CN116925953A - Fermentation method of stink mandarin fish and strain used by same - Google Patents

Fermentation method of stink mandarin fish and strain used by same Download PDF

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CN116925953A
CN116925953A CN202310217694.3A CN202310217694A CN116925953A CN 116925953 A CN116925953 A CN 116925953A CN 202310217694 A CN202310217694 A CN 202310217694A CN 116925953 A CN116925953 A CN 116925953A
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mandarin fish
fermentation
inoculum
staphylococcus
agar
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杨柳
徐宝才
孙汉巨
吴永祥
陈亚林
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Hefei University of Technology
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Hefei University of Technology
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Abstract

The invention provides a fermentation method of mandarin fish and a strain used by the same, and provides a lactobacillus plantarum and a staphylococcus soil; the lactobacillus plantarum is added to change the community composition of microorganisms in the fermentation process of the mandarin fish, reduce the growth of other spoilage microorganisms and inhibit the spoilage of the mandarin fish; the staphylococcus aureus can produce extracellular protease and lipase to promote the decomposition of mandarin fish protein and fat; the two strains are mixed and inoculated to ferment the mandarin fish, so that not only can the growth of other spoilage microorganisms be inhibited, but also the decomposition of the fish meat can be promoted, biogenic amine of the mixed inoculation and fermentation fish meat can be inhibited, and the sulfur-containing compounds in the volatile flavor compounds of the mixed inoculation and fermentation fish meat can be reduced, thereby improving the flavor of the mandarin fish.

Description

Fermentation method of stink mandarin fish and strain used by same
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a fermentation method of stinky mandarin fish and a strain used by the fermentation method.
Background
The fermented mandarin fish is also called barren fresh fish, smelly mandarin fish, etc. and is one of the representative dishes of Huizhou dishes, and the traditional fermented mandarin fish is produced through pickling fresh or frozen mandarin fish, sealing and fermenting at room temperature for 7-15 days. The stink mandarin fish prepared by fermentation has the characteristics of smelling and smelling, and the stink-like and non-stink flavor is the important biochemical reaction in the fermentation maturation process because protein is decomposed under the action of endogenous enzyme and microbial enzyme, amino acid is released by proteolysis, free fatty acid is produced by lipolysis and the like. However, the complex microbial community in the fermentation system brings great challenges for identifying the fermentation process and realizing the quality control of the product.
In the traditional mandarin fish fermentation process, because of the existence of self-contained mandarin fish and complex microorganisms in the environment, uncontrollability and instability of products in the fermentation process can be caused, and the flavor and quality of the fermented products can be influenced. Research shows that the spoilage microorganisms such as psychrophilic bacillus, acinetobacter and vibrio in the traditional mandarin fish fermentation process can increase the decomposition of proteins and fats, so that the oxidation of proteins and lipids can be accelerated, the excessive oxidation of proteins can generate ammonia, amines and other alkaline nitrogen-containing substances, and the excessive decomposition and oxidation of fats can generate micromolecular aldehyde, ketone and fatty acid compounds, so that bad flavor is generated, commonly called rancidity.
Biogenic amine is one of main safety hazard factors of aquatic products, and is mainly divided into tryptamine, beta-phenethylamine, cadaverine, putrescine, tyramine, histamine, spermine, spermidine and the like, the biogenic amine in the aquatic products is mainly produced by degrading protein of the biogenic amine by microorganisms, the formation mechanism can be used as an important detection index for quality and safety of chilled fresh aquatic products such as minced fillet, fish block and the like, the biogenic amine content of salted or fermented fish products is determined by microbial flora, and spoilage bacteria are important causes for reducing the quality of the aquatic products. Some biogenic amines in trace amounts are normal active ingredients in the human body and have important physiological effects, but once the human body ingests excessively, serious harm and even poisoning are caused, biogenic Amines (BAs) are organic nitrogen-containing compounds, mainly formed by decarboxylation of corresponding amino acids. If the biogenic amine concentration reaches a threshold level, this can lead to mild to severe health problems in humans
Therefore, how to control spoilage microorganisms and reduce the biogenic amine content in fermented fish products is the key to fermenting stinky mandarin fish.
Disclosure of Invention
The invention aims to provide a saccule-covered yeast strain and application thereof in yellow wine brewing.
To achieve the above and other related objects, the present invention provides the following technical solutions: a lactobacillus plantarum strain is lactobacillus plantarum (Lactiplantibacillus plantarum) YR07 and is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022, 8 months and 18 days, and the strain preservation number is CCTCCNO: M20221303.
To achieve the above and other related objects, the present invention provides the following technical solutions: a staphylococcus aureus is staphylococcus aureus (Staphylococcus edaphicus) T-F, and is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022, 12 months and 19 days, and the strain preservation number is CCTCCNO: M20221987.
To achieve the above and other related objects, the present invention provides the following technical solutions: a fermentation method of stink mandarin fish comprises the following steps:
step 1: inoculating Lactobacillus plantarum (Lactiplantibacillus plantarum) YR07 of claim 1 to an MRS agar medium with an inoculating loop, activating, culturing at 30-37deg.C for 36-48 hr, inoculating the obtained culture with an inoculating loop, culturing at 30-37deg.C for 24-28 hr to obtain a first inoculum;
step 2: transferring the staphylococcus soil (Staphylococcus edaphicus) T-F of claim 1 from the inclined plane to the MSA agar culture medium by using an inoculating loop for activation, culturing at 30-37 ℃ for 36-48h, transferring the obtained culture to the MSA culture medium by using the inoculating loop, and culturing at 30-37 ℃ for 28-36h to obtain a second inoculum;
step 3: removing scales and viscera of fresh mandarin fish, cleaning, placing in a container, and adding sterile saline water;
step 4: adding the first inoculum and the second inoculum, capping, and fermenting at 8-12deg.C.
The preferable technical scheme is as follows: in the step 1, the obtained first inoculum was liquid, and the first inoculum was centrifuged at 3500rpm/min to obtain wet cells.
The preferable technical scheme is as follows: in step 2, the obtained second inoculum was washed with sterile physiological saline on an agar plate, followed by centrifugation at 3500rpm/min to obtain wet cells.
The preferable technical scheme is as follows: in the step 3, the mass fraction of the sterile saline is 5-10%, and the ratio of the sterile saline to the fish meat is as follows: 1L, 1.2-1.6kg.
The preferable technical scheme is as follows: in step 4, the inoculum size of the first inoculum and the second inoculum is 1.0-1.5g/kg according to the mass ratio of wet bacteria to fish meat. Namely: the inoculum size of the first inoculum is 1.0-1.5g/kg according to the ratio of wet thallus to fish meat mass, and the inoculum size of the second inoculum is the same as that of the first inoculum.
The preferable technical scheme is as follows: every liter of MRS agar medium contains: 10.0g of peptone, 8.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 1.0g of tween and 15.0-20.0g of agar; sterilizing at 121deg.C for 20min at pH 6.5+ -0.2; MRS liquid medium differs from MRS agar medium in that no agar is added.
The preferable technical scheme is as follows: the formula of the MSA agar medium is as follows: 10g of peptone, 1g of beef extract powder, 10g of mannitol, 25g of sodium chloride, 15-20g of agar powder and 1L of distilled water; sterilizing at 121deg.C for 20min at pH 7.0+ -2.
Due to the application of the technical scheme, compared with the prior art, the invention has the advantages that:
1. the plant milk stalk can inhibit the increase of other putrefying bacteria, the staphylococcus aureus can generate protease and lipase, promote the decomposition of protein and fat to form good flavor, reduce the bad smell of sulfide, and the flavor of the final fermentation product is more easily accepted by consumers.
2. The strain does not produce amino acid decarboxylase in metabolism, controls the biogenic amine content of the product, and is beneficial to improving the safety of the product.
3. The mixed inoculation fermentation can improve the content of the delicious taste and sweet amino acid in the free amino acid of the mandarin fish flesh, reduce the content of bitter amino acid and improve the taste of the product.
Drawings
FIG. 1 morphology of Lactobacillus plantarum (left panel) and Staphylococcus aureus (right panel) under an optical microscope.
Fig. 2 glucose fermentation experiments, a staphylococcus soil group, a lactobacillus plantarum group and a control group are sequentially carried out from left to right.
The bacterial strain of FIG. 3 produces the biogenic amine experiment, which is a staphylococcus soil group, a lactobacillus plantarum group, a blank control group and a positive control group from left to right.
The hemolysin production experiment of the strain in figure 4 is carried out by lactobacillus plantarum and staphylococcus soil sequentially from left to right.
Fig. 5 shows indole production experiments of lactobacillus plantarum, staphylococcus soil, blank control and positive control groups from left to right.
FIG. 6 texture of fish after 7 days of fermentation between the inoculation group and the control group.
Detailed Description
Further advantages and effects of the present invention will be readily apparent to those skilled in the art from the following disclosure of the present invention by reference to the specific embodiments.
Please refer to fig. 1-6. It should be understood that the structures, proportions, sizes, etc. shown in the drawings are shown only in the drawings and should not be taken as limiting the invention to those having ordinary skill in the art, since modifications, changes in proportions, or adjustments of sizes, etc. could be made without departing from the spirit or essential characteristics of the invention. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the examples described below were purchased from conventional biochemical reagent stores unless otherwise specified.
Preservation of biological material:
lactobacillus plantarum (Lactiplantibacillus plantarum) YR07 has been deposited with China Center for Type Culture Collection (CCTCC) at month 8 and 18 of 2022, with a deposit number of CCTCC NO: M20221303 and a strain of staphylococcus aureus (Staphylococcus edaphicus) T-F, and has been deposited with China Center for Type Culture Collection (CCTCC) at month 12 and 19 of 2022, with a deposit number of CCTCC NO: M20221987. Chinese typical culture collection address: no. 299 is eight paths in Wuchang district of Wuhan, hubei province.
The reagents and materials described in the examples below are commercially available unless otherwise indicated.
Example 1: fermentation method of stink mandarin fish and strain used by same
The screening procedure for Lactobacillus plantarum (Lactiplantibacillus plantarum) YR07 and Staphylococcus aureus (Staphylococcus edaphicus) T-F was as follows:
1. the method for separating and screening lactobacillus plantarum (Lactiplantibacillus plantarum) YR07 and staphylococcus soil (Staphylococcus edaphicus) T-F from natural fermentation stink mandarin fish comprises the following specific steps: 10g of fish meat is taken, homogenized for 10min in a sterile homogenizing bag filled with 90mL of sterile normal saline, 1mL of homogenized solution is taken for gradient dilution for 5 to 6 times, the final diluted solution is coated on an MRS agar medium plate containing light calcium carbonate and an MSA agar medium plate added with a phenol red indicator, and colonies with transparent rings in the MRS medium and yellowish colonies in the MSA medium are taken for further screening after culturing for 24 to 48h in an incubator. And purifying the bacterial colony obtained by primary screening for 3 times to obtain a pure bacterial colony, and then carrying out gram staining, microscopic examination, contact enzyme experiment and 6% salt tolerance experiment to carry out the next screening to obtain the bacterial strain with gram positive, contact enzyme negative and salt tolerance.
2. According to the microorganism identification technology, a physiological and biochemical experiment is carried out on the provided strain, and the physicochemical characteristics of the strain, such as a glucose fermentation experiment, a biogenic amine experiment, a hemolysis experiment and an indole experiment, which are favorable for the fermentation of the siniperca chuatsi are judged; the strain obtained by identification is subjected to DNA extraction and PCR amplification to obtain an amplified product, and then the amplified product is sent to a biological company to identify the sequence, and the sequence result is that: SEQ No.1 and SEQ No.2.
(1) Acid and gas production experiment of fermentation glucose
Inoculating Lactobacillus plantarum culture on MRS agar medium and Staphylococcus aureus culture on MSA agar medium to glucose fermentation tube, culturing a group of non-inoculated bacteria as control at 35deg.C for 24-48 hr, and observing the color of the culture medium and whether bubbles are generated in Du Shixiao tube; if the strain produces acid, the indicator in the culture medium changes from purple to yellow, if the strain ferments glucose to produce gas, bubbles are generated in the Du Shixiao tube, otherwise, the indicator does not exist. As a result, as shown in FIG. 2, lactobacillus plantarum and Staphylococcus aureus were found to ferment glucose but not produce gas.
Glucose fermentation medium composition: beef extract 5.0g, peptone 10.0g, naCl 3g and Na 2 HPO 4 ·12H 2 O2 g, 12mL of 0.2% bromocresol purple ethanol solution, 5g of glucose, 1L of distilled water to a constant volume, pH7.4; preparing hair: after the components are well distributed, the components are packaged in test tubes with inverted Du's small tubes, each tube is packaged with 10mL, and the tubes are sterilized for 15min at 121 ℃ for later use.
(2) Experiment of producing biogenic amine
Lactobacillus plantarum and Staphylococcus soil on agar medium are cultured in amine production liquid medium at 35deg.C for 24-48 hr, and the color change of indicator in the medium is observed, so that neither strain produces amine, as shown in figure 3.
Production of biogenic amine liquid medium: beef extract powder 5.0g, tryptone 5.0g, yeast extract powder 5.0g, glucose 0.5g, naCl2.5g, ammonium citrate 2.0g and K 2 HPO 4 2.0g, 0.1g CaCO3, 0.05g manganese sulfate, 0.04g ferrous sulfate, 0.01g thiamine, 0.05g pyridoxal phosphate, 1mL Tween-80, 0.06g bromocresol purple, amino acid (1 mL each of arginine, histidine, tyrosine and lysine), adding distilled water to 1L, adjusting pH to 5.3, and sterilizing at 121deg.C for 15min; the indicator in the culture medium is yellow in color at pH5.3, and if amine is produced after the growth of the inoculation, the pH of the culture medium can be increased, and the indicator in the culture medium can be changed from yellow to light purple.
(3) Hemolysis experiment
Some bacteria can generate hemolysin in the growth process, so that erythrocytes are broken and dissolved, transparent or semitransparent hemolytic rings are observed around colonies when the bacteria grow on a blood plate, alpha hemolysis (grass green), beta hemolysis (complete hemolysis) and gamma hemolysis (no hemolysis or hemolysis ring) can be formed on the plate due to different hemolysin generated by different bacteria, and many bacterial hemolysis can be pathogenic. Lactobacillus plantarum and Staphylococcus aureus on agar medium are streaked on blood plates, and cultured at 35deg.C for 24-48 hr, and both strains are observed to have no hemolysis, as shown in figure 4.
(4) Indole production experiment
Some bacteria have tryptophan hydrolase, and can decompose tryptophan in protein to produce indole, which may cause diseases such as intraperitoneal infection and endocarditis. Indole itself is not colored and cannot be seen directly, but by the addition of a para-dimethylaminobenzaldehyde reagent, it reacts with indole to give a red rose indole. The cultures on the culture medium were inoculated with a needle to obtain appropriate cultures in an indole kit, and the indole kit was cultured in an incubator at 35℃for 24-48 hours with non-inoculated bacteria as a negative control group and inoculated with E.coli as a positive control group, respectively, and it was found that Lactobacillus plantarum and Staphylococcus aureus did not produce indole, and the results are shown in FIG. 5.
Example 2: fermentation method of stink mandarin fish and strain used by same
The present example provides Lactobacillus plantarum (Lactiplantibacillus plantarum) YR07, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 8 and 18 of 2022, with a deposit number of CCTCCNO: M20221303 and a strain of staphylococcus soil (Staphylococcus edaphicus) T-F, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 12 and 19 of 2022, with a deposit number of CCTCC NO: M20221987.
The method is applied to the fermentation of the stinky mandarin fish, and comprises the following steps:
1. inoculating lactobacillus plantarum on an inclined plane, inoculating to an MRS agar culture medium for activation, culturing at 35 ℃ for 36-48h, inoculating the obtained culture to an MRS liquid culture medium again, culturing at 35 ℃ for 24-28h to obtain an inoculum, centrifuging the liquid culture medium at 3500rpm/min to obtain wet thalli, and inoculating as a starter.
2. Transferring staphylococcus soil from an inclined plane to an MSA agar culture medium for activation, culturing at 35 ℃ for 36-48h, transferring the obtained culture to the MSA culture medium again by using the inoculating loop, culturing at 35 ℃ for 28-36h to obtain an inoculum, flushing the obtained inoculum on an agar plate by using sterile normal saline, and centrifuging at 3500rpm/min to obtain wet thalli serving as a starter.
3. Preparing and treating mandarin fish raw materials: removing scales and viscera of fresh mandarin fish, cleaning, placing in a container, adding 6% sterile saline, and adding the sterile saline according to the water volume (L): the fish meat mass (kg) is 1:1.5.
4. Lactobacillus plantarum and staphylococcus are added into the prepared mandarin fish raw materials and marked as LS group, the inoculation amount of the fermentation strain is added according to the proportion of the mass of wet thalli and fish meat of 1.25g/kg, the addition amount of single strain is halved when the two strains are inoculated together, the container is covered after the fermentation agent is added, and the container is placed at 10+/-2 ℃ for fermentation for one week.
Example 3: fermentation method of stink mandarin fish and strain used by same
The present example provides Lactobacillus plantarum (Lactiplantibacillus plantarum) YR07, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 8 and 18 of 2022, with a deposit number of CCTCCNO: M20221303 and a strain of staphylococcus soil (Staphylococcus edaphicus) T-F, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 12 and 19 of 2022, with a deposit number of CCTCC NO: M20221987.
The method is applied to the fermentation of the stinky mandarin fish, and comprises the following steps:
1. inoculating lactobacillus plantarum on an inclined plane, inoculating to an MRS agar culture medium for activation, culturing at 35 ℃ for 36-48h, inoculating the obtained culture to an MRS liquid culture medium again, culturing at 35 ℃ for 24-28h to obtain an inoculum, centrifuging the liquid culture medium at 3500rpm/min to obtain wet thalli, and inoculating as a starter.
2. Preparing and treating mandarin fish raw materials: removing scales and viscera of fresh mandarin fish, cleaning, placing in a container, adding 6% sterile saline, and adding the sterile saline according to the water volume (L): the fish meat mass (kg) is 1:1.5.
3. Adding lactobacillus plantarum into the prepared mandarin fish raw material, marking the lactobacillus plantarum as L groups, adding the inoculum size of the fermentation strain according to the proportion of wet thalli and fish meat mass of 1.25g/kg, inoculating the two strains together, halving the addition size of a single strain, adding a starter, covering a container, and fermenting at 10+/-2 ℃ for one week.
Example 4: fermentation method of stink mandarin fish and strain used by same
The present example provides Lactobacillus plantarum (Lactiplantibacillus plantarum) YR07, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 8 and 18 of 2022, with a deposit number of CCTCCNO: M20221303 and a strain of staphylococcus soil (Staphylococcus edaphicus) T-F, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 12 and 19 of 2022, with a deposit number of CCTCC NO: M20221987.
The method is applied to the fermentation of the stinky mandarin fish, and comprises the following steps:
1. transferring staphylococcus soil from an inclined plane to an MSA agar culture medium for activation, culturing at 35 ℃ for 36-48h, transferring the obtained culture to the MSA culture medium again by using the inoculating loop, culturing at 35 ℃ for 28-36h to obtain an inoculum, flushing the obtained inoculum on an agar plate by using sterile normal saline, and centrifuging at 3500rpm/min to obtain wet thalli serving as a starter.
2. Preparing and treating mandarin fish raw materials: removing scales and viscera of fresh mandarin fish, cleaning, placing in a container, adding 6% sterile saline, and adding the sterile saline according to the water volume (L): the fish meat mass (kg) is 1:1.5.
3. Adding staphylococcus into the prepared mandarin fish raw material, marking as S group, adding the inoculation amount of the fermentation strain according to the proportion of wet thalli and fish meat mass of 1.25g/kg, inoculating two strains together, halving the addition amount of a single strain, adding a starter, covering a container, and fermenting at 10+/-2 ℃ for one week.
Example 5: fermentation method of stink mandarin fish and strain used by same
The present example provides Lactobacillus plantarum (Lactiplantibacillus plantarum) YR07, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 8 and 18 of 2022, with a deposit number of CCTCCNO: M20221303 and a strain of staphylococcus soil (Staphylococcus edaphicus) T-F, which has been deposited at China Center for Type Culture Collection (CCTCC) at month 12 and 19 of 2022, with a deposit number of CCTCC NO: M20221987.
The method is applied to the fermentation of the stinky mandarin fish, and comprises the following steps:
1. preparing and treating mandarin fish raw materials: removing scales and viscera of fresh mandarin fish, cleaning, placing in a container, adding 6% sterile saline, and adding the sterile saline according to the water volume (L): the fish meat mass (kg) is 1:1.5.
2. Adding no fermentation strain into the prepared mandarin fish raw material, marking as group C, covering the container, and fermenting at 10+ -2deg.C for one week.
Example 6: fermentation method of stink mandarin fish and strain used by same
1. Determination of fish malondialdehyde content: the fish meat during fermentation was taken and the fish meat on day 1, day 3, day 5 and day 7 were taken as samples, and the content of malondialdehyde during fish fermentation was measured according to the second spectrophotometry in GB 5009.181-2016, "determination of malondialdehyde in food safety national Standard food", with the following results.
The malondialdehyde content (unit: mg/kg) of fish during fermentation of each group of stinky mandarin fish (note: group C is non-inoculating fermentation;
l group is single lactobacillus plantarum fermentation; s group is fermentation of staphylococcus soil singly; LS group is mixed inoculation fermentation
Group C L group S group LS group
Day 1 of fermentation 0.282±0.009 0.278±0.012 0.277±0.023 0.264±0.045
Day 3 of fermentation 0.582±0.018 0.387±0.014 0.447±0.021 0.273±0.009
Day 5 of fermentation 0.741±0.032 0.678±0.008 0.765±0.019 0.582±0.027
Day 7 of fermentation 0.914±0.014 0.787±0.034 0.792±0.026 0.618±0.027
2. Determination of fish texture: taking fermented fish meat, peeling the back part near the head, and cutting into blocks with thickness of 10mm, length of 15mm and width of 15 mm. The samples were subjected to a texture multi-faceted analysis (TPA) mode test using a TA-XTPLUS physical property apparatus using a flat-bottomed cylindrical probe p36/R using a simulated human tooth to chew food, under conditions of 2mm/s pre-test rate, 1mm/s post-test rate, 50% compression, 5s dwell interval, 3 samples each, 6 times each, averaged, and texture results shown in FIG. 6.
Texture results: the hardness, the mastication degree and the elasticity of the fish meat of the three groups of bacteria groups are higher than those of the control group, the restoring force and the adhesive force of the fish meat of the single bacteria group (the L group and the S group) are lower than those of the mixed bacteria group, and the texture of the fish meat of the mixed bacteria group is best, which indicates that the mixed bacteria can promote the fermentation of mandarin fish and improve the texture of the fish meat.
3. Determination of fish biogenic amine: the method adopts High Performance Liquid Chromatography (HPLC) to measure the biogenic amine content in the fermentation process of the stinky mandarin fish, and comprises the following specific steps:
sample treatment: taking 2.5g of fish, cutting, adding 10mL of 0.6M HClO4 and 125 mu L of internal standard, homogenizing, centrifuging, taking supernatant after centrifuging, repeating the steps, and finally merging the supernatant to a 25mL volumetric flask to obtain a sample liquid;
sample and standard derivatization: adding 50 mu L of an internal standard into 0.2mL of a standard series, adding 40 mu L of 2MNaOH, 60 mu L of saturated NaHCO3 and 400 mu L of 10mg/mL of dansyl chloride, shaking uniformly, adding 20 mu L of NH4OH, carrying out water bath at 40 ℃ for 45min, reacting in the dark for 30min, adding acetonitrile to 1mL, filtering, and carrying out sample injection;
conditions for HPLC: reverse phase HPLC was used, ammonium acetate (0.1M; solvent A) and acetonitrile (solvent B) were used as mobile phases. Elution was performed using a gradient of 0min, 50% B;25 minutes, 90% B;35 minutes, 90% B;45,50% B. The flow rate was 0.8 ml/min and the temperature was 30 ℃. The sample was detected at 254nm and the sample volume was 10. Mu.L.
Only 3 biogenic amines, aniline, putrescine and histamine were detected during the fermentation of the stink mandarin fish, of which histamine is the most toxic biogenic amine common in aquatic products and its content is shown in Table 2.
TABLE 2 fish biogenic amine content (unit: mg/kg) during fermentation of various groups of stinky Mandarin fish
Note that: ND in the table indicates that no such material was detected.
Biogenic amine is a low molecular mass nitrogen-containing organic compound with biological activity, and biogenic amine in food is mainly formed by decarboxylation of amino acid by microbial amino acid decarboxylase. Production strains or environmental microorganisms in the production of fermented foods and fermented alcoholic beverages often cause the accumulation of biogenic amines in the products due to the activity of amino acid decarboxylase. Proper amounts of biogenic amine in the organism promote normal physiological activities, while excessive biogenic amine can cause adverse reactions in the organism. From the above table, it can be seen that aniline, putrescine and histamine are gradually accumulated as fermentation time is prolonged in the fermentation process of the mandarin fish samples, and aniline is detected on the 5 th day of group C and then on the 7 th day of group LS, in addition, the content of putrescine and histamine in the mixed inoculation LS group is obviously lower than that in the control group C and lower than that in the single inoculation L group and S group at the end of the 7 th day of fermentation, which indicates that the accumulation of biogenic amine is inhibited by the inoculated strain, and the mixed inoculation group is more favorable for inhibiting the accumulation of biogenic amine.
4. Determination of fish volatile compounds: the volatile flavor substances in the process of the fermentation of the stink mandarin fish are determined by adopting a solid phase microextraction gas chromatography-mass spectrometry (HPMS-HS-GC-MS), 5g of each sample is taken, the samples are crushed in a 20mL headspace bottle, and 10 mu L of an internal standard (2, 4, 6-trimethylpyridine) with the concentration of one ten thousandth is added. The extraction head was inserted into a headspace bottle at 70℃for 40min of extraction time, 3 replicates per sample.
The chromatographic column is DB-WAX capillary chromatographic column (30mx0.20 mm0.25 m), the temperature of the sample inlet is 250 ℃, the initial temperature of the programmed temperature is 30 DEG, and the temperature is kept for 1min. Raising the temperature to 92 ℃ at 4 ℃/min, and keeping the temperature for 2min, raising the temperature to 200 ℃ at 5 ℃/min, and raising the temperature to 240 ℃ at 6 ℃/min, and keeping the temperature for 6min. The carrier gas flow rate is 1mL/min, and the sample injection is not split. Mass spectrometry conditions ionization mode (EI); electron energy 70eV; the interface temperature is 250 ℃ and the ion source temperature is 250 ℃; the mass spectrum scanning range is 29-450m/z, and the acquisition mode is full scanning.
TABLE 3 content of various volatile flavor substances in fish meat after 7 days of fermentation of various groups of smelly Mandarin fish (unit: ng/g)
Note that: group C is non-inoculation fermentation; l group is single lactobacillus plantarum fermentation; s group is fermentation of staphylococcus soil singly; LS group is mixed inoculation fermentation.
TABLE 4 alcohol content in fish meat after 7 days of fermentation of the groups of stinky Mandarin fish (unit: ng/g)
Note that: ND in the table indicates that the substance was not detected; group C is non-inoculation fermentation; l group is single lactobacillus plantarum fermentation; s group is fermentation of staphylococcus soil singly; LS group is mixed inoculation fermentation.
TABLE 5 aldehyde and ketone Compound content in fish meat after 7 days of fermentation of the groups of stinky Mandarin fish (unit: ng/g)
Note that: ND in the table indicates that the substance was not detected; and (3) injection: group C is non-inoculation fermentation; l group is single lactobacillus plantarum fermentation; s group is fermentation of staphylococcus soil singly; LS group is mixed inoculation fermentation.
TABLE 6 content of Sulfur-containing and Nitrogen-containing Compounds in fish meat after 7 days of fermentation of the groups of stinky Mandarin fish (unit: ng/g)
Note that: ND in the table indicates that the substance was not detected; group C is non-inoculation fermentation; l group is single lactobacillus plantarum fermentation; s group is fermentation of staphylococcus soil singly; LS group is mixed inoculation fermentation.
The table shows that the total content of sulfur-containing and nitrogen-containing compounds after the fermentation of the inoculation group is obviously lower than that of the C group, the sulfur-containing and nitrogen-containing compounds are main substances which generate putrefactive flavor in the fermented mandarin fish, the substances of the mixed inoculation LS group have obvious inhibition effect, except indole, but the substances have smaller difference from the comparison group, wherein three groups of inoculation groups have inhibition effect on trimethylamine, the inhibition effect of the mixed inoculation group is more obvious, the trimethylamine content of the comparison C group is 415.61 +/-27.06 ng/g, the content of trimethylamine of the mixed inoculation group LS group is the lowest, and only 177.14 +/-35.56 ng/g, so that the content of trimethylamine is obviously reduced, and the content of the produced bad flavor substances is reduced.
5. Determination of free amino acids in fish: measuring free amino acid in fermentation process by using a full-automatic amino acid analyzer, taking 5.0g of fish meat of the control group and the inoculation group in 1, 3, 5 and 7 days of fermentation process, stirring, adding about 15mL of 5% trichloroacetic acid solution for homogenizing, fixing the volume to 25mL, carrying out ultrasonic treatment for 2 times for 15min each time, filtering by using a needle filter of 0.22 mu m, bottling, and carrying out on-machine analysis.
TABLE 7 content of Free Amino Acids (FAA) in fish meat after 7 days of fermentation of the groups of stinky Mandarin fish (unit: mg/100 g)
(note: group C is non-inoculating fermentation, group L is single inoculating Lactobacillus plantarum fermentation, group S is single inoculating Staphylococcus aureus fermentation;
LS group is mixed inoculation fermentation
Note that: ND in the table indicates that no such material was detected.
From the above table, it can be seen that the umami amino acid and the sweet amino acid of the mixed inoculation group are obviously increased relative to the control group after fermentation is finished, and the bitter amino acid of the mixed inoculation group is reduced relative to the control group, which indicates that the inoculation of lactobacillus plantarum and staphylococcus soil can promote the formation of amino acid in the mandarin fish and improve the umami taste of the mandarin fish meat.
SEQ No.1 Lactobacillus plantarum molecular sequence:
CAGGGTTATGGCTGACAGCTCGTACATAACGGGTCGAACAGAACTCTGGTATTGATTGGTGCTTGCATCATGATTTACATTTGAGTGAGTGGCGAACTGGTGAGTAACACGTGGGAAACCTGCCCAGAAGCGGGGGATAACACCTGGAAACAGATGCTAATACCGCATAACAACTTGGACCGCATGGTCCGAGCTTGAAAGATGGCTTCGGCTATCACTTTTGGATGGTCCCGCGGCGTATTAGCTAGATGGTGGGGTAACGGCTCACCATGGCAATGATACGTAGCCGACCTGAGAGGGTAATCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGACGAAAGTCTGATGGAGCAACGCCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAACTCTGTTGTTAAAGAAGAACATATCTGAGAGTAACTGTTCAGGTATTGACGGTATTTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTTTTAAGTCTGATGTGAAAGCCTTCGGCTCAACCGAAGAAGTGCATCGGAAACTGGGAAACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGTATGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCATACCGTAAACGATGAATGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCTAACGCATTAAGCATTCCGCCTGGGGAGTACGGCCGCAAGGCTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCTACGCGAAGAACCTTACCAGGTCTTGACATACTATGCAAATCTAAGAGATTAGACGTTCCCTTCGGGGACATGGATACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTATCAGTTGCCAGCATTAAGTTGGGCACTCTGGTGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGATGGTACAACGAGTTGCGAACTCGCGAGAGTAAGCTAATCTCTTAAAGCCATTCTCAGTTCGGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGAGAGTTTGTAACACCCAAAGTCGGTGGGGTAACCTTTGAGGAACCAGCCTACCTCATCGAGTTTCCCCCG。
SEQ No.2 Staphylococcus aureus molecular sequence:
ACCTTCGACGGCTAGCTCCATAAATGGTTACTCCACCGGCTTCGGGTGTTACAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGTAGCATGCTGATCTACGATTACTAGCGATTCCAGCTTCATGTAGTCGAGTTGCAGACTACAATCCGAACTGAGAACAACTTTATGGGATTTGCATGACCTCGCGGTTTAGCTGCCCTTTGTATTGTCCATTGTAGCACGTGTGTAGCCCAAATCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCAACCTAGAGTGCCCAACTTAATGATGGCAACTAAGCTTAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCACTTTGTCCCCCGAAGGGGAAGGCTCTATCTCTAGAGTTTTCAAAGGATGTCAAGATTTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTAATGCGTTAGCTGCAGCACTAAGGGGCGGAAACCCCCTAACACTTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGATCCCCACGCTTTCGCACATCAGCGTCAGTTACAGACCAGAAAGTCGCCTTCGCCACTGGTGTTCCTCCATATCTCTGCGCATTTCACCGCTACACATGGAATTCCACTTTCCTCTTCTGCACTCAAGTTTCCCAGTTTCCAATGACCCTCCACGGTTGAGCCGTGGGCTTTCACATCAGACTTAAGAAACCGCCTACGCGCGCTTTACGCCCAATAATTCCGGATAACGCTTGCCACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTGGCTTTCTGATTAGGTACCGTCAAGACGTGCACAGTTACTTACACGTTTGTTCTTCCCTAATAACAGAGTTTTACGAGCCGAAACCCTTCATCACTCACGCGGCGTTGCTCCGTCAGGCTTTCGCCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGACCGTGTCTCAGTTCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGTATCGTCGCCTTGGTAAGCCGTTACCTTACCAACTAGCTAATACGGCGCGGGTCCATCTATAAGTGATAGCAAAACCATCTTTCACTTTAGAACCATGCGGTTCCAAATGTTATCCGGTATTAGCTCCGGTTTCCCGAAGTTATCCCAGTCTTATAGGTAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACGTCAAAGGAGCAAGCTCCTTATCTGTTCGCTCGACTtGCAtGtAtTAG。
the foregoing description of the preferred embodiment of the invention is not intended to be limiting in any way, but rather, it is intended to cover all modifications or variations of the invention which fall within the spirit and scope of the invention.

Claims (9)

1. A lactobacillus plantarum, characterized in that: the lactobacillus plantarum is lactobacillus plantarumLactiplantibacillus plantarum) YR07 is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022, 8 months and 18 days, and the strain preservation number is CCTCCNO: M20221303.
2. A staphylococcus soil strain, characterized in that: the staphylococcus soil is staphylococcus soilStaphylococcus edaphicus) T-F is preserved in China Center for Type Culture Collection (CCTCC), the preservation date is 2022, 12 months and 19 days, and the strain preservation number is CCTCC NO: M20221987.
3. A fermentation method of smelly mandarin fish is characterized in that: comprises the following steps:
step 1: plant milk as claimed in claim 1Bacillus [(s) ]Lactiplantibacillus plantarum) Transferring one loop of YR07 from the inclined plane to an MRS agar culture medium for activation by using an inoculating loop, culturing for 36-48h at 30-37 ℃, transferring the obtained culture to an MRS liquid culture medium by using the inoculating loop again, and culturing for 24-28h at 30-37 ℃ to obtain a first inoculum;
step 2: the staphylococcus aureus of claim 1Staphylococcus edaphicus) Transferring the T-F into an MSA agar culture medium from an inclined plane by using an inoculating loop for activation, culturing at 30-37 ℃ for 36-48h, transferring the obtained culture into the MSA agar culture medium by using the inoculating loop, and culturing at 30-37 ℃ for 28-36h to obtain a second inoculum;
step 3: removing scales, cheeks and viscera of fresh mandarin fish, cleaning, placing in a container, and adding sterile saline;
step 4: adding the first inoculum and the second inoculum, capping, and fermenting at 8-12deg.C.
4. A method of fermenting a stinky mandarin fish according to claim 3, characterized in that: in the step 1, the obtained first inoculum was liquid, and the first inoculum was centrifuged at 3500rpm/min to obtain wet cells.
5. A method of fermenting a stinky mandarin fish according to claim 3, characterized in that: in step 2, the obtained second inoculum was washed with sterile physiological saline on an agar plate, followed by centrifugation at 3500rpm/min to obtain wet cells.
6. A method of fermenting a stinky mandarin fish according to claim 3, characterized in that: in the step 3, the mass fraction of the sterile saline is 5-10%, and the ratio of the sterile saline to the fish meat is as follows: 1 L:1.2-1.6. 1.6kg.
7. A method of fermenting a stinky mandarin fish according to claim 3, characterized in that: in the step 4, the inoculation amount of the first inoculum and the second inoculum is 1.0-1.5g/kg according to the mass ratio of the wet thalli to the fish meat.
8. A method of fermenting a stinky mandarin fish according to claim 3, characterized in that: every liter of MRS agar medium contains: 10.0g of peptone, 8.0g of beef powder, 4.0g of yeast powder, 20.0g of glucose, 2.0g of dipotassium hydrogen phosphate, 2.0g of diammonium hydrogen citrate, 5.0g of sodium acetate, 0.2g of magnesium sulfate, 0.04g of manganese sulfate, 1.0g of tween and 15.0-20.0g of agar; sterilizing at 121deg.C for 20min at pH 6.5+ -0.2; MRS liquid medium differs from MRS agar medium in that no agar is added.
9. A method of fermenting a stinky mandarin fish according to claim 3, characterized in that: the formula of the MSA agar medium is as follows: 10g of peptone, 1g of beef extract powder, 10g of mannitol, 25g of sodium chloride, 15-20g of agar powder and 1L of distilled water; sterilizing at 121deg.C for 20min at pH 7.0+ -2.
CN202310217694.3A 2023-03-08 2023-03-08 Fermentation method of stink mandarin fish and strain used by same Pending CN116925953A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116622567A (en) * 2023-05-19 2023-08-22 合肥工业大学 Probiotics and application thereof in preparation of heme peptide iron

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116622567A (en) * 2023-05-19 2023-08-22 合肥工业大学 Probiotics and application thereof in preparation of heme peptide iron
CN116622567B (en) * 2023-05-19 2024-01-23 合肥工业大学 Probiotics and application thereof in preparation of heme peptide iron

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