WO2023050689A1 - 一种臭鳜鱼生物保鲜液及低盐臭鳜鱼的生产方法 - Google Patents

一种臭鳜鱼生物保鲜液及低盐臭鳜鱼的生产方法 Download PDF

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WO2023050689A1
WO2023050689A1 PCT/CN2022/077122 CN2022077122W WO2023050689A1 WO 2023050689 A1 WO2023050689 A1 WO 2023050689A1 CN 2022077122 W CN2022077122 W CN 2022077122W WO 2023050689 A1 WO2023050689 A1 WO 2023050689A1
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mandarin fish
stinky
fish
stinky mandarin
solution
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PCT/CN2022/077122
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English (en)
French (fr)
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吴永祥
陈向阳
周讯
杨柳
胡长玉
孙汉巨
佘新松
朱如瑾
董心雨
刘刚
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黄山学院
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/03—Drying; Subsequent reconstitution
    • A23B4/037—Freeze-drying, i.e. cryodessication, lyophilisation; Apparatus therefor
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A23L17/65—Addition of, or treatment with, microorganisms or enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/015—Preserving by irradiation or electric treatment without heating effect
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/12—Preserving with acids; Acid fermentation
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
    • A23B4/00—General methods for preserving meat, sausages, fish or fish products
    • A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
    • A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
    • A23B4/20—Organic compounds; Microorganisms; Enzymes
    • A23B4/22—Microorganisms; Enzymes; Antibiotics
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/015—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with pressure variation, shock, acceleration or shear stress or cavitation
    • A23L3/0155—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with pressure variation, shock, acceleration or shear stress or cavitation using sub- or super-atmospheric pressures, or pressure variations transmitted by a liquid or gas
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3571—Microorganisms; Enzymes
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/40—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by drying or kilning; Subsequent reconstitution
    • A23L3/44—Freeze-drying
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/125—Casei
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/157—Lactis
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2400/00—Lactic or propionic acid bacteria
    • A23V2400/11—Lactobacillus
    • A23V2400/179—Sakei

Definitions

  • the invention belongs to the technical field of food processing, and in particular relates to a biological preservation liquid for stinky mandarin fish and a production method for low-salt stinky mandarin fish.
  • Chinese patent application 2016101309986 announced "a method for continuous and rapid fermentation of stinky mandarin fish based on lactic acid bacteria", which specifically involves using the old soup (containing 4-15wt% of salt) fermented by stinky mandarin fish as an introduction, adding 0.005% to 0.03% Lactobacillus (combined with one or two of the following strains: Lactobacillus casei, Lactobacillus sake, Lactococcus gasseri or Lactobacillus thermophiles, the bacterial concentration is 10 7 ⁇ 10 9 CFU/mL), prepared by rapid fermentation Smelly mandarin fish.
  • Chinese patent 201811371639.5 announced "a method for rapidly fermenting stinky mandarin fish by mixing Lactococcus lactis M10 and Weissella angustiformis M3", which specifically involved the use of two strains of lactic acid bacteria isolated from stinky mandarin fish - lactic acid milk Cocci M10 and Weissella esophagus M3 are prepared by inoculating the two together, adding 3-10 wt% of salt, and fermenting at 10°C-15°C for 5-7 days. Both of these two patent applications use lactic acid bacteria to inoculate and rapidly ferment stinky mandarin fish, which shortens the fermentation time and improves product safety to a certain extent.
  • both of the two patent applications did not carry out effective pre-sterilization treatment on the body of mandarin fish in the early stage of fermentation.
  • the miscellaneous bacteria or harmful bacteria in the smelly mandarin fish can still affect the reproduction and growth of dominant lactic acid bacteria, resulting in the deterioration of the fermentation quality and flavor of the product. unstable.
  • These two patent applications are only a preliminary study on the application of lactic acid bacteria in the fermentation of stinky mandarin fish.
  • the fermentation performance of different lactic acid bacteria varies widely. Many aspects such as stage fermentation, pressure size and pressure setting time have not been disclosed.
  • Chinese patent application 2017105286691 discloses "a preparation method of instant stinky mandarin fish and its fresh-keeping solution", which specifically involves frying stinky mandarin fish, preparing stinky mandarin fish fresh-keeping solution, soaking half-cooked stinky mandarin fish in the fresh-keeping solution and sealing it in bags, Then, the sealed and packaged stinky mandarin fish was sterilized at a high temperature of 115°C for 15 ⁇ 5min, and then kept at 121°C for 10 ⁇ 3min.
  • the main components of the preservation solution are 0.01%-0.1% potassium sorbate, 0.005%-0.02% sodium pyrophosphate, 0.1%-0.4% TBHQ, 0.15%-0.5% hydroxylated lecithin, 0.08%-0.15% tea polyphenols, etc.
  • This application is mainly for the research on the instant fresh-keeping solution of stinky mandarin fish, but no research on the fresh-keeping solution of refrigerated stinky mandarin fish has been seen; and the main components of the fresh-keeping solution are mostly chemically synthesized additives rather than biologically active substances.
  • the present invention develops a biological fresh-keeping liquid for fresh-keeping stinky mandarin fish and a production method for low-salt stinky mandarin fish.
  • the present invention adopts the following technical solutions:
  • a preparation method of stinky mandarin fish biological preservation liquid comprising the following steps:
  • Step 1 pretreatment: washing the collected mandarin fish scales with water, then vacuum freeze-drying, and then pulverizing at -5°C to 10°C to obtain coarse scale powder;
  • Step 2 decalcification and impurity removal: decalcify the coarse fish scale powder obtained in step 1 with HCl solution, wash with water to neutrality and then centrifuge, add NaOH solution to the precipitate to soak and remove impurities, wash with water to neutrality, then centrifuge, and vacuum freeze-dry;
  • Step 3 enzymatic hydrolysis: resuspend the fish scale powder processed in step 2 in the enzymatic hydrolysis buffer, add protease to carry out enzymatic hydrolysis, and obtain the enzymatic hydrolysis solution;
  • Step 4 ultra-high pressure treatment the enzymolysis liquid obtained after the completion of step 3 enzymatic hydrolysis is placed in a high-pressure resistant packaging bag and sealed for ultra-high pressure treatment;
  • Step 5 purification: filter the enzymatic hydrolysis solution after the ultra-high pressure treatment in step 4 to obtain fish scale collagen peptide concentrate, and then perform vacuum freeze-drying to obtain mandarin fish scale collagen peptide powder;
  • Step 6 Maillard peptide reaction: mix and dissolve the mandarin fish scale collagen peptide powder and Polygonatum polysaccharide purified in step 5 into pure water to form a mixed solution, and perform the Maillard reaction. After the reaction, the ice bath is rapidly cooled to terminate the reaction. After the reaction solution is concentrated, Maillard peptide is obtained;
  • Step 7 preparation prepare a chitosan solution, add glycerin and mix it with the Maillard peptide prepared in step 6, and then homogenize after mixing to obtain the stinky mandarin fish biological preservation solution.
  • the decalcification condition is soaking with 0.2-0.5 mol/L HCl for 1-2 hours, and the impurity removal condition is adding 0.1-0.2 mol/L NaOH solution to the precipitate and soaking for 0.5-2 hours.
  • the enzymolysis condition is to resuspend the fish scale powder treated in step 2 in the enzymolysis buffer at a ratio of 1:5 to 1:10 (w/v), and then add protease for enzymolysis, and the protease is At least one of neutral protease, compound flavor protease, papain, trypsin, and pepsin, wherein the enzyme activity is 10,000 to 25,000 U/g, the amount of enzyme added is 0.2% to 1% by weight, and the enzymatic hydrolysis temperature is 37°C-55°C, enzymolysis pH 3.0-9.0, enzymolysis time 2-5h.
  • the ultra-high pressure treatment condition is: the set pressurized pressure value is 100 ⁇ 500MPa, the holding time is 5 ⁇ 20min, and the boosting rate is 15 ⁇ 45MPa/ s, the temperature used by ultra-high pressure equipment is 20-25°C, and the pressure transmission medium is water; after the set pressure holding time is reached, within 0.4-5s, that is, at the pressure relief rate of 100-250Mpa/s, the Take the pressure off.
  • Maillard peptide reaction The preferred conditions of the above scheme step 6 Maillard peptide reaction are as follows: the mass ratio of the mandarin fish scale collagen peptide powder and Polygonatum polysaccharide prepared in step 5 is 10:1 ⁇ 10:3, and the mixed solution obtained by dissolving in pure water The concentration is 10% to 18%, the pH value of the mixed solution is adjusted to 5.5 to 8.5, the mixed solution is subjected to Maillard reaction at 100°C to 125°C for 80 to 120min, and the reaction is terminated by rapid cooling in an ice bath. Concentrate the obtained reaction liquid to 15wt%-25wt%, which is Maillard peptide.
  • a preferred version of the preparation method is: dissolving chitosan in 1% (v/v) acetic acid to prepare 0.5% to 2.5% (w/v) shell Glycan solution, add 5% ⁇ 25% (w/v) glycerol and 15% ⁇ 25% (w/v) of Maillard peptide prepared in step 6, mix and homogenize, and place at 4°C for disinfection Soak it to get a kind of biological fresh-keeping solution for refrigerated stinky mandarin fish.
  • the invention also discloses a production method of low-salt smelly mandarin fish, comprising the following steps:
  • Step 1 cleaning remove the scales and gills of the fresh mandarin fish, remove the viscera by laparotomy, wash and drain;
  • Step 2 Soaking: first prepare the plant antibacterial combination liquid, take 8-12 parts of rhizoma rhizome powder after drying, 6-10 parts of protein mulberry leaf powder, 5-8 parts of ginger powder, and 3-5 parts of garlic in parts by weight Powder, 100-150 parts of liquor with an alcohol content of 40°-55° or equivalent edible alcohol solution, stir and mix evenly, under the negative pressure strength of -0.02Mpa-0.08Mpa, stirring speed of 100-150rpm, temperature of 30 ⁇ 55°C, extraction time is 0.5 ⁇ 1h, and the extraction frequency is 1 ⁇ 3 times.
  • the extracts are combined, which is the plant antibacterial combination liquid;
  • the mandarin fish treated in step 1 is immersed in the plant antibacterial Put it in the combination solution for 5-20 minutes, so as to play the dual role of deodorization and synergistic sterilization; after taking out and draining, put it into a high-pressure-resistant packaging bag, remove the air in the packaging bag, and seal the mouth of the bag tightly;
  • Step 3 ultra-high pressure sterilization put the mandarin fish processed in step 2 into an ultra-high pressure pressurized cabin for ultra-high pressure sterilization;
  • Step 4 Pickling: put the mandarin fish processed in step 3 into the fermenter, pour sterilized salt water into the fermenter, add seasoning to the fermenter at the same time, stir evenly, and adjust the temperature to 10°C to 16°C Marinate in water for 5-10 hours;
  • Step 5 inoculation after the water pickling in step 4 is completed, inoculate the dominant lactic acid bacteria isolated from stinky mandarin fish into the fermenter, stir evenly, and the bacterial concentration is 10-50 billion CFU/g, and the inoculation amount is 0.01%-0.3 %(w/w);
  • Step 6 Fermentation Fermentation is carried out in stages. In the first stage, the fermentation temperature is set at 20°C-32°C, and the fermentation time is 1-2d; in the second stage, the fermentation temperature is set at 12°C-18°C, and the fermentation time is 2-3d , and at the same time set the pressure to 10-50Mpa in the second stage;
  • Step 7 Biological preservation and storage: soak the stinky mandarin fish fermented in step 6 in the biological preservation solution prepared according to the above-mentioned preparation method of the stinky mandarin fish biological preservation solution for 5-30 minutes, drain and vacuum pack, and place at 4°C Refrigerate under conditions.
  • the ultra-high pressure used in step 3 is preferably a pulse type ultra-high pressure sterilization, wherein the pressure peak value is set to be 200MPa ⁇ 500MPa when the pressure is boosted, and the pulse type pressurized discharge Press 1 to 3 times.
  • the temperature used by the ultra-high pressure equipment is room temperature, that is, 20-25°C, and the pressure transmission medium is water.
  • the preferred pickling conditions of the production method of the above-mentioned low-salt smelly mandarin fish are: calculated by every 100kg of mandarin fish, the sterilized brine poured into the fermenter is 0.8 ⁇ 1.5kg of refined salt and is completely dissolved in 50 ⁇ 75L of sterilized treatment.
  • the seasonings added to the fermentation tank are 0.02-0.1kg of perilla, 0.2-1.0kg of onion, 0.5-1.5kg of chili powder, and 0.5-1.2kg of Chinese prickly ash.
  • the dominant lactic acid bacteria isolated from stinky mandarin fish used for inoculation is preferably at least one of the following: Lactobacillus sakei ATCC 15521, Lactococcus.Lactis ATCC 49032, Lactobacillus casei ATCC393 .
  • the step of collecting the fish scales produced during descaling is used for the preparation of the above-mentioned stinky mandarin fish biological preservation solution.
  • the present invention provides a method for producing low-salt smelly mandarin fish by artificial inoculation and fermentation, which realizes the production of low-salt smelly mandarin fish (salt concentration is reduced by 5 to 10 times), and reduces the generation of nitrite in fish fermentation , improve the safety of stinky mandarin fish; effectively shorten the fermentation period of 4 to 7 days, promote the production of flavor substances of stinky mandarin fish, and have good sensory evaluation; it is beneficial to the standardized and industrialized production of stinky mandarin fish;
  • the present invention provides a method for preparing stinky mandarin fish biological fresh-keeping solution.
  • mandarin fish scale by-products as raw materials, adopting bio-enzymatic hydrolysis coupling ultra-high pressure extraction technology, the prepared fish scale collagen peptide has high purity, anti-oxidation, The antibacterial biological activity is good, the production technology is friendly to the environment, and industrial production can be realized.
  • the resource utilization rate of the by-products of stinky mandarin fish processing is greatly improved, and the problems of environmental pollution and resource waste caused by direct discarding of fish scales are solved;
  • the present invention has carried out the application of stinky mandarin fish preservation to the prepared Maillard peptide biological preservation solution, which significantly prolongs the shelf life of stinky mandarin fish, and avoids adverse effects on the color, sensory quality, flavor substances, etc. of stinky mandarin fish It also effectively reduces the generation of thiobarbituric acid reactants and histamine during the preservation of stinky mandarin fish, has the dual functions of antibacterial, anti-corrosion and anti-oxidation, and greatly improves the quality and safety quality of stinky mandarin fish during preservation.
  • Fig. 1 is the influence of artificial inoculation fermentation method on the change of total number of lactic acid bacteria colonies in the fermentation of stinky mandarin fish;
  • Fig. 2 is the influence of artificial inoculation fermentation method on the change of nitrous hydrochloric acid content in the fermentation of stinky mandarin fish;
  • Figure 3 shows the effect of biological fresh-keeping solution on the total number of microbial colonies of stinky mandarin fish at 4°C and different storage times.
  • the direct refrigeration of the stinky mandarin fish did not kill the microorganisms in the stinky mandarin fish.
  • the microorganisms will continue to ferment, causing the stinky mandarin fish to have unpleasant odors and rotten meat.
  • intense heat treatment can effectively kill the microorganisms in the fermented stinky mandarin fish and prolong the shelf life of the stinky mandarin fish, it has adverse effects on the color, sensory quality, and flavor substances of the stinky mandarin fish, and also reduces its nutritional and edible value.
  • Adopting the method of traditional preservation can not fully satisfy consumers' demand to smelly mandarin fish sense organ, local flavor, nutrition.
  • the invention provides a biological fresh-keeping method for stinky mandarin fish.
  • the scales of the fish body are used as the raw material of the biological fresh-keeping agent for stinky mandarin fish, and a green and safe active ingredient is prepared by adopting biological enzymatic hydrolysis coupling ultra-high pressure extraction technology.
  • Maillard peptide biological fresh-keeping liquid has significant antibacterial, antiseptic and anti-oxidation dual effects, prolongs the shelf life, avoids adverse effects on the color, sensory quality, and flavor substances of stinky mandarin fish, and effectively reduces the smell of stinky mandarin fish.
  • the generation of thiobarbituric acid reactants and histamine during the preservation process improves the safety and quality of the stinky mandarin fish during preservation, and at the same time greatly improves the resource utilization rate of the by-products of stinky mandarin fish processing, and solves the problem of direct discarding of fish scales The resulting environmental pollution and waste of resources and other issues.
  • the present invention adopts the combination of plant antibacterial combination liquid and pulsed ultra-high pressure technology to carry out non-thermal sterilization treatment on mandarin fish before fermentation, and selects the superior lactic acid bacteria with good performance separated from stinky mandarin fish as a starter and adds them to fresh mandarin fish.
  • artificial inoculation technology was used to ferment stinky mandarin fish.
  • the salt concentration, optimum fermentation temperature, optimum fermentation time, staged fermentation conditions, pressure and pressure The fermentation process such as setting time has been optimized, so as to maximize the fermentation effect, and realize high-quality, high-safety and repeatable industrial production of stinky mandarin fish.
  • the present invention uses mandarin fish scales as raw materials, adopts bio-enzymatic hydrolysis coupling ultra-high pressure extraction technology, and the prepared fish scale collagen peptide has high purity, good anti-oxidation and antibacterial biological activities, the production technology is friendly to the environment, and industrial production can be realized.
  • the resource utilization rate of the by-products of stinky mandarin fish processing has been greatly improved, and high-value products have been developed.
  • the invention discloses a method for preparing a biological fresh-keeping liquid for stinky mandarin fish, which uses fish scales produced in the production of stinky mandarin fish, and undergoes pretreatment, decalcification and impurity removal, enzymatic hydrolysis, ultra-high pressure treatment, purification, and Maillard peptide. Reaction and preparation to obtain biological fresh-keeping solution for refrigerated stinky mandarin fish.
  • the invention also discloses a production method of low-salt stinky mandarin fish, which includes cleaning, soaking in plant antibacterial combination liquid, ultra-high pressure sterilization, pickling, inoculating dominant lactic acid bacteria isolated from stinky mandarin fish, fermenting and using
  • the stinky mandarin fish biological fresh-keeping liquid prepared by the preparation method of the stinky mandarin fish biological fresh-keeping liquid is kept fresh to obtain low-salt stinky mandarin fish.
  • a kind of artificial inoculation fermentation comprises the steps:
  • Step 1 Cleaning: Remove the scales and gills of the fresh mandarin fish, remove the viscera by laparotomy, wash and drain. More preferably, in this step, a step of collecting fish scale by-products for subsequent preparation of fish scale collagen peptides is also included.
  • Step 2 Soaking: In parts by weight, take 8 to 12 parts of rhizoma rhizome powder, 6 to 10 parts of protein mulberry leaf powder, 5 to 8 parts of ginger powder, 3 to 5 parts of garlic powder, and 100 to 150 parts of alcohol after drying It is 40° ⁇ 55° liquor or equivalent edible alcohol solution, stir and mix evenly. Extract under the conditions of negative pressure strength of -0.02Mpa ⁇ -0.08Mpa, stirring speed of 100 ⁇ 150rpm, temperature of 30 ⁇ 55°C, extraction time of 0.5 ⁇ 1h, extraction times of 1 ⁇ 3 times, and combine the extracts after filtration , which is the plant antibacterial combination liquid.
  • Step 3 Pulse ultra-high pressure sterilization: Put the mandarin fish treated in step 2 into the ultra-high pressure pressurization chamber, set the peak pressure (that is, the highest pressure value during boosting) to 200MPa ⁇ 500MPa, pulse pressurization and pressure relief 1 to 3 times, the temperature used by the ultra-high pressure equipment is room temperature (ie 20-25°C), and the pressure transmission medium is water (water of the ultra-high pressure equipment).
  • Step 4 Pickling: Put the mandarin fish treated in step 3 into the fermenter according to the standard of 100kg, completely dissolve 0.8-1.5kg of refined salt in 50-75L of sterilized clean water, and then pour it into the fermenter . At the same time, 0.02-0.1 kg of perilla, 0.2-1.0 kg of onion, 0.5-1.5 kg of chili powder, and 0.5-1.2 kg of Chinese prickly ash are put into the fermenter, and stirred evenly. Adjust the temperature to 10°C to 16°C for water pickling, and the time is 5 to 10 hours.
  • Step 5 inoculation Inoculate 0.01% ⁇ 0.3% (w/w) dominant lactic acid bacteria isolated from mandarin fish in the fermenter in step 4 (the bacterial species is at least one of the following: Lactobacillus sakei ATCC 15521, Lactococcus.Lactis ATCC 49032, Lactobacillus casei ATCC393, the bacterial concentration is 10-50 billion CFU/g), and stir evenly.
  • Step 6 Fermentation Fermentation is carried out in stages. In the first stage, the fermentation temperature is set at 20°C-32°C, and the fermentation time is 1-2 days; in the second stage, the fermentation temperature is set at 12°C-18°C, the fermentation time is 2-3 days, and the pressure is set at 10-3 days in the second stage. 50Mpa.
  • Step 7 Biological fresh-keeping and storage: soak the stinky mandarin fish fermented in step 6 in biological fresh-keeping solution for 5-30 minutes, drain, pack in vacuum, and refrigerate at 4°C.
  • Step 1 Pretreatment: Wash the collected fresh mandarin fish scales with water, freeze-dry them in vacuum, and transfer them to a wall breaking machine for airflow crushing. 0.5MPa ⁇ 2.5MPa, crushing time is 5 ⁇ 15min, to get fish scale coarse powder.
  • Step 2 Decalcification and impurity removal: Soak the coarse fish scale powder treated in step 1 with 0.2-0.5mol/L HCl for 1-2 hours to decalcify, wash with water until neutral, then centrifuge, add 0.1-0.2mol/L NaOH solution to the precipitate for soaking Remove impurities for 0.5-2 hours, wash with water until neutral, centrifuge, and vacuum freeze-dry.
  • Step 3 enzymatic hydrolysis: resuspend the fish scale powder treated in step 2 in the buffer at a ratio of 1:5 to 1:10 (w/v), and then add protease for enzymatic hydrolysis (the protease is at least one of the following: protease, compound flavor protease, papain, trypsin, pepsin, the enzyme activity is 10000 ⁇ 25000U/g), the amount of enzyme added is 0.2% ⁇ 1%, the enzymolysis temperature is 37°C ⁇ 55°C, the enzymolysis pH 3.0 ⁇ 9.0, and the enzymatic hydrolysis time is 2 ⁇ 5h.
  • protease is at least one of the following: protease, compound flavor protease, papain, trypsin, pepsin, the enzyme activity is 10000 ⁇ 25000U/g
  • the amount of enzyme added is 0.2% ⁇ 1%
  • the enzymolysis temperature is 37°C ⁇ 55°C
  • Step 4 Ultra-high pressure treatment: After the enzymatic hydrolysis is completed, put it in a high-pressure resistant packaging bag and seal it, and then perform ultra-high pressure treatment to play the dual role of increasing product yield and inactivating enzymes.
  • Set the pressurized pressure value to 100-500MPa, the pressure holding time to 5-20min, the pressurization rate to 15-45MPa/s, the temperature used by the ultra-high pressure equipment is room temperature (20-25°C), and the pressure transmission medium It is water; after reaching the set pressure holding time, quickly release the pressure within 0.4 ⁇ 5s (that is, the pressure relief rate is 100 ⁇ 250Mpa/s).
  • Step 5 Purification: The enzymolysis solution treated in step 4 is subjected to ceramic membrane microfiltration, activated carbon fiber membrane ultrafiltration, and roll membrane nanofiltration to obtain fish scale collagen peptide concentrate, and finally vacuum freeze-dried to obtain mandarin fish scale collagen Protein peptide powder.
  • Step 6 Maillard peptide reaction: select mandarin fish scale collagen peptide powder and Polygonatum polysaccharide (ratio is 10:1 ⁇ 10:3) prepared in step 5, dissolve in pure water, concentration is 10% ⁇ 18% Mixture. The pH value is adjusted to 5.5-8.5, and the solution is subjected to Maillard reaction at 100° C.-125° C. for 80-120 minutes, and is rapidly cooled in an ice bath to terminate the reaction. Concentrate the obtained reaction liquid to 15wt%-25wt%, which is Maillard peptide.
  • Step 7 preparation dissolving chitosan in 1% (v/v) acetic acid prepares 0.5% ⁇ 2.5% (w/v) chitosan solution, adds 5% ⁇ 25% (w/v) glycerin and step 6
  • the prepared Maillard peptide is 15% to 25% (w/v), homogenized after mixing, and defoamed at 4°C to obtain a biological preservation solution for refrigerated stinky mandarin fish.
  • Embodiment 1 a kind of artificial inoculation fermentation produces the method for low-salt smelly mandarin fish
  • Step 1 Cleaning Remove the scales and gills of the fresh mandarin fish, remove the viscera by laparotomy, wash and drain. Fish scale by-products were collected for subsequent preparation of fish scale collagen peptides.
  • Step 2 Soaking: In parts by weight, take dried 10 parts of Polygonatum powder, 8 parts of protein mulberry leaf powder, 5 parts of ginger powder, 4 parts of garlic powder, and 120 parts of white wine with an alcohol content of 52°, and stir and mix evenly. Extract under the conditions of negative pressure strength of -0.05Mpa, stirring speed of 120rpm, temperature of 45°C, and extraction time of 0.5h. The number of extractions is 3 times. After filtering, the extracts are combined to form the plant antibacterial combination liquid. Submerge the mandarin fish treated in step 1 in the plant antibacterial combination liquid for 15 minutes, so as to play the dual functions of deodorization and synergistic sterilization. After taking out and draining, put it into a high-pressure-resistant packaging bag, remove the air in the packaging bag, and seal the mouth of the bag tightly.
  • Step 3 Pulse ultra-high pressure sterilization: Put the mandarin fish treated in step 2 into the ultra-high pressure pressurization chamber, set the peak pressure (that is, the highest pressure value during boosting) to 250MPa, and pulse pressurization and decompression twice , the temperature used in ultra-high pressure equipment is room temperature (ie 20-25°C), and the pressure transmission medium is water (water in ultra-high pressure equipment).
  • Step 4 marinating put the mandarin fish processed in step 3 into the fermenter according to the standard of every 100kg, completely dissolve 1.0kg of refined salt in 60L of sterilized clear water and then pour into the fermenter. At the same time, put 0.02kg of perilla, 0.2kg of onion, 0.5kg of chili powder, and 0.8kg of Chinese prickly ash into the fermenter, and stir evenly. Adjust the temperature to 12°C for water pickling for 6 hours.
  • Step 5 inoculation and fermentation first inoculate 0.1% Lactobacillus sakei ATCC 15521 (bacterial concentration: 20 billion CFU/g) into the fermenter in step 4, stir evenly, and ferment for 2 days at a fermentation temperature of 25°C On the 3rd day, continue to inoculate 0.15% Lactococcus.Lactis ATCC 49032 (bacterial concentration: 20 billion CFU/g), stir evenly, and ferment for 2 days at a fermentation temperature of 15°C, while setting a pressure of 12Mpa.
  • Step 6 Biological preservation and storage: soak the stinky mandarin fish fermented in step 6 in the biological preservation solution for 10 minutes, drain and pack in vacuum, and refrigerate at 4°C.
  • Embodiment 2 a kind of preparation method of stinky mandarin fish biological preservation liquid
  • Step 1 Pretreatment: Wash the collected fresh mandarin fish scales, vacuum freeze-dry them, and transfer them to a wall breaker for airflow crushing.
  • the temperature inside the machine is adjusted to 4°C, and a high-speed airflow is introduced.
  • the pressure of the airflow crushing is 1MPa.
  • the time is 10 minutes, and the fish scale coarse powder is obtained.
  • Step 2 Decalcification and impurity removal: Soak the coarse fish scale powder treated in step 1 with 0.4mol/L HCl for 2 hours to decalcify, wash with water until neutral, then centrifuge, add 0.15mol/L NaOH solution to the precipitate, soak for 1 hour to remove impurities, and wash with water until After neutralization, centrifuge and vacuum freeze-dry.
  • Step 3 enzymatic hydrolysis: resuspend the fish scale powder treated in step 2 in the buffer at a ratio of 1:10 (w/v), add 1% of compound flavor protease with an enzyme activity of 20000U/g, and set the enzymatic hydrolysis temperature to 50°C, enzymolysis pH 6.5, enzymolysis time 4h.
  • Step 4 Ultra-high pressure treatment: After the enzymatic hydrolysis is completed, put it in a high-pressure resistant packaging bag and seal it, and then perform ultra-high pressure treatment to play the dual role of increasing the yield and inactivating the enzyme.
  • Step 5 Purification: The enzymolysis solution treated in step 4 is subjected to ceramic membrane microfiltration, activated carbon fiber membrane ultrafiltration, and roll membrane nanofiltration to obtain fish scale collagen peptide concentrate, and finally vacuum freeze-dried to obtain mandarin fish scale collagen Protein peptide powder.
  • Step 6 Maillard peptide reaction: select mandarin fish scale collagen peptide powder and Polygonatum polysaccharide (ratio: 10:3) prepared in step 5, dissolve in pure water, and form a mixed solution with a concentration of 15%. The pH value was adjusted to 7.0, and the solution was subjected to Maillard reaction at 110° C. for 90 minutes, and cooled rapidly in an ice bath to terminate the reaction. Concentrate the resulting reaction solution to 20 wt%, which is Maillard peptide.
  • Step 7 Preparation: Dissolve chitosan in 1% (v/v) acetic acid to prepare 2.2% (w/v) chitosan solution, add 20% (w/v) glycerin and Maillard prepared in step 6 Peptide 20% (w/v), homogenized after mixing, and placed at 4°C to defoam, a biological fresh-keeping solution for refrigerated stinky mandarin fish was obtained.
  • Embodiment 3 comparative example
  • Comparative example 1 the traditional natural fermentation of stinky mandarin fish disclosed in patent application 2018113413585 was used as a control group, and the specific steps were: cleaning mandarin fish, removing fishy smell, dry curing at room temperature for 5 to 7 days, and natural fermentation (at a temperature of 18 ° C to 20 ° C). °C, ferment for 5-7 days), vacuum pack, and refrigerate at 4 °C to obtain traditional naturally fermented stinky mandarin fish.
  • Comparative example 2 take the stinky mandarin fish that has not been treated with biological fresh-keeping solution as a blank control group.
  • Comparative example 3 the Maillard peptide that does not add the present invention to make in embodiment 3 fresh-keeping solution is as control group, promptly fresh-keeping solution is only 2.2% (w/v) chitosan solution and 20% (w/v) Made from glycerin.
  • lactic acid bacteria content Refer to GB4789.35-2016 "Food Microbiological Examination of Lactic Acid Bacteria” to measure the total number of lactic acid bacteria colonies in the stinky mandarin fish fermentation broth with fermentation time of 1st, 3rd, 5th, and 7th days.
  • Figure 1 is the effect of the artificial inoculation fermentation method on the change of the total number of lactic acid bacteria colonies in the fermentation of stinky mandarin fish.
  • the results show that, adopting the artificial inoculation fermentation method of the present invention, the total number of lactic acid bacteria colonies increases first and then basically maintains a stable trend with the prolongation of fermentation time.
  • the artificial inoculation fermentation method significantly increased the total number of lactic acid bacteria colonies in the stinky mandarin fish. It was determined that on the fifth day of fermentation, the total number of lactic acid bacteria colonies increased by 268 times.
  • Determination of nitrite content refer to the national standard GB5009.33-2016 "Determination of Nitrite and Nitrate in Food" by naphthalene ethylenediamine hydrochloride method to determine the stinky mandarin fish with fermentation time of 1st, 3rd, 5th and 7th days Residual content of NaNO 2 in fish, the experiment was carried out in 3 parallels.
  • Figure 2 is the effect of the artificial inoculation fermentation method on the content of nitrous hydrochloric acid in the fermentation of stinky mandarin fish.
  • the result shows that adopting the artificial inoculation fermentation method of the present invention significantly reduces the generation of nitrite content in the fermentation of stinky mandarin fish. It has been determined that, compared with the traditional natural fermentation of the stinky mandarin fish disclosed in patent application 2018113413585, the nitrite content of the present invention is reduced by 4.52 times on the 7th day of fermentation of the stinky mandarin fish. The results confirmed that the artificial inoculation of the screened lactic acid bacteria into the fermented stinky mandarin fish significantly reduced the nitrite content and improved the food safety of the stinky mandarin fish.
  • Sensory evaluation A sensory evaluation team is composed of 10 professionally trained personnel. The fish samples of each group were selected for cooking, put in the same container and coded, and evaluated by five indicators including color, smell, taste, tissue state and acceptance. The evaluation criteria are shown in Table 1.
  • Table 2 shows the effect of artificial inoculation and fermentation on the sensory quality of stinky mandarin fish.
  • the sensory evaluation is mainly based on the comprehensive evaluation of stinky mandarin fish by the members of the evaluation team through vision, smell, taste and overall acceptance.
  • the stinky mandarin fish produced by the artificial inoculation fermentation method of the present invention has a salt concentration reduced by 5 to 10 times, a higher sensory acceptance, and white and shiny flesh , has the special smell of fermented fish meat, the fish meat is firm but has a certain degree of elasticity, the meat quality is neat, the bones and flesh are separated, and the garlic clove shape is more obvious.
  • Determination of the total number of microbial colonies refer to GB4789.2-2016 "National Food Safety Standard Food Microbiology Inspection - Determination of Total Bacterial Colonies”.
  • the plate counting method was used to determine the total number of microbial colonies in stinky mandarin fish at 4°C and different storage times, and the total number of colonies was counted after cultured in a biochemical incubator at (36 ⁇ 1)°C for (48 ⁇ 2) hours.
  • Figure 3 shows the effect of biological fresh-keeping solution on the total number of microbial colonies of stinky mandarin fish at 4°C and different storage times.
  • the total number of bacterial colonies is an effective indicator for judging the degree of contamination and hygienic quality of food.
  • the International Committee on Microbiological Standards for Food stipulates that when the total number of bacterial colonies in fish exceeds 6 (logCFU/g), it is spoilage.
  • Fig. 1 The International Committee on Microbiological Standards for Food
  • the total number of colonies of the stinky mandarin fish of different treatment groups all increases significantly (P ⁇ 0.05) along with the prolongation of storage time, and wherein the growth rate of comparative example 2 is obviously higher than other treatment groups (P ⁇ 0.05 ), at the 2nd week of storage, the total number of colonies of stinky mandarin fish in comparative example 2 reached 6.14 (logCFU/g), which exceeded the edible standard stipulated by ICMSF; The total also reached 6.91 (logCFU/g). And the growth rate of the total number of colonies in the stinky mandarin fish processed by the Maillarde peptide biological fresh-keeping solution prepared by the present invention is the slowest, and only reaches 6.32 (logCFU/g) in the 5th week, and the shelf life is extended by 2 to 3 weeks.
  • TBA 2-thiobarbituric acid
  • Table 3 shows the effect of biological preservation solution on the content of thiobarbituric acid in stinky mandarin fish at 4 °C and different storage times.
  • Thiobarbituric acid value (TBA) is an effective method to detect oil oxidation products, and is widely used to determine the degree of fat oxidation and rancidity of meat and aquatic products, and is an important indicator for judging the degree of fat oxidation in fish.
  • TBA Thiobarbituric acid value
  • the TBA value of the stinky mandarin fish in the maillarde peptide biological preservation solution and chitosan co-treatment group remained at a lower level, which may be related to the antioxidant properties of the Maillarde peptide biological preservation solution. function related.
  • Table 3 shows the effect of biological fresh-keeping solution on the change of histamine content of stinky mandarin fish at 4°C and different storage time. Histamine is the most toxic biogenic amine, and excessive intake of histamine can cause severe food poisoning. my country stipulates that the limit standard of marine fish histamine is 300mg/kg. As can be seen from Table 3, the histamine content of the stinky mandarin fish of different treatment groups all increased significantly (P ⁇ 0.05) with the prolongation of storage time, and the growth rate of the comparative example 2 was significantly higher than that of other treatment groups (P ⁇ 0.05).
  • the histamine contents of the stinky mandarin fish of Comparative Example 2 and Comparative Example 3 were 345.55mg/kg and 318.34mg/kg respectively, all exceeding the national limit standard.
  • the histamine content of stinky mandarin fish treated with Maillard peptide biological fresh-keeping solution and chitosan increased the slowest, reaching 297.73 mg/kg at the fifth week, and the shelf life was extended by 1 to 2 weeks, which had a better effect. fresh-keeping effect.

Abstract

本发明公开了一种臭鳜鱼生物保鲜液的制备方法，通过采用臭鳜鱼生成中产生的鱼鳞，经过预处理、脱钙与除杂、酶解、超高压处理、纯化、美拉德肽反应及制备，获得用于冷藏臭鳜鱼的生物保鲜液。本发明还公开了一种低盐臭鳜鱼的生产方法，通过清理、在植物抑菌组合液中浸泡、超高压杀菌、腌制、接种从臭鳜鱼中分离出的优势乳酸菌、发酵并用通过上述臭鳜鱼生物保鲜液的制备方法制备的臭鳜鱼生物保鲜液进行保鲜，获得低盐臭鳜鱼。

Description

一种臭鳜鱼生物保鲜液及低盐臭鳜鱼的生产方法 技术领域
本发明属于食品加工技术领域，具体来说涉及一种臭鳜鱼生物保鲜液及低盐臭鳜鱼的生产方法。
背景技术
“初闻有异味，深嗅奇香，肉质白嫩如蒜瓣，入口润滑，生津吐香”，描述的就是我国黄山地区的传统发酵食品臭鳜鱼(stinky mandarin fish)，其具有悠久的历史传承、丰富的营养价值、独特的地域风味。黄山臭鳜鱼已成为市场高销量产品，2020年单品销售额高达40亿。但黄山臭鳜鱼生产过程存在以下问题：传统自然发酵导致臭鳜鱼品质不稳定，人工经验的依赖性强、还没有实现质量标准化；臭鳜鱼保质时间短，不宜流通及贮藏；生产过程中产生大量的鳜鱼鱼鳞等下脚料，每年约有200t～300t，且呈逐年递增的趋势，造成了较大的资源浪费和环境污染。因此，建立黄山臭鳜鱼的人工接种定向调控发酵模式，提高产品品质，延长产品货架期，综合利用与开发臭鳜鱼副产物，是黄山臭鳜鱼行业亟待解决的关键问题。
中国专利申请2016101309986公布了“一种基于乳酸菌的连续快速发酵臭鳜鱼方法”，具体涉及了以臭鳜鱼发酵的老汤(含4～15wt％的盐)作为引子，通过添加0.005％～0.03％的乳酸菌(为以下菌种的一种或两种复配：干酪乳杆菌、清酒乳杆菌、格氏乳球菌或嗜热乳杆菌，菌浓度为10 7～10 9CFU/mL)，快速发酵制备臭鳜鱼。同时，中国专利201811371639.5公布了“一种利用乳酸乳球菌M10和食窦魏斯氏菌M3混合接种快速发酵臭鳜鱼的方法”，具体涉及了利用两株从臭鳜鱼中分离的乳酸菌—乳酸乳球菌M10和食窦魏斯氏菌M3，采用二者混合接种的方法，添加3～10wt％的盐，在10℃～15℃下发酵5～7天而成。该两件专 利申请均采用乳酸菌接种快速发酵臭鳜鱼，缩短了发酵时间，一定程度上提升了产品安全性。然而两件专利申请均在鳜鱼发酵前期未对鱼体内进行有效的杀菌前处理，臭鳜鱼体内的杂菌或有害菌仍能影响优势乳酸菌的繁殖与生长，从而导致产品发酵品质与风味的不稳定。该两件专利申请只是初步研究了乳酸菌在臭鳜鱼发酵中的应用，然而不同的乳酸菌，其发酵性能千差万别，在发酵条件优化如盐浓度、最适发酵温度、最适发酵时间、是否需分段发酵、压力大小及压力设置时间等多个方面未见揭示。
中国专利申请2017105286691公布了“一种即食臭鳜鱼的制备方法及其保鲜液”，具体涉及了油炸臭鳜鱼、配制臭鳜鱼保鲜液、保鲜液浸渍半熟臭鳜鱼并装袋密封，再将密封封装后的臭鳜鱼在115℃的条件下高温灭菌处理，保持15±5min，再在121℃的温度下保持10±3min。保鲜液主要成分为0.01％～0.1％山梨酸钾、0.005％～0.02％焦磷酸钠、0.1％～0.4％TBHQ、0.15％～0.5％羟化卵磷脂、0.08％～0.15％茶多酚等。该申请主要是对即食臭鳜鱼保鲜液的研究，未见在冷藏臭鳜鱼保鲜液的研究；且保鲜液的主要成分多为化学合成添加剂，而非生物活性物质。
发明内容
针对现有技术的不足，本发明开发了一种用于臭鳜鱼保鲜的生物保鲜液以及低盐臭鳜鱼的生产方法。
具体来说，本发明采用了以下技术方案：
一种臭鳜鱼生物保鲜液的制备方法，包含以下步骤：
步骤1预处理：将收集的鳜鱼鱼鳞水洗干净后真空冷冻干燥，之后在-5℃～10℃下粉碎得鱼鳞粗粉末；
步骤2脱钙与除杂：将步骤1获得的鱼鳞粗粉末用HCl溶液脱钙，水洗至中性后离心，沉淀中加入NaOH溶液浸泡除杂，水洗至中性后离心，真空冷冻干燥；
步骤3酶解：将步骤2处理的鱼鳞粉末重悬于酶解缓冲液中，加入蛋白酶进 行酶解，获得酶解液；
步骤4超高压处理：步骤3酶解完成后获得的酶解液置于耐高压的包装袋中密封，进行超高压处理；
步骤5纯化：将经过步骤4超高压处理后的酶解液进行过滤，获得鱼鳞胶原蛋白肽浓缩液，然后进行真空冷冻干燥，获得鳜鱼鱼鳞胶原蛋白肽粉；
步骤6美拉德肽反应：将步骤5纯化获得的鳜鱼鱼鳞胶原蛋白肽粉和黄精多糖于纯水中混合溶解为混合液，进行美拉德反应，反应后冰浴迅速冷却以终止反应，反应液浓缩后获得美拉德肽；
步骤7制备：制备壳聚糖溶液，添加甘油并与步骤6制得的美拉德肽混合，混匀后均质，即得臭鳜鱼生物保鲜液。
在一个优选方案中，脱钙条件为用0.2～0.5mol/L HCl浸泡1～2h，除杂条件为沉淀中加入0.1～0.2mol/L NaOH溶液浸泡0.5～2h。
在优选方案中，酶解条件为将步骤2处理的鱼鳞粉末按1:5～1:10(w/v)的比例重悬于酶解缓冲液中，然后加入蛋白酶酶解，所述蛋白酶为中性蛋白酶、复合风味蛋白酶、木瓜蛋白酶、胰蛋白酶、胃蛋白酶中的至少一种，其中酶活为10000～25000U/g，设置加酶量按重量计为0.2％～1％、酶解温度为37℃～55℃、酶解pH为3.0～9.0、酶解时间为2～5h。
在上述臭鳜鱼生物保鲜液的制备方法在一个优选方案中，超高压处理条件为：设定加压压力数值为100～500MPa，保压时间为5～20min，升压速率为15～45MPa/s，超高压设备所采用的温度为20～25℃，传压介质为水；达到设定的保压时间后，在0.4～5s内，即在100～250Mpa/s的卸压速率下，快速卸掉压力。
上述方案步骤6美拉德肽反应的优选条件为：步骤5制得的鳜鱼鱼鳞胶原蛋白肽粉和黄精多糖的质量比例为10:1～10:3，溶解于纯水获得的混合液的浓度为10％～18％，将混合液的pH值调节为5.5～8.5，将该混合液在100℃～125℃的条件下进行美拉德反应80～120min，冰浴迅速冷却终止反应后，将所得反应 液浓缩至15wt％～25wt％，即为美拉德肽。
在上述臭鳜鱼生物保鲜液制备方法的步骤7中，制备方法的一个优选方案为：将壳聚糖溶于1％(v/v)乙酸中制备0.5％～2.5％(w/v)壳聚糖溶液，添加5％～25％(w/v)甘油和步骤6制得的美拉德肽15％～25％(w/v)，混匀后均质，并置于4℃下消泡，即得一种冷藏臭鳜鱼的生物保鲜液。
本发明还公开了一种低盐臭鳜鱼的生产方法，包含以下步骤：
步骤1清理：将鲜活的鳜鱼去鳞、去除鱼鳃、并剖腹去除内脏，洗净后沥水；
步骤2浸泡：首先制备植物抑菌组合液，按重量份数计，取干燥后的8～12份黄精粉末、6～10份蛋白桑叶粉末、5～8份生姜粉末、3～5份大蒜粉末、100～150份酒精度为40°～55°的白酒或等同的食用酒精溶液，搅拌混合均匀，在负压强度为-0.02Mpa～-0.08Mpa、搅拌速度为100～150rpm、温度为30～55℃、提取时间为0.5～1h条件下提取，提取次数为1～3次，过滤后合并提取液，即为植物抑菌组合液；将经步骤1处理后的鳜鱼浸没在植物抑菌组合液中5～20min，以起到去腥及协同杀菌的双重作用；取出沥水后，装入耐高压的包装袋中，排除包装袋中的空气，封严袋口；
步骤3超高压杀菌：将经步骤2处理后的鳜鱼放入超高压加压舱进行超高压杀菌；
步骤4腌制：将经步骤3处理后的鳜鱼放入发酵罐内，向发酵罐中倒入灭菌盐水，同时向发酵罐中加入调料，搅拌均匀，调控温度为10℃～16℃进行水腌，时间为5～10h；
步骤5接种：步骤4处理的水腌完成后向发酵罐中接种从臭鳜鱼中分离出的优势乳酸菌，搅拌均匀，其中菌浓度为100～500亿CFU/g，接种量为0.01％～0.3％(w/w)；
步骤6发酵：采用分段式进行发酵，第一阶段设置发酵温度为20℃～32℃、发酵时间为1～2d；第二阶段设置发酵温度为12℃～18℃、发酵时间为2～3d， 同时在第二阶段设置压力为10～50Mpa；
步骤7生物保鲜与贮藏：将经步骤6发酵后的臭鳜鱼浸泡于根据上述臭鳜鱼生物保鲜液的制备方法制备的生物保鲜液中5～30min，沥干后真空包装，置于4℃条件下冷藏。
在上述低盐臭鳜鱼的生产方法中，步骤3中所用的超高压优选为脉冲式超高压杀菌，其中设定压力峰值即升压时的压力最高值为200MPa～500MPa，脉冲式增压泄压1～3次，超高压设备所采用的的温度为室温即20～25℃，传压介质为水。
上述低盐臭鳜鱼的生产方法的优选腌制条件为：按每100kg的鳜鱼计算，倒入发酵罐中的灭菌盐水为精盐0.8～1.5kg完全溶解于50～75L的灭菌处理后的清水中所得，向发酵罐中加入的调料为紫苏0.02～0.1kg、洋葱0.2～1.0kg、辣椒粉0.5～1.5kg、花椒0.5～1.2kg。
上述方法的步骤5中，接种所用的从臭鳜鱼中分离出的优势乳酸菌优选为如下至少一种：清酒乳杆菌Lactobacillus sakei ATCC 15521、乳酸乳球菌Lactococcus.Lactis ATCC 49032、干酪乳杆菌Lactobacillus casei ATCC393。
在另一个优选实施方案中，优选在上述低盐臭鳜鱼的生产方法的步骤1清理中还包括对去鳞时产生的鱼鳞进行收集以用于上述臭鳜鱼生物保鲜液的制备的步骤。
有益效果：
1、采用植物抑菌组合液结合脉冲式超高压技术对鳜鱼发酵前进行非热杀菌，保存了臭鳜鱼产品的口感和营养，提升了产品品质；
2、选择从臭鳜鱼中分离的优势乳酸菌作为发酵剂添加到新鲜鳜鱼中进行人工接种定向调控发酵，并依据不同菌株的发酵特性，探索出了最佳的发酵技术；
3、以鳜鱼鱼体鱼鳞用于臭鳜鱼的生物保鲜剂的原料，采用生物酶解耦合超 高压提取技术，制得了一种绿色安全的活性美拉德肽生物保鲜液，提高臭鳜鱼加工副产物的资源利用率，并且保存了臭鳜鱼的风味；
4、对制得的美拉德肽生物保鲜液进行了臭鳜鱼保藏的应用，延长了货架期，提高了臭鳜鱼保藏过程中的安全质量；
5、本发明提供了一种人工接种发酵生产低盐臭鳜鱼的方法，实现了低盐臭鳜鱼的生产(盐浓度降低了5～10倍)，降低了鱼肉发酵中亚硝酸盐的生成，提高了臭鳜鱼的安全性；有效缩短发酵周期4～7天的同时，促进臭鳜鱼风味物质的产生，感官评价好；有益于臭鳜鱼标准化、工业化的生产；
6、本发明提供了一种臭鳜鱼生物保鲜液的制备方法，以鳜鱼鱼鳞副产物为原料，采用生物酶解耦合超高压提取技术，制得的鱼鳞胶原蛋白肽纯度高，抗氧化、抑菌生物活性好，生产技术对环境友好，能够实现工业化生产，同时大大提高了臭鳜鱼加工副产物的资源利用率，解决了鱼鳞被直接丢弃所造成的环境污染和资源浪费等问题；
7、本发明对制得的美拉德肽生物保鲜液进行了臭鳜鱼保藏的应用，显著延长了臭鳜鱼货架期，避免了对臭鳜鱼色泽、感官品质、风味物质等产生不良影响，还有效降低臭鳜鱼保藏过程中硫代巴比妥酸反应物、组胺的生成，具有抑菌防腐和抗氧化的双重作用，大大提高了臭鳜鱼保藏过程中的品质和安全质量。
附图说明
图1为人工接种发酵法对臭鳜鱼发酵中乳酸菌菌落总数变化的影响；
图2为人工接种发酵法对臭鳜鱼发酵中亚硝盐酸含量变化的影响；
图3为生物保鲜液对臭鳜鱼在4℃、不同保藏时间内微生物菌落总数的影响。
具体实施方式
在臭鳜鱼的传统加工生产中，采用自然发酵，其微生物组成复杂且不稳定、杂菌繁多、产品风味质量可控性差、安全隐患多，给准确认识发酵过程与实现产 品品质调控带来了巨大的挑战。
臭鳜鱼的直接冷藏并没有杀灭臭鳜鱼体内的微生物，当环境回到适宜温度，微生物仍会继续发酵，导致臭鳜鱼出现不愉快的臭味、肉质变烂等问题。强烈的热处理虽能有效杀灭发酵后臭鳜鱼中的微生物、延长臭鳜鱼保质期，但对臭鳜鱼色泽、感官品质、风味物质等产生不良影响，也降低了其营养及食用价值。采用传统保藏的方法已不能完全满足广大消费者对臭鳜鱼感官、风味、营养的需求。本发明提供了一种臭鳜鱼的生物保鲜方法，以鱼体自身的鱼鳞用于臭鳜鱼的生物保鲜剂的原料，采用生物酶解耦合超高压提取技术，制得了一种绿色安全的活性美拉德肽生物保鲜液，具有显著的抑菌防腐和抗氧化双重作用，延长了货架期，同时避免了对臭鳜鱼色泽、感官品质、风味物质等产生不良影响，还有效降低臭鳜鱼保藏过程中硫代巴比妥酸反应物和组胺的生成，提高了臭鳜鱼保藏过程中的安全质量，同时大大提高了臭鳜鱼加工副产物的资源利用率，解决了鱼鳞被直接丢弃所造成的环境污染和资源浪费等问题。
本发明采用植物抑菌组合液结合脉冲式超高压技术，对鳜鱼发酵前进行了非热杀菌处理，选择从臭鳜鱼中分离得到性能良好的优势乳酸菌作为发酵剂添加到新鲜鳜鱼中，在低盐的条件下，采用人工接种技术对臭鳜鱼进行发酵处理，依据不同菌种的发酵特性，对盐浓度、最适发酵温度、最适发酵时间、分段发酵条件、压力大小及压力设置时间等发酵工艺进行了优化，从而将发酵效果发挥到最大化，实现了臭鳜鱼高品质、高安全性和可重复工业化生产。
另外据统计，仅黄山地区在臭鳜鱼加工过程中产生鱼鳞副产物每年约有200t～300t，且呈逐年递增的趋势，而鱼鳞中富含50％～70％的高蛋白等可利用资源，目前大部分被直接丢弃，造成潜在的环境污染和极大的资源浪费等问题。本发明以鳜鱼鱼鳞为原料，采用生物酶解耦合超高压提取技术，制得的鱼鳞胶原蛋白肽纯度高，抗氧化、抑菌生物活性好，生产技术对环境友好，能够实现工业化生产，还大大提高臭鳜鱼加工副产物的资源利用率，开发了高值化产品。
本发明公开了一种臭鳜鱼生物保鲜液的制备方法，通过采用臭鳜鱼生成中产生的鱼鳞，经过预处理、脱钙与除杂、酶解、超高压处理、纯化、美拉德肽反应 及制备，获得用于冷藏臭鳜鱼的生物保鲜液。本发明还公开了一种低盐臭鳜鱼的生产方法，通过清理、在植物抑菌组合液中浸泡、超高压杀菌、腌制、接种从臭鳜鱼中分离出的优势乳酸菌、发酵并用通过上述臭鳜鱼生物保鲜液的制备方法制备的臭鳜鱼生物保鲜液进行保鲜，获得低盐臭鳜鱼。
下面将结合代表性实施例及具体实施例对本发明的技术方案做进一步详细的描述。
在一个代表性实施例中，提供了一种人工接种发酵生产低盐臭鳜鱼的方法，包括如下步骤：
步骤1清理：将鲜活的鳜鱼去鳞、去除鱼鳃、并剖腹去除内脏，洗净后沥水。更优选在该步骤中，还包含收集鱼鳞副产物用于后续鱼鳞胶原蛋白肽的制备的步骤。
步骤2浸泡：按重量份数计，取干燥后的8～12份黄精粉末、6～10份蛋白桑叶粉末、5～8份生姜粉末、3～5份大蒜粉末、100～150份酒精度为40°～55°的白酒或等同的食用酒精溶液，搅拌混合均匀。在负压强度为-0.02Mpa～-0.08Mpa、搅拌速度为100～150rpm、温度为30～55℃、提取时间为0.5～1h条件下提取，提取次数为1～3次，过滤后合并提取液，即为植物抑菌组合液。将经步骤1处理后的鳜鱼浸没在植物抑菌组合液中5～20min，以起到去腥及协同杀菌的双重作用。取出沥水后，装入耐高压的包装袋中，排除包装袋中的空气，封严袋口。
步骤3脉冲式超高压杀菌：将经步骤2处理后的鳜鱼放入超高压加压舱，设定压力峰值(即升压时的压力最高值)为200MPa～500MPa，脉冲式增压泄压1～3次，超高压设备所采用的的温度为室温(即20～25℃)，传压介质为水(超高压设备的水)。
步骤4腌制：将经步骤3处理后的鳜鱼按照每100kg的标准放入发酵罐内，将精盐0.8～1.5kg完全溶解于50～75L的灭菌处理后的清水中后倒入发酵罐。同时，取紫苏0.02～0.1kg、洋葱0.2～1.0kg、辣椒粉0.5～1.5kg、花椒0.5～ 1.2kg放入发酵罐中，搅拌均匀。调控温度为10℃～16℃进行水腌，时间为5～10h。
步骤5接种：向步骤4中的发酵罐中接种0.01％～0.3％(w/w)的从臭鳜鱼中分离出的优势乳酸菌(所述菌种为如下至少一种：清酒乳杆菌Lactobacillus sakei ATCC 15521、乳酸乳球菌Lactococcus.Lactis ATCC 49032、干酪乳杆菌Lactobacillus casei ATCC393，菌浓度为100～500亿CFU/g)，搅拌均匀。
步骤6发酵：采用分段式进行发酵。第一阶段设置发酵温度为20℃～32℃、发酵时间为1～2d；第二阶段设置发酵温度为12℃～18℃、发酵时间为2～3d，同时在第二阶段设置压力为10～50Mpa。
步骤7生物保鲜与贮藏：将经步骤6发酵后的臭鳜鱼浸泡于生物保鲜液中5～30min，沥干后真空包装，置于4℃条件下冷藏。
在另一个代表性实施例中，还公开了一种臭鳜鱼生物保鲜液的制备方法，具体包括如下步骤：
步骤1预处理：将收集的新鲜鳜鱼鱼鳞水洗干净后真空冷冻干燥，并转移至破壁机中进行气流粉碎，机内温度调节至-5℃～10℃，通入高速气流，气流粉碎压力为0.5MPa～2.5MPa，粉碎时间为5～15min，得鱼鳞粗粉末。
步骤2脱钙与除杂：将步骤1处理的鱼鳞粗粉末用0.2～0.5mol/L HCl浸泡1～2h脱钙，水洗至中性后离心，沉淀中加入0.1～0.2mol/L NaOH溶液浸泡0.5～2h除杂，水洗至中性后，离心，真空冷冻干燥。
步骤3酶解：将步骤2处理的鱼鳞粉末按1:5～1:10(w/v)的比例重悬于缓冲液中，然后加入蛋白酶酶解(所述蛋白酶为如下至少一种：中性蛋白酶、复合风味蛋白酶、木瓜蛋白酶、胰蛋白酶、胃蛋白酶，酶活为10000～25000U/g)，设置加酶量为0.2％～1％、酶解温度为37℃～55℃、酶解pH为3.0～9.0、酶解时间为2～5h。
步骤4超高压处理：酶解完成后，置于耐高压的包装袋中密封，在进行超高压处理，以起到提高产品得率和灭酶的双重作用。设定加压压力数值为100～500MPa，保压时间为5～20min，升压速率为15～45MPa/s，超高压设备所采用的的温度为室温(即20～25℃)，传压介质为水；达到设定的保压时间后，在0.4～5s内(即卸压速率为100～250Mpa/s)，快速卸掉压力。
步骤5纯化：将步骤4处理后的酶解液进行陶瓷膜微滤、活性炭纤维膜超滤、卷式膜纳滤，即得鱼鳞胶原蛋白肽浓缩液，最后真空冷冻干燥，获得鳜鱼鱼鳞胶原蛋白肽粉。
步骤6美拉德肽反应：选用步骤5制得的鳜鱼鱼鳞胶原蛋白肽粉和黄精多糖(比例为10:1～10:3)，溶解于纯水中，浓度为10％～18％的混合液。调节pH值为5.5～8.5，将该溶液在100℃～125℃的条件下进行美拉德反应80～120min，冰浴迅速冷却终止反应。将所得反应液浓缩至15wt％～25wt％，即为美拉德肽。
步骤7制备：将壳聚糖溶于1％(v/v)乙酸中制备0.5％～2.5％(w/v)壳聚糖溶液，添加5％～25％(w/v)甘油和步骤6制得的美拉德肽15％～25％(w/v)，混匀后均质，并置于4℃下消泡，即得一种冷藏臭鳜鱼的生物保鲜液。
下面将借助于一些具体实施例来对上述方法进行更具体的说明。
实施例1：一种人工接种发酵生产低盐臭鳜鱼的方法
步骤1清理：将鲜活的鳜鱼去鳞、去除鱼鳃、并剖腹去除内脏，洗净后沥水。收集鱼鳞副产物用于后续鱼鳞胶原蛋白肽的制备。
步骤2浸泡：按重量份数计，取干燥后的10份黄精粉末、8份蛋白桑叶粉末、5份生姜粉末、4份大蒜粉末、120份酒精度为52°的白酒，搅拌混合均匀。在负压强度为-0.05Mpa、搅拌速度为120rpm、温度为45℃、提取时间为0.5h条件下提取，提取次数为3次，过滤后合并提取液，即为植物抑菌组合液。将经步骤1处理后的鳜鱼浸没在植物抑菌组合液中15min，以起到去腥及协同杀菌的双重作用。取出沥水后，装入耐高压的包装袋中，排除包装袋中的空气，封严袋口。
步骤3脉冲式超高压杀菌：将经步骤2处理后的鳜鱼放入超高压加压舱，设定压力峰值(即升压时的压力最高值)为250MPa，脉冲式增压泄压2次，超高压设备所采用的的温度为室温(即20～25℃)，传压介质为水(超高压设备的水)。
步骤4腌制：将经步骤3处理后的鳜鱼按照每100kg的标准放入发酵罐内，将精盐1.0kg完全溶解于60L的灭菌处理后的清水中后倒入发酵罐。同时，取紫苏0.02kg、洋葱0.2kg、辣椒粉0.5kg、花椒0.8kg放入发酵罐中，搅拌均匀。调控温度为12℃进行水腌，时间为6h。
步骤5接种与发酵：向步骤4的发酵罐中先接种0.1％的清酒乳杆菌Lactobacillus sakei ATCC 15521(菌浓度为200亿CFU/g)，搅拌均匀，在发酵温度为25℃条件下，发酵2d；第3d，继续接种0.15％乳酸乳球菌Lactococcus.Lactis ATCC 49032(菌浓度为200亿CFU/g)，搅拌均匀，在发酵温度为15℃条件下，发酵2d，同时设置压力12Mpa。
步骤6生物保鲜与贮藏：将经步骤6发酵后的臭鳜鱼浸泡于生物保鲜液中10min，沥干后真空包装，置于4℃条件下冷藏。
实施例2：一种臭鳜鱼生物保鲜液的制备方法
步骤1预处理：将收集的新鲜鳜鱼鱼鳞水洗干净后真空冷冻干燥，并转移至破壁机中进行气流粉碎，机内温度调节至4℃，通入高速气流，气流粉碎压力为1MPa，粉碎时间为10min，得鱼鳞粗粉末。
步骤2脱钙与除杂：将步骤1处理的鱼鳞粗粉末用0.4mol/L HCl浸泡2h脱钙，水洗至中性后离心，沉淀中加入0.15mol/L NaOH溶液浸泡1h除杂，水洗至中性后，离心，真空冷冻干燥。
步骤3酶解：将步骤2处理的鱼鳞粉末按1:10(w/v)的比例重悬于缓冲液中，加入1％的酶活为20000U/g的复合风味蛋白酶，设置酶解温度为50℃、酶解pH为6.5、酶解时间为4h。
步骤4超高压处理：酶解完成后，置于耐高压的包装袋中密封，在进行超高压处理，以起到提高产率和灭酶的双重作用。设定加压压力数值为350MPa，保压时间为10min，升压速率为35MPa/s，超高压设备所采用的的温度为室温(即20～25℃)，传压介质为水；达到设定的保压时间后，在1.75s内(即卸压速率为200Mpa/s)，快速卸掉压力。
步骤5纯化：将步骤4处理后的酶解液进行陶瓷膜微滤、活性炭纤维膜超滤、卷式膜纳滤，即得鱼鳞胶原蛋白肽浓缩液，最后真空冷冻干燥，获得鳜鱼鱼鳞胶原蛋白肽粉。
步骤6美拉德肽反应：选用步骤5制得的鳜鱼鱼鳞胶原蛋白肽粉和黄精多糖(比例为10:3)，溶解于纯水中，浓度为15％的混合液。调节pH值为7.0，将该溶液在110℃的条件下进行美拉德反应90min，冰浴迅速冷却终止反应。将所得反应液浓缩至20wt％，即为美拉德肽。
步骤7制备：将壳聚糖溶于1％(v/v)乙酸中制备2.2％(w/v)壳聚糖溶液，添加20％(w/v)甘油和步骤6制得的美拉德肽20％(w/v)，混匀后均质，并置于4℃下消泡，即得一种冷藏臭鳜鱼的生物保鲜液。
实施例3：对比实施例
对比例1：将专利申请2018113413585公开的臭鳜鱼传统自然发酵作为对照组，具体步骤为：鳜鱼清理、去腥、干法常温腌制5～7d、自然发酵(在温度为18℃～20℃下，发酵5～7d)、真空包装、4℃冷藏，即得传统自然发酵的臭鳜鱼。
对比例2：以未进行生物保鲜液处理的臭鳜鱼作为空白对照组。
对比例3：将实施例3保鲜液中未添加本发明制得的美拉德肽作为对照组，即保鲜液仅为2.2％(w/v)壳聚糖溶液和20％(w/v)甘油制得而成。
实验结果：
乳酸菌含量的测定：参照GB4789.35—2016《食品微生物学检验乳酸菌检验》测定发酵时间为第1、3、5、7天的臭鳜鱼发酵液中乳酸菌菌落总数。
图1为人工接种发酵法对臭鳜鱼发酵中乳酸菌菌落总数变化的影响。结果表明，采用本发明的人工接种发酵法，乳酸菌菌落总数随发酵时间延长均呈先增大后基本保持稳定的变化趋势。相对于专利申请2018113413585公开的所述臭鳜鱼传统自然发酵，人工接种发酵法显著提高了臭鳜鱼中乳酸菌菌落总数，经测定，在发酵第5天，乳酸菌菌落总数提高了268倍。
亚硝酸盐含量的测定：参考国标GB5009.33—2016《食品中亚硝酸盐与硝酸盐的测定》中的盐酸萘乙二胺法测定发酵时间为第1、3、5、7天的臭鳜鱼中NaNO 2的残留含量，实验进行3个平行。
图2为人工接种发酵法对臭鳜鱼发酵中亚硝盐酸含量变化的影响。结果表明，采用本发明的人工接种发酵法显著降低了臭鳜鱼发酵中亚硝酸盐含量的生成。经测定，相对于专利申请2018113413585公开的所述臭鳜鱼传统自然发酵，本发明在臭鳜鱼发酵第7天，亚硝酸盐含量降低了4.52倍。结果证实，将筛选的乳酸菌作为发酵剂，人工接种到臭鳜鱼发酵中，显著降低亚硝酸盐含量，提高了臭鳜鱼的食用安全性。
感官评价：由10名经过专业训练人员组成感官评定小组。分别选取各组样品鱼肉进行烹饪制熟，盛放在相同的容器中编码，通过色泽、气味、口感、组织状态与接受程度等5个指标进行评定。评定标准如表1所示。
表1臭鳜鱼的感官评分标准
Figure PCTCN2022077122-appb-000001
表2为人工接种发酵法对臭鳜鱼感官品质的影响。感官评定主要根据评定小组成员通过视觉、嗅觉、味觉及整体接受度对臭鳜鱼进行的综合评价。相对于专利申请2018113413585公开的所述臭鳜鱼传统自然发酵，采用本发明的人工接种发酵法生产的臭鳜鱼，盐浓度降低了5～10倍，感官接受度较高，肉质白皙且有光泽，具有鱼肉发酵后的特殊臭味，鱼肉较紧致但有一定弹性，肉质块形较整齐，骨肉分离，蒜瓣状较明显。结果表明，臭鳜鱼发酵中加入乳酸菌作发酵剂，促进了低盐臭鳜鱼的生产，能够保持臭鳜鱼的感官品质，得到风味和品质俱佳的臭鳜鱼产品。
表2
Figure PCTCN2022077122-appb-000002
微生物菌落总数的测定：参照GB4789.2-2016《食品安全国家标准食品微生物学检验—菌落总数测定》操作。采用平板计数法测定臭鳜鱼在4℃、不同保藏时间中的微生物菌落总数值，在(36±1)℃生化培养箱培养(48±2)h后计数得菌落总数。
图3为生物保鲜液对臭鳜鱼在4℃、不同保藏时间内微生物菌落总数的影响。 菌落总数是用来判断食品的污染程度及卫生质量的有效指标，国际食品微生物标准委员会(ICMSF)规定鱼肉中菌落总数超过6(logCFU/g)时即为腐败变质。由图3可以看出，不同处理组的臭鳜鱼的菌落总数均随着贮藏时间的延长而显著增加(P＜0.05)，其中对比例2的增长速度明显高于其他处理组(P＜0.05)，在贮藏第2周时，对比例2的臭鳜鱼菌落总数达到6.14(logCFU/g)，超过了ICMSF规定的可食用标准；对比例3在贮藏第3周时，臭鳜鱼的菌落总数也达到6.91(logCFU/g)。而经本发明制备的美拉德肽生物保鲜液处理后的臭鳜鱼中菌落总数增长速度最慢，在第5周时才达到6.32(logCFU/g)，货架期延长了2～3周。由此可见，美拉德肽生物保鲜液和壳聚糖协同处理具有一定的增效作用，可以增强对微生物生长繁殖的抑制能力，起到更好的保鲜效果，延长鱼肉的贮藏期。
硫代巴比妥酸的测定：取10g不同处理组及不同保藏时间内的臭鳜鱼鱼肉于烧杯中，加入25mL蒸馏水，均质后加入10g/100mL三氯乙酸25mL，混匀后静置30min，过滤，取5mL上清液，加入5mL 0.02mol/L 2-硫代巴比妥酸(TBA)溶液，将混合液在80℃恒温水浴锅中加热40min，取出冷却至室温后在532nm波长处测其吸光度。TBA值以每千克鱼肉中所含丙二醛质量表示，单位为mg/kg。
组胺含量的测定：GB5009.208-2016《食品安全国家标准食品中生物胺的测定》中分光光度法测定臭鳜鱼在4℃、不同保藏时间内的组胺含量。
表3为生物保鲜液对臭鳜鱼在4℃、不同保藏时间内硫代巴比妥酸含量变化的影响。硫代巴比妥酸值(TBA)是检测油脂氧化产物的有效方法，被广泛用于测定肉类和水产品脂肪氧化酸败程度，是判断鱼肉脂肪氧化程度的重要指标。结果表明，随着贮藏时间的延长，各组的TBA值明显增加(P＜0.05)，美拉德肽生物保鲜液处理组上升速率低于对照组。相比对比例2和对比例3，美拉德肽生物保鲜液和壳聚糖协同处理组的臭鳜鱼的TBA值保持较低的水平，这可能与美拉德肽生物保鲜液的抗氧化作用有关。
表3为生物保鲜液对臭鳜鱼在4℃、不同保藏时间内组胺含量变化的影响。组胺是毒性最强的生物胺，摄入过量的组胺能够造成严重的食物中毒。我国规定 海产鱼类组胺的限量标准为300mg/kg。由表3可以看出，不同处理组的臭鳜鱼的组胺含量均随着贮藏时间的延长而显著增加(P＜0.05)，其中对比例2的增长速度明显高于其他处理组(P＜0.05)，在贮藏第4周时，对比例2和对比例3的臭鳜鱼组胺含量分别为345.55mg/kg、318.34mg/kg，均超过了国家限量标准。而经美拉德肽生物保鲜液和壳聚糖协同处理的臭鳜鱼组胺含量增长速度最慢，在第5周时才达到297.73mg/kg，货架期延长了1～2周，起到更好的保鲜效果。
表3
Figure PCTCN2022077122-appb-000003
数据处理：数据间的显著差异运用统计软件SPSS 18.0中的Duncan’s多重比较法进行方差分析，P＜0.05表示显著性差异。结果以平均值±标准差表示。
上面结合附图和具体实施例对本发明的实施方式作了详细的说明，但是本发明不限于上述实施方式，在所属技术领域普通技术人员所具备的知识范围内，还可以在不脱离本发明宗旨的前提下做出各种变化。

Claims (10)

  1. 一种臭鳜鱼生物保鲜液的制备方法，其特征在于，包含以下步骤：
    步骤1预处理：将收集的鳜鱼鱼鳞水洗干净后真空冷冻干燥，之后在-5℃～10℃下粉碎得鱼鳞粗粉末；
    步骤2脱钙与除杂：将步骤1获得的鱼鳞粗粉末用HCl溶液脱钙，水洗至中性后离心，沉淀中加入NaOH溶液浸泡除杂，水洗至中性后离心，真空冷冻干燥；
    步骤3酶解：将步骤2处理的鱼鳞粉末重悬于酶解缓冲液中，加入蛋白酶进行酶解，获得酶解液；
    步骤4超高压处理：步骤3酶解完成后获得的酶解液置于耐高压的包装袋中密封，进行超高压处理；
    步骤5纯化：将经过步骤4超高压处理后的酶解液进行过滤，获得鱼鳞胶原蛋白肽浓缩液，然后进行真空冷冻干燥，获得鳜鱼鱼鳞胶原蛋白肽粉；
    步骤6美拉德肽反应：将步骤5纯化获得的鳜鱼鱼鳞胶原蛋白肽粉和黄精多糖于纯水中混合溶解为混合液，进行美拉德反应，反应后冰浴迅速冷却以终止反应，反应液浓缩后获得美拉德肽；
    步骤7制备：制备壳聚糖溶液，添加甘油并与步骤6制得的美拉德肽混合，混匀后均质，即得臭鳜鱼生物保鲜液。
  2. 如权利要求1所述的臭鳜鱼生物保鲜液的制备方法，其特征在于，脱钙条件为用0.2～0.5mol/L HCl浸泡1～2h，除杂条件为沉淀中加入0.1～0.2mol/L NaOH溶液浸泡0.5～2h。
  3. 如权利要求1所述的臭鳜鱼生物保鲜液的制备方法，其特征在于，酶解条件为将步骤2处理的鱼鳞粉末按1:5～1:10(w/v)的比例重悬于酶解缓冲液中， 然后加入蛋白酶酶解，所述蛋白酶为中性蛋白酶、复合风味蛋白酶、木瓜蛋白酶、胰蛋白酶、胃蛋白酶中的至少一种，其中酶活为10000～25000U/g，设置加酶量按重量计为0.2％～1％、酶解温度为37℃～55℃、酶解pH为3.0～9.0、酶解时间为2～5h。
  4. 如权利要求1所述的臭鳜鱼生物保鲜液的制备方法，其特征在于，超高压处理条件为：设定加压压力数值为100～500MPa，保压时间为5～20min，升压速率为15～45MPa/s，超高压设备所采用的温度为20～25℃，传压介质为水；达到设定的保压时间后，在0.4～5s内，即在100～250Mpa/s的卸压速率下，快速卸掉压力。
  5. 如权利要求1所述的臭鳜鱼生物保鲜液的制备方法，其特征在于，美拉德肽反应条件为：步骤5制得的鳜鱼鱼鳞胶原蛋白肽粉和黄精多糖的质量比例为10:1～10:3，溶解于纯水获得的混合液的浓度为10％～18％，将混合液的pH值调节为5.5～8.5，将该混合液在100℃～125℃的条件下进行美拉德反应80～120min，冰浴迅速冷却终止反应后，将所得反应液浓缩至15wt％～25wt％，即为美拉德肽。
  6. 如权利要求1所述的臭鳜鱼生物保鲜液的制备方法，其特征在于，步骤7的制备方法为：将壳聚糖溶于1％(v/v)乙酸中制备0.5％～2.5％(w/v)壳聚糖溶液，添加5％～25％(w/v)甘油和步骤6制得的美拉德肽15％～25％(w/v)，混匀后均质，并置于4℃下消泡，即得一种冷藏臭鳜鱼的生物保鲜液。
  7. 一种低盐臭鳜鱼的生产方法，其特征在于，包含以下步骤：
    步骤1清理：将鲜活的鳜鱼去鳞、去除鱼鳃、并剖腹去除内脏，洗净后沥水；
    步骤2浸泡：首先制备植物抑菌组合液，按重量份数计，取干燥后的8～12份黄精粉末、6～10份蛋白桑叶粉末、5～8份生姜粉末、3～5份大蒜粉末、100～150份酒精度为40 o～55 o的白酒或等同的食用酒精溶液，搅拌混合均匀，在负压强度为-0.02Mpa～-0.08Mpa、搅拌速度为100～150rpm、温度为30～55℃、提取时间为0.5～1h条件下提取，提取次数为1～3次，过滤后合并提取液，即为植 物抑菌组合液；将经步骤1处理后的鳜鱼浸没在植物抑菌组合液中5～20min，以起到去腥及协同杀菌的双重作用；取出沥水后，装入耐高压的包装袋中，排除包装袋中的空气，封严袋口；
    步骤3超高压杀菌：将经步骤2处理后的鳜鱼放入超高压加压舱进行超高压杀菌；
    步骤4腌制：将经步骤3处理后的鳜鱼放入发酵罐内，向发酵罐中倒入灭菌盐水，同时向发酵罐中加入调料，搅拌均匀，调控温度为10℃～16℃进行水腌，时间为5～10h；
    步骤5接种：步骤4处理的水腌完成后向发酵罐中接种从臭鳜鱼中分离出的优势乳酸菌，搅拌均匀，其中菌浓度为100～500亿CFU/g，接种量为0.01％～0.3％(w/w)；
    步骤6发酵：采用分段式进行发酵，第一阶段设置发酵温度为20℃～32℃、发酵时间为1～2d；第二阶段设置发酵温度为12℃～18℃、发酵时间为2～3d，同时在第二阶段设置压力为10～50Mpa；
    步骤7生物保鲜与贮藏：将经步骤6发酵后的臭鳜鱼浸泡于根据权利要求1至6中任一项所述的臭鳜鱼生物保鲜液的制备方法制备的生物保鲜液中5～30min，沥干后真空包装，置于4℃条件下冷藏。
  8. 如权利要求7所述的低盐臭鳜鱼的生产方法，其特征在于，在步骤3中，采用脉冲式超高压杀菌，其中设定压力峰值即升压时的压力最高值为200MPa～500MPa，脉冲式增压泄压1～3次，超高压设备所采用的的温度为室温即20～25℃，传压介质为水。
  9. 如权利要求7所述的低盐臭鳜鱼的生产方法，其特征在于，步骤4腌制中，按每100kg的鳜鱼计算，倒入发酵罐中的灭菌盐水为精盐0.8～1.5kg完全溶解于50～75L的灭菌处理后的清水中所得，向发酵罐中加入的调料为紫苏0.02～0.1kg、洋葱0.2～1.0kg、辣椒粉0.5～1.5kg、花椒0.5～1.2kg。
  10. 如权利要求7所述的低盐臭鳜鱼的生产方法，其特征在于，步骤5接种中所用的从臭鳜鱼中分离出的优势乳酸菌为如下至少一种：清酒乳杆菌Lactobacillus sakei ATCC 15521、乳酸乳球菌Lactococcus.Lactis ATCC 49032、干酪乳杆菌Lactobacillus casei ATCC393。
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