Lactobacillus brevis SMF L6 for improving characteristic flavor of fermented fish and application of lactobacillus brevis SMF L6

Abstract

The invention discloses a lactobacillus brevis SMF L6 strain for improving the characteristic flavor of fermented fish and application thereof, and belongs to the technical field of aquatic product processing. According to the method disclosed by the invention, low-temperature fermentation is carried out by utilizing a sake lactobacillus wide cloth SMF L6 direct vat set fermentation inoculant to regulate and control the flavor; the method can effectively promote the production of flavor substances such as n-hexyl alcohol, eudesmol, 2-ethylhexyl alcohol, 4-platycladi alcohol, linalool, 4-terpene alcohol, alpha-terpilenol, piperonone, beta-myrcene, alpha-terpinene, D-limonene, beta-phellandrene, gamma-terpinene and the like in the fermented fish. The content of pungent smell and fishy smell substances such as acetic acid, p-xylene, o-xylene, trimethylamine, methyl mercaptan and carbon disulfide is reduced.

Description

Technical Field

[0001] The present invention relates to a Lactobacillus sakei SMF L6 for enhancing the characteristic flavor of fermented fish and its application, belonging to the fields of microbial technology and food fermentation technology. Background Art

[0002] Fermented fish products have a long history in China. The famous Anhui cuisine, stinky mandarin fish, is one of the typical representatives. After heating, stinky mandarin fish has the characteristics of smelling stinky but tasting fragrant, having separated bones and meat, and the fish meat being in the shape of garlic cloves, which is deeply loved by consumers. The processing clusters of stinky mandarin fish are mainly distributed in the Huangshan area of Anhui. In 2023, the processing volume was nearly 55,000 tons, and the processing output value has exceeded 5 billion yuan. In recent years, with the continuous maturity of the fermentation technology of stinky mandarin fish, more and more stinky mandarin fish processing enterprises have extended it to other fish species, forming new products, including stinky perch, stinky yellow croaker, etc.

[0003] Fermentation is a traditional way of storing and processing fish. During the fermentation process, various microorganisms through complex metabolic processes synergistically degrade macromolecular substances such as proteins and lipids in fish meat, produce flavor precursor substances, and further produce a series of low-molecular-weight flavor compounds, forming the unique flavors of different fermented fish. The contents of volatile flavor substances in different fermented fish products vary greatly, but the compositions are similar, all including alcohols, aldehydes, ketones, acids, esters, sulfur-containing compounds, nitrogen-containing compounds, olefins, alkanes, etc. Research shows that aroma-active substances such as 1-octen-3-ol, eucalyptol, linalool, piperitone, β-myrcene, D-limonene, terpinolene, etc. make important contributions to the characteristic flavor of fish, and odor components such as methanethiol, trimethylamine, propionic acid, indole, etc. are important sources of the special odor of fish. Traditional fermented fish products are naturally fermented by the microorganisms in the fish body itself and the fermentation environment. The products are prone to problems such as insufficient fragrance and strong fishy and stinky smells, thus seriously restricting the development of the fermented fish industry. Therefore, it is urgent to change the traditional fermentation method to improve the flavor quality of products and promote the development of the industry.

[0004] Traditional fermented fish contains rich lactic acid bacteria resources, including Lactobacillus, Lactococcus, Leuconostoc, Weissella, etc. Among them, related functional strains such as Lactobacillus plantarum, Lactobacillus sakei, Lactococcus lactis, Leuconostoc mesenteroides, Weissella cibaria, etc. have properties such as fermenting to produce acid, inhibiting the growth of harmful bacteria, and metabolizing to produce flavor substances, which play an important role in improving product quality and promoting flavor formation. In recent years, inoculation fermentation as a technical method for improving the flavor quality of fermented fish has received attention. Screening suitable lactic acid bacteria for inoculation fermentation is of great significance for improving the preparation process of fermented fish. Summary of the Invention

[0005] The object of the present invention is to provide a Latilacotobacillus sakei SMF L6 for enhancing the characteristic flavor of fermented fish and its application, so as to solve the technical problems in the prior art that the processing technology of fermented fish follows the traditional natural fermentation method, resulting in problems such as insufficient fragrance and strong fishy and stinky odors in the products, which seriously restricts the development of the fermented fish industry. The strain or starter provided by the invention is beneficial to improving the hardness and elasticity of the flesh of fermented mandarin fish and fermented large yellow croaker, increasing the content of volatile aroma substances in the fish body, and reducing the content of fishy and stinky flavor substances.

[0006] The first technical solution provided by the present invention is a strain of Latilacotobacillus sakei SMF L6. The original strain name of the Latilacotobacillus sakei is Lacotobacillus sakei, which was deposited at the Guangdong Microbial Culture Collection Center on December 7, 2020, with the deposit number GDMCC NO: 61346.

[0007] The Latilacotobacillus sakei SMF L6 is isolated and screened from fermented fish. The Latilacotobacillus sakei SMF L6 is cultured on MRS solid medium containing bromocresol green for 48 h, and the colonies are round, with a smooth surface, neat edges, a raised center, and a light green color around and a dark green center.

[0008]

[0009] The Lactobacillus sakei SMF L6 is anaerobically cultured in MRS liquid medium at 30 °C for 24 h, and the viable cell count reaches 1×10 9 ~1×10 10 CFU / mL, and it has good growth and acid production, salt tolerance, antibacterial ability and does not produce biogenic amines.

[0010] The second technical solution provided by the present invention is a microbial preparation containing the Lactobacillus sakei SMF L6 described in the first technical solution.

[0011] In one embodiment, the viable cell count of the Lactobacillus sakei SMF L6 in the microbial preparation is at least 1×10 10 CFU / mL.

[0012] The third technical solution provided by the present invention is a direct vat inoculant, and the fermenting agent contains the Lactobacillus sakei SMF L6 described in the first technical solution or the microbial preparation described in the second technical solution.

[0013] In one embodiment, the viable cell count of the Lactobacillus sakei SMF L6 in the direct vat inoculant is at least 1×10 11 CFU / g.

[0014] The fourth technical solution provided by the present invention is the application of the Lactobacillus sakei SMF L6 described in the first technical solution, or the microbial preparation described in the second technical solution, or the direct vat inoculant described in the third technical solution in the preparation of fermented fish products.

[0015] In one embodiment, the fermented fish products include fermented mandarin fish and fermented large yellow croaker.

[0016] The fifth technical solution provided by the present invention is the application of the Lactobacillus sakei SMF L6 described in the first technical solution, or the microbial preparation described in the second technical solution, or the direct vat inoculant described in the third technical solution in enhancing the flavor of fermented fish, and the enhancement of the flavor of fermented fish includes enhancing the flavor substances in fermented fish and reducing the pungent odor and fishy smell substances in fermented fish.

[0017] In one embodiment, the flavor substances include n-hexanol, 1-octen-3-ol, eucalyptol, 2-ethylhexanol, 4-thujanol, linalool, 4-terpineol, α-terpineol, piperitone, β-myrcene, α-terpinene, D-limonene, β-phellandrene, γ-terpinene, etc.

[0018] In one embodiment, the pungent odor and fishy odor substances include acetic acid, propionic acid, butyric acid, p-xylene, o-xylene, trimethylamine, methanethiol, carbon disulfide, dimethyl disulfide, etc.

[0019] The sixth technical solution provided by the present invention is a fermentation regulation method for enhancing the characteristic flavor of fermented fish. The method is to use Lactobacillus sakei SMF L6 described in the first technical solution, or the microbial preparation described in the second technical solution, or the direct vat inoculum described in the third technical solution to carry out low-temperature fermentation regulation of the flavor of mandarin fish and large yellow croaker respectively, enhance the flavor substances in the fermented fish, and reduce the pungent odor and fishy odor substances in the fermented fish.

[0020] In one embodiment, the flavor substances include n-hexanol, 1-octen-3-ol, eucalyptol, 2-ethylhexanol, 4-thujanol, linalool, 4-terpineol, α-terpineol, piperitone, β-myrcene, α-terpinene, D-limonene, β-phellandrene, γ-terpinene, etc.

[0021] In one embodiment, the pungent odor and fishy odor substances include acetic acid, p-xylene, o-xylene, trimethylamine, methanethiol, carbon disulfide, dimethyl disulfide, etc.

[0022] In one embodiment, in the fermentation system, the concentration of Lactobacillus sakei SMF L6 is 1×10 7 ~1×10 8 CFU / g.

[0023] In one embodiment, it includes the following steps:

[0024] 1. Raw material sorting

[0025] Live mandarin fish and ice fresh large yellow croaker are sorted by a sorting machine, and fish with a weight of 0.5 - 0.6 kg per tail are used as raw materials for fermented fish;

[0026] 2. Slaughter

[0027] The raw material fish is placed in clean water for cleaning to remove surface dirt, then slaughtered, and scales, gills and internal organs are removed, and then it is cleaned and drained;

[0028] 3. Fermentation

[0029] Place the fish after slaughtering and draining in a saline solution of 6% - 8% for 20 - 30 minutes, then take out and drain. Evenly stack the processed mandarin fish and large yellow croaker in different fermentation devices, with 40 - 60 kg of fish stacked in each device. For each layer stacked, evenly sprinkle stir-fried Chinese prickly ash (the total amount of Chinese prickly ash used is 12 - 18 g) on the surface of the fish. After stacking is completed, place a stainless steel pressing plate on the upper layer of fish, and add the pre-prepared pickling solution (containing 8% salt and 0.025% direct vat set fermentation starter of Lactiplantibacillus sakei) along the inner wall of the device. The total amount of pickling solution is 14 - 21 kg. Place a stone slab on the stainless steel pressing plate (the weight of the stone slab is 40% of the weight of the fish) to immerse all the fish below the liquid level of the pickling solution. Cover the top cover of the fermentation device and ferment in an anaerobic fermentation manner at 8 - 10 °C for 12 - 16 days.

[0030] 4. Packaging

[0031] After fermentation is completed, take out the fermented fish, wash it with clean water and then perform vacuum packaging, and store it frozen at -18 °C.

[0032] The technical effects of the present invention are as follows:

[0033] The present invention provides a fermentation regulation method for enhancing the characteristic flavor of fermented fish. Using Lactiplantibacillus sakei SMF L6 for low-temperature fermentation to regulate flavor can effectively promote the generation of flavor substances in fermented fish such as hexanol, 1-octen-3-ol, eucalyptol, 2-ethylhexanol, 4-thujanol, linalool, 4-terpineol, α-terpineol, piperitone, β-myrcene, α-terpinene, D-limonene, β-phellandrene, γ-terpinene, etc., and reduce the content of irritating odors and fishy and stinky substances such as acetic acid, p-xylene, o-xylene, trimethylamine, methanethiol, carbon disulfide, dimethyl disulfide, etc.

[0034] Biological material preservation

[0035] The Lactiplantibacillus sakei SMF L6 of the present invention, classified and named as Latilacotobacillus sakei, with the original strain name being Lactobacillus sakei, has been preserved in the Guangdong Provincial Microbial Culture Collection Center, with the preservation number GDMCC NO: 61346, the preservation date being December 7, 2020, and the preservation address being the 5th floor, Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Guangdong Province. Description of the drawings

[0036] Figure 1 It is a morphological characteristic diagram of the lactic acid bacteria strain;

[0037] Figure 2 It is an identification result diagram of the lactic acid bacteria strain;

[0038] Figure 3 Graph showing the results of measuring the growth and acid production ability of lactic acid bacteria strains;

[0039] Figure 4 Graph showing the results of measuring the antibacterial ability of lactic acid bacteria strains;

[0040] Figure 5 Graph showing the results of measuring the biogenic amine production of lactic acid bacteria strains;

[0041] Figure 6 Graph showing the detection results of volatile flavor substances in mandarin fish and large yellow croaker fermented by SMF L6 strain; Detailed implementation manners

[0042] The following describes the preferred embodiments of the present invention. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0043] Isolation and preservation of strains

[0044] A strain of Lactiplantibacillus sakei isolated and screened from traditional Huizhou stinky mandarin fish samples in the present invention.

[0045] This strain is named Lactiplantibacillus sakei SMF L6, and its taxonomic name is Latilacotobacillus sakei. The original strain name is Lactobacillus sakei. It was deposited at the Guangdong Provincial Microbial Culture Collection Center on December 7, 2020, with the deposit number GDMCC NO: 61346, and the deposit address is the 5th floor of Building 59, No. 100 Yard, Xianlie Middle Road, Guangzhou, Guangdong Province, Institute of Microbiology, Guangdong Academy of Sciences.

[0046] The 16S rDNA gene sequence of the strain SMF L6 of the present invention has a similarity of 100% with Latilacotobacillus sakei and is identified as Latilacotobacillus sakei, which can be used in food production and processing.

[0047]

[0048] The colony characteristics of Lactobacillus sakei SMF L6 are as follows: After culturing on MRS solid medium containing bromocresol green for 48 h, the colonies are round, with a smooth surface, regular edges, a raised center, and a light green color around the center and a dark green color in the center.

[0049] The biological characteristics of Lactobacillus sakei SMF L6 are as follows: Anaerobic culture in MRS liquid medium at 30 °C for 24 h, the viable count reaches 1×10 8 ~1×10 9 CFU / mL, with good growth and acid production, salt tolerance, antibacterial ability, and no biogenic amine production.

[0050] Medium formulation

[0051] 1. MRS solid medium (g / L): Peptone 10.0, Beef extract powder 5.0, Yeast extract powder 4.0, Glucose 20.0, Dipotassium hydrogen phosphate 2.0, Ammonium citrate dibasic 2.0, Sodium acetate 5.0, Magnesium sulfate 0.2, Manganese sulfate 0.05, Agar 15.0, Tween 80 1.0, Calcium carbonate 10, Bromocresol green 0.1, pH value 6.2 ± 0.2.

[0052] 2. MRS liquid medium (g / L): Peptone 10.0, Beef extract powder 8.0, Yeast extract powder 4.0, Glucose 20.0, Dipotassium hydrogen phosphate 2.0, Ammonium citrate dibasic 2.0, Sodium acetate 5.0, Magnesium sulfate 0.2, Manganese sulfate 0.04, Tween 80 1.0, pH value 5.7 ± 0.2.

[0053] 3. Water agar medium (g / L): Agar 15 g.

[0054] 4. Nutrient agar medium (g / L): Tryptone 10.0, Beef extract powder 3.0, Sodium chloride 5.0, Agar 15.0, pH value 7.3 ± 0.1.

[0055] 5. Biogenic amine detection (color development) medium (g / L): Peptone 10.0, Beef extract powder 8.0, Yeast extract powder 4.0, Glucose 20.0, Dipotassium hydrogen phosphate 2.0, Ammonium citrate dibasic 2.0, Sodium acetate 5.0, Magnesium sulfate 0.2, Manganese sulfate 0.04, Tween 80 1.0, Bromocresol purple 0.06, Amino acids (phenylalanine, histidine, arginine, ornithine, lysine, tryptophan) 10.0, Tyrosine 0.4, pH 5.5.

[0056] Determination of volatile compounds in fermented fish

[0057] The volatile substances in fermented fish were extracted by headspace solid-phase microextraction. The fermented fish flesh was dissected with a scalpel and minced with a meat grinder. 5 g of minced fish and 20 μL of internal standard substance (chlorobenzene-d5 dissolved in methanol, 1.0 mg / L) were placed in a 20 mL headspace vial and equilibrated in a 60 °C water bath for 60 min. Then, an extraction head equipped with 2 cm-50 / 30 DVB / CAR / PDMS StableFlex was inserted into the headspace vial, and headspace adsorption was carried out in a 60 °C water bath for 50 min. After the adsorption was completed, the extraction head was immediately taken out and inserted into the gas chromatography injection port for 6 min of desorption. The determination of volatile substances was carried out using a gas chromatography-mass spectrometer (GC-MS). Gas chromatography conditions: DB-WAX capillary column (60 m × 0.25 mm, 0.25 μm); temperature programming: held at 40 °C for 3 min, gradually increased to 90 °C at a rate of 5 °C / min, and then gradually increased to 230 °C at a rate of 10 °C / min and held for 7 min; carrier gas (He) flow rate 1.0 mL / min, pressure 2.4 kPa, injection volume 0.5 μL; splitless. Mass spectrometry conditions: electron impact ion source; electron energy 70 eV; scanning mass range 30-500 m / z; detector voltage 1000 v; injector temperature 250 °C.

[0058] Data analysis: Each experiment was repeated three times. The identification of volatile compounds was carried out by comparing with the NIST 14 and NIST14S mass spectrometry libraries, and the compounds with a similarity greater than 80% were screened for identification. The internal standard method (chlorobenzene-d5) was used to quantitatively calculate the identified substances in each sample, and the content was expressed as ng / 100 g.

[0059] Example 1 Isolation, identification and biological characteristic analysis of lactic acid bacteria

[0060] 1. Strain isolation

[0061] a. Pretreatment of separation matrix

[0062] Under aseptic operation conditions, the collected Huizhou stinky mandarin fish samples were cut off the heads, fins and tails, the fish bones were removed, and the remaining parts were minced with a meat grinder.

[0063] b. Isolation and purification of strains

[0064] Accurately weigh 1.0 g of the treated sample, perform gradient dilution with sterile normal saline, and select 10 -6 ~10 -9The gradient dilution solution was spread on MRS solid medium and anaerobically cultured at 30 °C for 48 h. Colonies that could produce a calcium dissolution zone on the plate and could change the color of the bromocresol green medium from blue-green to light green or yellow were picked and streaked on MRS solid medium by the three-zone streaking method for strain purification. Four suspected lactic acid bacteria strains were initially obtained by Gram staining and catalase test, numbered as: SMF L6, SMF L16, F55, FL86. The morphological characteristics of the strains are shown in Figure 1 .

[0065] c. Preservation of strains

[0066] The purified strains were respectively inoculated into MRS liquid medium and anaerobically cultured at 30 °C for 24 h. The culture solution and sterilized 60% glycerol were mixed at a volume ratio of 1:1, aliquoted into sterile cryotubes, and stored at -80 °C for a long time.

[0067] 2. Strain identification

[0068] The purified strains were subjected to 16S rRNA molecular biological identification. The sequencing primers were 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-CTACGGCTACCTTGTTACGA-3’). The sequencing results were aligned and an evolutionary tree was constructed using the website of the National Center for Biotechnology Information (NCBI) (http: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The identification results are shown in Figure 2 .

[0069] 3. Analysis of strain biological characteristics

[0070] a. Determination of growth ability

[0071] The pure strain was inoculated into MRS liquid medium for activation culture (30 °C, 24 h). The culture solution was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and cultured for 48 h. The bacterial solution was taken every 6 h to measure its OD 600 value. The measurement results are shown in Figure 3 A.

[0072] b. Determination of acid production ability

[0073] The activated bacterial solution was taken and inoculated into the corresponding liquid medium at an inoculation amount of 1% (v / v) and cultured for 48 h. The bacterial solution was taken every 6 h, and after centrifugation, the acid production amount of the strain was determined by the sodium hydroxide solution titration method. The measurement results are shown in Figure 3 B.

[0074] c. Determination of antibacterial ability

[0075] The antibacterial characteristics of the strains were detected by the Oxford cup double-layer agar diffusion method. The indicator bacteria were Staphylococcus saparophytics, Pseudomonas syringae, and Pseudomonas brenneri isolated from fermented mandarin fish. The measurement results are shown in Figure 4 .

[0076] First, 10 mL of sterilized water agar was poured onto the bottom layer of the petri dish. After it dried, Oxford cups were placed on it, with 4 cups placed in each petri dish. The indicator bacteria suspension (100 μL) was mixed with the nutrient agar (20 mL) pre-warmed to 46 °C, and then poured into the petri dish with the Oxford cups placed. After it dried, the Oxford cups were removed. One well was used as a control, and 100 μL of the uninoculated MRS liquid medium was added to it. The other 3 wells were used as parallel samples, and 100 μL of the test bacterial liquid was added to them. Diffusion was carried out at 4 °C for 2 h, and then static culture was carried out at 30 °C for 48 h. The diameter of the antibacterial zone was observed and measured with a vernier caliper, and the antibacterial performance of the strain was judged according to the size of the antibacterial zone diameter.

[0077] d. Determination of biogenic amine production

[0078] The activated bacterial liquid was streaked on the solid medium for biogenic amine detection (color development). It was cultured at 30 °C for 48 h. The color change of the medium was observed. If the medium turned blue-violet, it indicated the production of biogenic amines. If the color of the medium remained unchanged or turned yellow, it indicated less or no production of biogenic amines. The measurement results are shown in Figure 5 .

[0079] Comprehensively Figure 3 , Figure 4 , Figure 5 From the measurement results, the growth, acid production, and antibacterial ability of the strain SMF L6 were better than those of other isolated strains, and it had the ability not to produce biogenic amines, so it could be used as a source of excellent fermentation inoculant.

[0080] Example 2 Preparation of direct vat set (DVS) fermentation inoculant of Lactobacillus sakei subsp. sakei SMF L6

[0081] 1. Activation and culture of the strain

[0082] Lactobacillus sakei subsp. sakei SMF L6 (GDMCC NO: 61346) obtained in Example 1 was streaked and inoculated onto the MRS solid medium and anaerobically cultured at 30 °C for 36 h. Single colonies were picked and inoculated into a triangular flask containing 200 mL of MRS liquid medium, and activated and cultured at 30 °C for 24 - 36 h, with continuous culture carried out 2 times.

[0083] 2. Strain expansion and cultivation

[0084] The bacterial liquid from the second activation culture was inoculated into MRS liquid culture medium at a 1% inoculum size for expansion culture (50 L) and cultured for 24 to 36 h. The bacterial sludge was collected by centrifugation at 6500 rpm / min for 15 min, and sterile physiological saline was added at 10 times the volume of the bacterial sludge. The culture was centrifuged and washed, and the bacterial sludge was collected again.

[0085] 3. Freeze Drying

[0086] The protective agent (12% skim milk powder, 6% trehalose, 3% glycerol) was fully mixed with the bacterial sludge in a ratio of 2:1 (w / w), transferred to a disposable sterile culture dish (pour thickness of about 0.5 cm), covered with a sterile culture dish cover, placed in a -40℃ refrigerator, pre-frozen for 12 h, and then vacuum-frozen (cold trap temperature of -60℃, vacuum degree of 1 Pa), vacuum-frozen for 48 h, and the bacterial powder was collected to obtain the sake Lactobacillus SMF L6 direct-injection fermentation agent, with a live cell count of at least 1×10 11 CFU / g.

[0087] Example 3 Preparation of fermented mandarin fish using direct fermentation agent of Lactobacillus sakei SMF L6

[0088] 1. Raw material sorting

[0089] Fresh mandarin fish were sorted by a sorting machine, and 0.5-0.6 kg / fish was used as the fermented fish raw material;

[0090] 2. Slaughter

[0091] The raw fish is washed in clean water to remove surface dirt, then slaughtered, the scales, gills and internal organs are removed, and then washed and drained;

[0092] 3. Fermentation

[0093] Soak the slaughtered and drained fish in 6% to 8% salt water for 20 to 30 min, then remove and drain. Stack the processed mandarin fish and large yellow croaker flatly in different fermentation devices, with 40 to 60 kg of fish stacked in each device, one layer per stack, and evenly sprinkle fried peppercorns (the total amount of peppercorns is 12 to 18 g) on ​​the surface of the fish. After stacking, place a stainless steel pressing plate on the upper layer of the fish, and add the pre-prepared pickling liquid (containing 8% salt and 0.025% sake-wide Lactobacillus SMF L6 direct-injection fermentation agent, the total amount of pickling liquid is 14 to 21 kg) along the inner wall of the device. Place a stone plate on the stainless steel pressing plate (the weight of the stone plate is 40% of the weight of the fish) to immerse the fish below the surface of the pickling liquid. Cover the top cover of the fermentation device and ferment it at 8 to 10 ° C for 12 to 16 days by anaerobic fermentation.

[0094] 4. Packaging

[0095] After fermentation, take out the fermented fish, wash it with clean water and then perform vacuum packaging, and store it frozen at -18°C.

[0096] Example 4 Preparation of fermented yellow croaker using the direct vat inoculation fermentation agent of Lactobacillus sakei subsp. sakei SMF L6

[0097] Replace the raw materials in Example 3 with chilled yellow croaker, and keep the other conditions and steps the same as in Example 3 to prepare fermented yellow croaker.

[0098] Comparative Example 1 Preparation of fermented mandarin fish by natural fermentation

[0099] 1. Raw material sorting

[0100] Sort the live mandarin fish with a sorting machine, and use fish with a weight of 0.5 - 0.6 kg per tail as the raw material for fermented fish;

[0101] 2. Slaughter

[0102] Place the raw material fish in clean water for washing to remove surface dirt, then slaughter it, remove scales, gills and internal organs, and then wash and drain the water;

[0103] 3. Fermentation

[0104] Soak the slaughtered and drained fish in 6% - 8% brine for 20 - 30 min, then take it out and drain the water. Place the processed mandarin fish and yellow croaker separately and neatly stack them in different fermentation devices. Stack 40 - 60 kg of fish in each device. For each layer stacked, evenly sprinkle stir-fried Chinese prickly ash (the total amount of Chinese prickly ash used is 12 - 18 g) on the surface of the fish. After stacking is completed, place a stainless steel pressing plate on the upper layer of fish, and add the pre-prepared pickling solution (containing 8% salt, and the total amount of pickling solution is 14 - 21 kg) along the inner wall of the device. Place a stone slab (the weight of the stone slab is 40% of the weight of the fish) above the stainless steel pressing plate to completely immerse the fish body below the liquid level of the pickling solution. Cover the top cover of the fermentation device and ferment anaerobically at 8 - 10°C for 12 - 16 days.

[0105] 4. Packaging

[0106] After fermentation, take out the fermented fish, wash it with clean water and then perform vacuum packaging, and store it frozen at -18°C.

[0107] Comparative Example 2 Preparation of fermented yellow croaker by natural fermentation

[0108] Replace the raw materials in Comparative Example 1 with chilled yellow croaker, and keep the other conditions and steps the same as in Comparative Example 1 to prepare fermented yellow croaker.

[0109] Test Example: Detection of Volatile Flavor Compounds in Naturally and Inoculated Fermented Fermented Fish

[0110] After the fermentation was completed, the volatile flavor compounds in the fermented fish fermented naturally and inoculated with the direct-fed starter culture of Lactobacillus sakei subsp. sakei SMF L6 were detected.

[0111] Table 1 Composition and Content of Volatile Flavor Compounds in Naturally and Inoculated Fermented Fermented Fish (ng / 100 g)

[0112] Note: " / " indicates that the odor description was not found; "ND" indicates not detected.

[0113] The results are shown in Table 1 and Figure 6 As shown, inoculating with the direct-fed starter culture of Lactobacillus sakei subsp. sakei SMF L6 can significantly increase the contents of alcohols, ketones, esters, and olefins in the fermented mandarin fish, and reduce the contents of acids and other compounds including sulfur-containing, nitrogen-containing, and aromatic compounds. Among them, the contents of aroma substances such as n-hexanol, n-heptanol, 1-octen-3-ol, eucalyptol, 2-ethylhexanol, 4-thujanol, linalool, 4-terpineol, α-terpineol, piperitone, β-myrcene, α-terpinene, D-limonene, β-phellandrene, γ-terpinene, etc. increased significantly, while the contents of irritating odor and fishy odor substances such as acetic acid, propionic acid, butyric acid, p-xylene, o-xylene, methanethiol, trimethylamine, carbon disulfide, dimethyl disulfide, etc. decreased significantly, thus promoting the formation of the characteristic flavor of the fermented mandarin fish and reducing the fishy odor. Inoculating with the direct-fed starter culture of Lactobacillus sakei subsp. sakei SMF L6 can promote the increase in the contents of aroma substances such as n-hexanol, n-heptanol, 1-octen-3-ol, 2-ethylhexanol, α-thujene, sabinene, α-phellandrene, etc. in the fermented large yellow croaker, and the decrease in the contents of irritating odor and fishy odor substances such as acetic acid, propionic acid, butyric acid, p-xylene, o-xylene, methanethiol, trimethylamine, carbon disulfide, dimethyl disulfide, etc. Generally speaking, inoculating with the direct-fed starter culture of Lactobacillus sakei subsp. sakei SMF L6 has a promoting effect on the formation of the characteristic flavor of the fermented fish.

[0114] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A strain of Latilacotobacillus sakei SMF L6, characterized in that, The original strain name of Lactobacillus sakei var. sakei is Lactobacillus sakei, which was deposited in the Guangdong Microbial Culture Collection Center on December 7, 2020, with the deposit number GDMCC NO: 61346.

2. A microbial preparation containing Lactobacillus sakei SMF L6 described in claim 1.

3. The microbial preparation according to claim 2, wherein The viable count of Lactobacillus sakei SMF L6 in the microbial preparation is at least 1×10 10 CFU / mL.

4. A direct-injection fermentation inoculant, characterized in that, The direct-fed fermentation starter contains Lactobacillus sakei var. sakei SMF L6 described in claim 1 or the microbial preparation described in claim 2 or 3.

5. The application of Lactobacillus sakei var. sakei SMF L6 described in claim 1, or the microbial preparation described in claim 2 or 2, or the direct-fed fermentation starter described in claim 4 in the preparation of fermented fish products.

6. The application according to claim 5, wherein The fermented fish products include fermented mandarin fish and fermented large yellow croaker.

7. The application of Lactobacillus sakei var. sakei SMF L6 described in claim 1, or the microbial preparation described in claim 2 or 3, or the direct-fed fermentation starter described in claim 4 in improving the flavor of fermented fish, where improving the flavor of fermented fish includes enhancing the flavor substances in fermented fish and reducing the pungent and fishy odor substances in fermented fish.

8. The application according to claim 7, wherein The flavor substances include n-hexanol, 1-octen-3-ol, eucalyptol, 2-ethylhexanol, 4-thujanol, linalool, 4-terpinenol, α-terpineol, piperitone, β-myrcene, α-terpinene, D-limonene, β-phellandrene, γ-terpinene, etc.; the pungent and fishy odor substances include acetic acid, propionic acid, butyric acid, p-xylene, o-xylene, trimethylamine, methanethiol, carbon disulfide, dimethyl disulfide, etc.

9. A fermentation regulation method for enhancing the characteristic flavor of stinky mandarin fish, characterized in that, The method is to regulate the flavor of mandarin fish and large yellow croaker by low-temperature fermentation using Lactobacillus sakei var. sakei SMF L6 described in claim 1, or the microbial preparation described in claim 2 or 3, or the direct-fed fermentation starter described in claim 4, enhancing the flavor substances in fermented fish and reducing the pungent and fishy odor substances in fermented fish.

10. The method according to claim 9, wherein The flavor substances include n-hexanol, 1-octen-3-ol, eucalyptol, 2-ethylhexanol, 4-thujanol, linalool, 4-terpinenol, α-terpineol, piperitone, β-myrcene, α-terpinene, D-limonene, β-phellandrene, γ-terpinene; the pungent and fishy odor substances include acetic acid, propionic acid, butyric acid, p-xylene, o-xylene, trimethylamine, methanethiol, carbon disulfide, dimethyl disulfide, etc.

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