared, casein solution with mass fraction of 1% was prepared as substrate with the buffer, and enzyme activity was measured with Folin-phenol method. The results are shown in FIG. 7. As can be seen from FIG. 7, CMC1-8 protease produced has an optimum pH of about 9; at pH 3, 632.06U/g of enzyme activity can be maintained; the high enzyme activity is maintained in the pH range of 7-9, belonging to neutral proteinase.

(6) Influence of different concentration of NaCl conditions on protease activity: preparing buffer solutions containing 3%, 5%, 7%, 9% and 11% of NaCl at different concentrations, preparing casein solution with mass fraction of 1% by using the buffer solution as a substrate, and measuring enzyme activity by Folin-phenol method. As shown in fig. 8. As can be seen from FIG. 8, the enzyme activity of 98.57% can be maintained at a NaCl mass concentration of 3%; enzyme activity was minimal at 11%. Although the protease is reduced at high salt concentration, the protease has higher enzyme activity when the NaCl mass concentration is 3-11%, so that the protease has salt tolerance to a certain extent.

EXAMPLE 3 evaluation of antioxidant and bacteriostatic Activity of the CMC1-8 metabolite of Strain

(1) Preparation of CMC1-8 metabolite: CMC1-8 strain is inoculated into MRS broth culture medium and cultured for 2-3 d at 12 ℃. Centrifuging the fermentation liquor at 4 ℃ at 10000rpm for 10min, collecting supernatant, filtering with a 0.22 μm filter membrane, extracting filtrate with ethyl acetate at a ratio of 1:1.5, evaporating and concentrating an organic layer, and diluting a sample with a certain concentration by using dimethyl sulfoxide (DMSO) as a solvent.

(2) ABTS radical scavenging energyForce measurement: diluting CMC1-8 metabolite to 1, 2, 4, 6, 8, 10mg/mL, taking 50 μl of each diluted sample solution, mixing 100 μl of ABTS solution in dark condition, reacting for 5min, and measuring absorbance A at 734nm ₀ The method comprises the steps of carrying out a first treatment on the surface of the Measurement of absorbance A Using equal volume of distilled Water instead of ABTS solution as blank group ₁ The method comprises the steps of carrying out a first treatment on the surface of the Measuring absorbance A with equal volume of DMSO instead of sample solution as control group ₂ The experiment was repeated three times with vitamin C as positive control and the average was taken. The clearance was calculated as follows:

table 1 shows the scavenging effect of CMC1-8 strain metabolites on ABTS free radicals

As can be seen from Table 1, metabolites at various concentrations of CMC1-8 have a significant effect on the radical scavenging ability of ABTS (P<0.05 The concentration of the metabolite is positively correlated with its clearance of ABTS, IC ₅₀ 1.05mg/mL was reached. When the metabolite concentration was 4mg/mL, the clearance reached (98.55.+ -. 0.68)%.

(3) Measurement of DPPH radical scavenging ability: diluting CMC1-8 metabolite to 1, 2, 4, 6, 8, 10mg/mL, taking 100 μl of each diluted sample solution, mixing with 50 μl DPPH solution in dark condition, reacting for 10min, and measuring absorbance A at 517nm ₀ The method comprises the steps of carrying out a first treatment on the surface of the Determination of absorbance A Using equal volume of absolute ethanol instead of DPPH solution as blank ₁ The method comprises the steps of carrying out a first treatment on the surface of the Measuring absorbance A with equal volume of DMSO instead of sample solution as control group ₂ The experiment was repeated three times with vitamin C as positive control and the average was taken. The clearance was calculated as follows:

table 2 shows the effect of CMC1-8 strain metabolite on DPPH radical scavenging

/>

As is clear from Table 2, metabolites of CMC1-8 at different concentrations have a significant influence on the free radical scavenging ability of DPPH (P<0.05 The concentration of the metabolite is positively correlated with its clearance of DPPH, IC ₅₀ 6.866mg/mL was reached. When the metabolite concentration was 10mg/mL, the clearance rate reached (63.54.+ -. 2.12)%.

(4) Measurement of reducing force: mixing 100 μL of each CMC1-8 metabolite of 1, 2, 4, 6, 8, 10mg/mL with 100 μL of phosphate buffer solution with pH of 6.6 and 100 μL of 1% potassium ferricyanide respectively, cooling in water bath at 85deg.C for 20min, adding 100 μL of 10% trichloroacetic acid, centrifuging at 3000r/min for 5min, collecting supernatant 100 μL, adding 100 μL of distilled water and 0.1% ferric trichloride for 10 μL, standing for 10min, and measuring absorbance A under 700nm ₀ . Determination of absorbance A Using DMSO as a control blank ₁ The experiment was repeated three times with vitamin C as positive control, and the average value was taken.

Table 3 shows the reducing power of the metabolites of CMC1-8 strain

As is clear from Table 3, the reducing power of the metabolites of CMC1-8 at different concentrations has a significant effect (P<0.05 Enhanced with increasing concentration, IC) ₅₀ Is 4.621mg/mL. OD when metabolite concentration was 10mg/mL _700nm And reaches 0.76 plus or minus 0.04. Sun Ning et al, "screening and identification of lactic acid bacteria in naturally fermented sour bamboo shoots and study of probiotic characteristics" studied the reducing ability of six lactic acid bacteria isolated from naturally fermented sour bamboo shoots, OD thereof _700nm Max 0.552.

(5) Determination of bacteriostatic Activity: adopting filter paper diffusion method, using Escherichia coli, staphylococcus aureus, bacillus subtilis and Pseudomonas aeruginosa as indicator pathogen, and dimethylSub-maple was used as a blank (CK) and propyl p-hydroxybenzoate as a positive (PP). Marking and activating the indicator bacteria on a slant culture medium, culturing at 37deg.C for 12 hr, preparing into bacterial suspension with physiological saline, and regulating OD _600nm At 0.1, 100. Mu.L of the bacterial suspension was spread evenly on the medium. The CMC1-8 metabolic product concentration is diluted to 2, 5, 10, 15 and 20mg/mL, 10 mu L of sample solution is dripped in the center of a sterile filter paper sheet on a culture medium, and the culture is inverted at 37 ℃ for 12 hours, and the diameter of a bacteriostasis zone is measured.

Table 4 shows the inhibition zone of CMC1-8 strain metabolite to 4 bacteria

/>

The bacteriostatic ability of metabolites of CMC1-8 at different concentrations was determined by the filter paper diffusion method, and the results are shown in Table 4. The metabolites with different concentrations have remarkable antibacterial activity (P < 0.05) on four pathogenic bacteria. For the same pathogenic bacteria, the diameter of the inhibition zone becomes larger along with the increase of the concentration of the metabolite.

(6) Statistics and analysis of data: experiments were repeated three times as an average, and the results are expressed as mean ± standard deviation. The phylogenetic tree is constructed by adopting MEGA11.0 software, the phylogenetic tree is plotted by adopting Origin 8.5 software, the analysis of variance is performed by adopting SPSS 18.0 software, and P <0.05 indicates that significant difference exists.

Example 4: application of strain CMC1-8 in fermentation of stink mandarin fish and effect evaluation

2) The drained mandarin fish is orderly placed in a fermentation tank, the mandarin fish is placed according to 200kg of standard of each tank, then 5kg of refined salt is completely dissolved in 100L of clear water and then poured into the fermentation tank, the mandarin fish is submerged, pickling auxiliary materials are added and uniformly mixed, the auxiliary materials at least comprise 1.2kg of chilli powder and 0.7kg of pepper, and conventional pickling auxiliary materials can be added according to requirements. Finally inoculating CMC1-8 bacterial suspension of lactobacillus sake subspecies with the mass percentage of 0.08% into a fermentation tank, and uniformly stirring;

3) Fermenting the fermentation tank in a dark place at 6-16 ℃ for 5-9 d to obtain a finished product of the stinky mandarin fish; the sectional fermentation is adopted: the first stage is to set the fermentation temperature at 16 ℃ and the fermentation time at 1d; after the completion of the fermentation, the mandarin fish is pressed at a weight 1.5 times of the weight of the mandarin fish, and the second stage fermentation is performed at 8deg.C for 5 days.

4) Taking out the mandarin fish fermented in the step 3), vacuum packaging, quick freezing in a freezer, and storing at-20deg.C.

The specific preparation method of the lactobacillus sake subspecies CMC1-8 bacterial suspension in the step 2) comprises the following steps:

s2: centrifuging the culture solution of the lactobacillus sake subspecies CMC1-8 obtained in the step S1 at the temperature of 10-15 ℃ for 5-8 min, wherein the rotation speed of a centrifuge is 6000-10000 rpm, and then standing for layering;

Comparative examples: the control group is the stinky mandarin fish which is not added with the strain CMC1-8 for fermentation treatment. Namely, the pickling step is the same as that of example 4, except that the comparative example does not add CMC1-8 bacterial suspension of lactobacillus sake subspecies, and pickling is carried out until the pickling of the stinky mandarin fish is completed.

Evaluation of the effect of the strain CMC1-8 in the fermentation application of stink mandarin fish:

(1) sensory evaluation: the sensory panel consisted of 10 trained individuals. And respectively selecting each group of sample fish meat to cook, and placing the sample fish meat in the same container for coding, and evaluating the sample fish meat by 5 indexes such as color, smell, taste, tissue state, acceptance degree and the like. The evaluation criteria are shown in Table 5.

TABLE 5 sensory scoring criteria for stink Mandarin fish

Sensory evaluation the sensory evaluation of the stinky mandarin fish was mainly based on the visual, olfactory, gustatory and overall acceptability of panelists, the results of which are shown in fig. 9. Compared with the comparative example, the addition of CMC1-8 strain has obvious influence on the color, smell, taste and tissue state of the stinky mandarin fish, the stinky mandarin fish after being added with CMC1-8 strain has better sensory acceptability, white and glossy meat quality, special odor after fish fermentation, compact and elastic fish meat, tidy meat mass, separated bones and meat and obvious garlic petal shape. Therefore, the sensory quality of the stinky mandarin fish after the CMC1-8 strain is added and treated is better.

(2) Determination of nitrite content: determination of NaNO in odor mandarin fish by naphthalene ethylenediamine hydrochloride method in national standard GB 5009.33-2016 determination of nitrite and nitrate in food ₂ The experiments were performed in 3 replicates.

(3) Measurement of histamine content: the histamine content in the stink mandarin fish is determined by spectrophotometry in GB 5009.208-2016 determination of biogenic amine in food safety national Standard food.

(4) Determination of thiobarbituric acid: taking 10g of stink mandarin fish flesh of different treatment groups, adding 25mL of distilled water into a beaker, homogenizing, adding 25mL of 10g/100mL of trichloroacetic acid, uniformly mixing, standing for 30min, filtering, taking 5mL of supernatant, adding 5mL of 0.02 mol/L2-thiobarbituric acid (TBA) solution, heating the mixed solution in a constant temperature water bath kettle at 80 ℃ for 40min, taking out, cooling to room temperature, and measuring the absorbance at 532nm wavelength. TBA is expressed as mass of malondialdehyde contained in fish meat per kg in mg/kg.

Table 6 shows the effect of CMC1-8 strain on the safety indexes of nitrite, histamine, thiobarbituric acid, etc. of fermented stink mandarin fish

As can be seen from Table 6, CMC1-8 strain has a significant effect on the content of safety indexes such as nitrite, histamine and thiobarbituric acid of the fermented stink mandarin fish. The results show that the artificial inoculation fermentation method of the invention obviously reduces the generation of nitrite, histamine and thiobarbituric acid content in the fermentation of the stinky mandarin fish by 6.17 times, 3.61 times and 2.45 times respectively. The result proves that the CMC1-8 strain is used as a starter, and is artificially inoculated into the fermentation of the stinky mandarin fish, so that the edible safety of the stinky mandarin fish is improved.

The above embodiments are merely exemplary and do not limit the scope of the present invention in any way. Therefore, the details and forms of the technical scheme of the invention can be modified or replaced by those skilled in the art without departing from the design spirit of the invention, and the invention falls within the protection scope of the invention.

Claims

1. Lactobacillus sake subspecies, designated CMC1-8, were preserved in chinese collection of typical cultures (CCTCC), address: the preservation number is CCTCC M2022621, and the preservation time is 2022, 5 and 13 days.

2. Use of lactobacillus sake subspecies according to claim 1 in pickling and fermentation of stinky mandarin fish.

3. Use of a metabolite of the species lactobacillus sake according to claim 1 in a bacteriostatic agent.

4. Use of a metabolite of the lactobacillus sake subspecies in accordance with claim 1 in an antioxidant.

5. Use of lactobacillus sake subspecies in curing and fermenting stinky mandarin fish as claimed in claim 2, comprising the steps of:

3) And (3) placing the fermentation tank in an environment of 6-16 ℃ to perform light-proof fermentation for 5-9 d to complete fermentation, thus obtaining the finished product of the stinky mandarin fish.

6. Use of lactobacillus sake subspecies in curing and fermenting stinky mandarin fish as claimed in claim 5, wherein: the specific preparation method of the lactobacillus sake subspecies bacterial suspension in the step 2) comprises the following steps:

7. Use of lactobacillus sake subspecies in curing and fermenting stinky mandarin fish as claimed in claim 5, wherein: step 3) adopts sectional fermentation, wherein the fermentation temperature is set to be 10-16 ℃ and the fermentation time is set to be 1-2 d in the first stage; then compacting the stinky mandarin fish by using a heavy object to ferment in the second stage, wherein the weight of the heavy object pressing the stinky mandarin fish is 1.5-3 times of the mass of the mandarin fish in the fermentation tank; the second stage fermentation temperature is 6-10 deg.c and fermentation time is 4-7 d.