WO2020107760A1 - 中度嗜盐菌及利用其发酵鱼肉酱的方法 - Google Patents

中度嗜盐菌及利用其发酵鱼肉酱的方法 Download PDF

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WO2020107760A1
WO2020107760A1 PCT/CN2019/080282 CN2019080282W WO2020107760A1 WO 2020107760 A1 WO2020107760 A1 WO 2020107760A1 CN 2019080282 W CN2019080282 W CN 2019080282W WO 2020107760 A1 WO2020107760 A1 WO 2020107760A1
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fish
fish paste
fermentation
paste
fermented
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French (fr)
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高瑞昌
郑志颖
袁丽
伍晓云
周巧珍
储倩
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Jiangsu University
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Jiangsu University
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Priority claimed from CN201811451711.5A external-priority patent/CN109456920B/zh
Priority claimed from CN201811452967.8A external-priority patent/CN109601965B/zh
Priority claimed from CN201811453568.3A external-priority patent/CN109593669B/zh
Priority claimed from CN201811452816.2A external-priority patent/CN109456922B/zh
Application filed by Jiangsu University filed Critical Jiangsu University
Priority to US16/761,132 priority Critical patent/US11974590B2/en
Publication of WO2020107760A1 publication Critical patent/WO2020107760A1/zh
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    • A23L17/00—Food-from-the-sea products; Fish products; Fish meal; Fish-egg substitutes; Preparation or treatment thereof
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
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    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/10—General methods of cooking foods, e.g. by roasting or frying
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    • C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
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Definitions

  • the invention relates to moderate halophilic bacteria and a method of using the same to ferment fish meat sauce, and belongs to the technical field of solid fermentation of aquatic products using strains.
  • moderate halophilic bacteria Compared with extreme halophilic archaea, moderate halophilic bacteria have a wider halophilic range than halophilic archaea, with wider distribution and sources, lower nutrient requirements, easier adaptation to the living environment, and more practical production Easy to be applied. Some moderate halophilic bacteria have strong extracellular protease-producing properties.
  • the invention will utilize the mixed strain of moderate halophilic bacteria to ferment fish meat sauce to obtain a fish meat sauce product with short fermentation time, low salinity and high flavor and nutritional value. It provides a theoretical basis and method for improving the fermentation quality of aquatic products.
  • the invention utilizes moderate halophilic bacteria, combined with the excellent fermentation characteristics of the strains, and uses the strain fermentation method to produce fish meat flavor sauce. This method can effectively improve the flavor and nutritional value.
  • the moderate halophilic bacteria JD (Bacillus aquimaris JD) provided by the present invention have been deposited on September 27, 2018 in the Chinese Microbial Strains, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing The Deposit Management Committee General Microbiology Center (CGMCC), numbered CGMCC NO. 16541, suggested classification name: Bacillus aquimaris.
  • CGMCC General Microbiology Center
  • a method for fermenting fish meat sauce using moderately halophilic strain Bacillus marinus according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria Bacillus aquimaris CGMCC 16541 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28-40°C for 5-30 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 to 40 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and then sterilize it at 120°C for 15 to 20 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the moderate halophilic bacteria JD (Bacillus hwajinpoensis JD) provided by the present invention have been deposited on September 27, 2018 in the Chinese Microbial Strains, Institute of Microbiology, Chinese Academy of Sciences, No. 3, Beichen West Road, Chaoyang District, Beijing The Deposit Management Committee General Microbiology Center (CGMCC), numbered CGMCC NO. 16542, suggested classification name: Bacillus hwajinpoensis.
  • a method for fermenting fish meat sauce using moderately halophilic strain Bacillus huajintan according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria Bacillus hwajinpoensis CGMCC 16542 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28-40°C for 5-30 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 to 40 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and then sterilize it at 120°C for 15 to 20 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the moderate halophilic bacteria JD (Halobacillus faecis JD) provided by the present invention has been deposited at the Chinese Academy of Sciences Microbiology, No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing on September 27, 2018
  • a method for fermenting fish paste with moderately halophilic strain Bacillus subtilis halodendron according to the following steps:
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28-40°C for 5-30 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 to 40 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and then sterilize it at 120°C for 15 to 20 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the present invention provides the following technical solution: A method for fermenting fish meat sauce using a mixed strain of moderate halophilic bacteria.
  • the invention includes the processing of raw materials, the preparation and addition of mixed strains, the fermentation of fish paste, the determination of amino acid nitrogen, and sensory evaluation.
  • the specific production process is as follows:
  • a method of fermentation using a mixed strain of moderately halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish sauce: The mixture obtained in step (3) is incubated and fermented at a temperature of 28-40°C for 5-30 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 to 40 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and then sterilize it at 120°C for 15 to 20 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the advantages of the present invention (1)
  • the fish sauce fermented by the moderate halophilic bacteria strain has short fermentation time, lower salinity, higher amino acid nitrogen content, delicious taste, rich nutritional value, and volatile base nitrogen.
  • the content is lower than the EU standard, and the content of histamine is far lower than the national standard. It is a fermented fish meat sauce product with high quality and safety.
  • the fermentation process of the inoculation ratio, inoculation amount, fermentation time, fermentation temperature, salt addition, etc. of the above mixed starter in fermentation was optimized, and the physical and chemical index evaluation and sensory evaluation of the comprehensive fermented fish paste were used to evaluate the final fish paste quality A comprehensive evaluation was carried out.
  • the fish paste obtained by this method has a short fermentation time, low salinity, high amino acid nitrogen content, delicious taste, and rich nutritional value.
  • the content of volatile basic nitrogen is lower than the EU standard, and the content of histamine is far lower than the national standard , Is a fermented fish meat sauce product with high quality and safety.
  • Figure 1 is the morphology of moderate halophilic bacteria Bacillus aquimaris JD colonies
  • Figure 2 is a phylogenetic tree constructed by the 16S rRNA sequence of the moderate halophilic bacterium Bacillus aquimaris JD. Note: zzy8 is the moderate halophilic bacteria Bacillus aquimaris JD of the present invention.
  • Figure 3 shows the colony morphology of moderate halophilic bacteria Bacillus hwajinpoensis JD;
  • Figure 4 is a phylogenetic tree constructed from the 16S rRNA sequence of the moderate halophilic bacterium Bacillus hwajinpoensis JD. Note: zzy9 is the moderate halophilic Bacillus hwajinpoensis JD of the present invention.
  • Figure 5 shows the colony morphology of the moderate halophilic bacteria Halobacillus faecis JD;
  • Figure 6 is a phylogenetic tree constructed by the 16S rRNA sequence of the moderate halophilic bacterium Halobacillus faecis JD. Note: zzy5 is Halobacillus faecis, the moderate halophilic bacteria of the present invention.
  • the shrimp paste comes from the homemade shrimp paste made by fishermen in Heiyanzi Town, Fengnan District, Tangshan City, Hebei province, and the moderate halophilic bacteria are separated and selected from the shrimp paste.
  • Isolation and purification of moderately halophilic bacteria use Gibbons medium supplemented with 1% skim milk powder, apply sterile water to shrimp paste after a certain multiple dilution on solid Gibbons medium, incubate at 30°C for 48h, pick The colonies with typical characteristics are continuously separated and purified on solid Gibbons medium supplemented with 1% skim milk powder to obtain pure cultured protease-producing halophilic bacteria strains.
  • the isolated and purified strains were inoculated on the Gibbons slant, cultured at 30°C for 48h, and placed in a refrigerator at 4°C for short-term storage for future use.
  • the colonies are pale yellow, with smooth surfaces, neat edges, opaqueness, and dense colonies.
  • the Gram stain is negative and the cells are rod-shaped. After culturing in liquid Gibbons medium for 24h, the bacterial solution was cloudy.
  • the strain can grow at a NaCl concentration of 0-15%; it grows faster at a pH of 7-9; it can grow well at a temperature range of 25°C-43°C;
  • the isolated and screened moderately halophilic bacteria were sequenced by 16SrRNA. After the gene sequence was sorted by operation, they were searched in Ezbiocloud and determined to be (Bacillus aquimaris). According to the retrieved homologous strains, the Mege 5.1 program of DNAStar software is used to construct a biological evolution relationship tree as shown in Fig. 2.
  • CGMCC General Microbiology Center
  • the shrimp paste comes from the homemade shrimp paste made by fishermen in Heiyanzi Town, Fengnan District, Tangshan City, Hebei province, and the moderate halophilic bacteria are separated and selected from the shrimp paste.
  • Isolation and purification of moderate halophilic bacteria use Gibbons medium supplemented with 1% skim milk powder, apply sterile water diluted shrimp paste to solid Gibbons medium, incubate at 30°C for 48h, pick The colonies with typical characteristics are continuously separated and purified on solid Gibbons medium supplemented with 1% skim milk powder to obtain pure cultured protease-producing halophilic bacteria strains.
  • the isolated and purified strains were inoculated on the Gibbons slant, cultured at 30°C for 48h, and placed in a refrigerator at 4°C for short-term storage for future use.
  • the colony is orange-yellow, with a smooth surface, neat edges, opaqueness, dense colonies, Gram-positive staining is positive, and the bacterial cells are rod-shaped. After culturing in liquid Gibbons medium for 24h, the bacterial solution was cloudy.
  • the strain can grow at a NaCl concentration of 0-20%; it grows faster at a pH of 7-9; it can grow well at a temperature range of 25°C-43°C;
  • the isolated and screened moderately halophilic bacteria were sequenced by 16SrRNA, and the gene sequence was sorted by operation, then searched in Ezbiocloud and determined as (Bacillus hwajinpoensis). According to the retrieved homologous strains, the Mege 5.1 program of DNAStar software is used to construct a biological evolution relationship tree as shown in Fig. 2.
  • CGMCC General Microbiology Center
  • the traditional fermented fresh shrimp paste was used as the sampling source of the strain.
  • the shrimp paste was derived from the traditional method of homemade shrimp paste made by fishermen in Heiyanzi Town, Fengnan District, Tangshan City, Hebei province.
  • Isolation and purification of moderate halophilic bacteria use Gibbons medium supplemented with 1% skim milk powder, apply sterile water diluted shrimp paste to solid Gibbons medium, incubate at 30°C for 48h, pick The colonies with typical characteristics are continuously separated and purified on solid Gibbons medium supplemented with 1% skim milk powder to obtain pure cultured protease-producing halophilic bacteria strains.
  • the isolated and purified strains were inoculated on the Gibbons slant, cultured at 30°C for 48h, and placed in a refrigerator at 4°C for short-term storage for future use.
  • the colonies were pale yellow, with smooth surfaces, regular edges, opaqueness, dense colonies, positive Gram staining, and the cells were rod-shaped. After culturing in liquid Gibbons medium for 24h, the bacterial solution was cloudy.
  • the strain can grow at a NaCl concentration of 0-15%; it grows faster at a pH of 7-9; it can grow well at a temperature range of 25°C-43°C;
  • the isolated and screened moderately halophilic bacteria were sequenced by 16SrRNA. After the gene sequence was sorted by operation, they were searched in Ezbiocloud and determined to be (Halobacillus faecis). According to the retrieved homologous strains, the Mege 5.1 program of DNAStar software is used to construct a biological evolution relationship tree as shown in Fig. 2.
  • CGMCC General Microbiology Center
  • a method for fermenting fish meat sauce using moderately halophilic strain Bacillus marinus according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria Bacillus aquimaris CGMCC 16541 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28°C for 30 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fish meat sauce fermented with this moderate halophilic bacteria strain is 0.643g/100g; the volatile basic nitrogen content is 25.32mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 3.77mg/ 100g, far below the national standard for food safety, the content of histamine in fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.27.
  • the sensory score of fish sauce without added strain was 5.69.
  • the earthy smell inherent in the raw materials of the fish meat sauce is greatly reduced, and the umami taste is obvious. Compared with the fish sauce without added strain, the flavor is significantly improved.
  • a method for fermenting fish meat sauce using moderately halophilic strain Bacillus marinus according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria Bacillus aquimaris CGMCC 16541 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 40°C for 5 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 40 minutes using steam to cooked fish paste.
  • Fish paste paste sterilization Fill the fish paste paste cooked in step (6) and seal it, and sterilize it at 120°C for 25 minutes to obtain the finished fish paste paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fish meat sauce fermented by this moderate halophilic bacteria strain is 0.307g/100g; the volatile basic nitrogen content is 7.57mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 2.01mg/ 100g, far lower than the national standard for food safety, the content of histamine in fish is 20mg/100g; the comprehensive score of sensory evaluation is 5.88.
  • the sensory score of fish sauce without added strain was 5.49.
  • the earthy smell inherent in the raw materials of the fish meat sauce is greatly reduced, and the umami taste is obvious. Compared with the fish sauce without added strain, the flavor is significantly improved.
  • a medium halophilic strain Bacillus huajintan bacillus fermented fish meat sauce according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria Bacillus hwajinpoensis CGMCC 16542 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28°C for 30 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fish meat sauce fermented with this moderate halophilic bacteria strain is 0.639g/100g; the volatile basic nitrogen content is 24.87mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 3.82mg/ 100g, far lower than the national standard for food safety, the content of histamine in fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.33.
  • the sensory score of fish sauce without added strain was 5.67.
  • the earthy smell inherent in the raw material of the fish meat sauce is greatly reduced, and the umami taste is obvious. Compared with the fish sauce without added strain, the flavor is significantly improved.
  • a medium halophilic strain Bacillus huajintan bacillus fermented fish meat sauce according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria Bacillus hwajinpoensis CGMCC 16542 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10 minutes at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 40°C for 5 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 40 minutes using steam to cooked fish paste.
  • Fish paste paste sterilization Fill the fish paste paste cooked in step (6) and seal it, and sterilize it at 120°C for 25 minutes to obtain the finished fish paste paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fish meat sauce fermented with this moderate halophilic bacteria strain is 0.312g/100g; the volatile basic nitrogen content is 7.80mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 1.98mg/ 100g, far lower than the national standard for food safety, the content of histamine in fish is 20mg/100g; the comprehensive score of sensory evaluation is 5.93.
  • the sensory score of fish sauce without added strain was 5.48.
  • the earthy smell inherent in the raw materials of the fish meat sauce is greatly reduced, and the umami taste is obvious. Compared with the fish sauce without added strain, the flavor is significantly improved.
  • a method for fermenting fish paste with moderately halophilic strain Bacillus subtilis halodendron according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria strain Halobacillus faecis CGMCC 16543 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28°C for 30 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fish meat sauce fermented with this moderate halophilic bacteria strain is 0.647g/100g; the volatile basic nitrogen content is 24.82mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 3.81mg/ 100g, far lower than the national standard for food safety, the content of histamine in fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.32.
  • the sensory score of fish paste without added strain was 5.66.
  • the earthy smell inherent in the raw materials of the fish meat sauce is greatly reduced, and the umami taste is obvious. Compared with the fish sauce without added strain, the flavor is significantly improved.
  • a method for fermenting fish paste with moderately halophilic strain Bacillus subtilis halodendron according to the following steps:
  • moderately halophilic bacteria strain Inoculate moderately halophilic bacteria strain Halobacillus faecis CGMCC 16543 on the medium to activate culture, and repeat the operation three times. The cultured strains were centrifuged at 10000r/min for 10min at 4°C, then washed twice with sterile physiological saline, and resuspended in sterile physiological saline. Finally, the bacterial solution concentration was adjusted to 10 5 ⁇ 10 6 CFU/mL, and the bacterial solution was stored at 4°C and used within 24 hours.
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 40°C for 5 days; the fermented fish paste is placed in a -20 refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 40 minutes using steam to cooked fish paste.
  • Fish paste paste sterilization Fill the fish paste paste cooked in step (6) and seal it, and sterilize it at 120°C for 25 minutes to obtain the finished fish paste paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fish meat sauce fermented with this moderate halophilic bacteria strain is 0.314g/100g; the volatile basic nitrogen content is 7.63mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 2.11mg/ 100g, far lower than the national standard for food safety, the content of histamine in fish is 20mg/100g; the comprehensive score of sensory evaluation is 5.94.
  • the sensory score of the fish sauce without the added strain was 5.53.
  • the earthy smell inherent in the raw materials of the fish meat sauce is greatly reduced, and the umami taste is obvious. Compared with the fish sauce without added strain, the flavor is significantly improved.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish sauce: The mixture obtained in step (3) is incubated and fermented at a temperature of 40°C for 5 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.311g/100g; the volatile base nitrogen content is 7.54mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 1.69mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 5.79.
  • the earthy smell inherent in the raw materials of the fish meat sauce is greatly reduced, slightly fishy, and the umami is slightly insufficient. Compared with the unfermented raw materials, the flavor is significantly improved.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish sauce: The mixture obtained in step (3) is incubated and fermented at a temperature of 37°C for 15 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.519g/100g; the volatile base nitrogen content is 23.86mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 2.17mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 7.20.
  • the fishy sauce inherent in the raw material has almost no earthy smell, no bad smell, delicious taste, high amino acid nitrogen content, rich nutritional value and high safety.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 31°C for 15 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.476g/100g; the volatile base nitrogen content is 20.11mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 1.92mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.62.
  • the fishy sauce inherent in the fish raw material has almost no earthy smell, no bad smell, delicious taste, high amino acid nitrogen content, rich nutritional value and high safety.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • the minced fish meat is chopped with a chopper, added with 20% (m/m) of pickled sea salt, and placed in a refrigerator at 4°C for 24 hours before fermentation.
  • step (3) Fermentation of fish sauce: The mixture obtained in step (3) is incubated and fermented at a temperature of 40°C for 30 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.621g/100g; the volatile base nitrogen content is 39.81mg/100g, which is higher than the EU standard 35mg/100g; the histamine content is 6.98mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 5.87.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 28°C for 5 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.362g/100g; the volatile base nitrogen content is 6.49mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 1.13mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.11.
  • This fish paste has less earthy smell inherent in the raw materials, and has a slightly unpleasant flavor.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish paste: The mixture obtained in step (3) is incubated and fermented at a temperature of 31°C for 20 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.501g/100g; the volatile base nitrogen content is 23.17mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 2.48mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.94.
  • the umami taste is outstanding, the taste is delicious, the saltiness is high, the amino acid nitrogen content is high, the nutrition value is rich, and the safety is high.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • the minced fish meat is chopped with a chopper, added with 20% (m/m) of pickled sea salt, and placed in a refrigerator at 4°C for 24 hours before fermentation.
  • step (3) Fermentation of fish sauce: The mixture obtained in step (3) is incubated and fermented at a temperature of 34°C for 10 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish meat sauce in this process is 0.287g/100g; the volatile base nitrogen content is 16.23mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 1.88mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 5.97.
  • the earthy smell inherent in the raw materials of the fish meat sauce is reduced, and the fishy smell is relatively obvious but the safety is high.
  • a method for fermenting fish meat sauce with a mixed strain of moderate halophilic bacteria according to the following steps:
  • step (3) Fermentation of fish sauce: The mixture obtained in step (3) is incubated and fermented at a temperature of 34°C for 15 days; the fermented product is placed in a -20°C refrigerator for use.
  • Cooked fish paste The fish paste obtained in step (4) is steamed in a steamer for 20 minutes with steam until the fish paste is fully cooked.
  • Sterilization of fish paste Fill and seal the cooked fish paste in step (6), and sterilize it at 120°C for 15 minutes to obtain the finished fish paste.
  • amino acid nitrogen content evaluation the method refers to GB5009.235-2016 acidity meter method.
  • volatile base nitrogen content refer to GB5009.228-2016 Micro Diffusion Method.
  • histamine content refer to GB5009.208-2016 spectrophotometry.
  • the sensory evaluation group is composed of 10 people. Before the evaluation, the team members need to be trained in flavor. The score includes 10 scores from 0 to 9, and a comprehensive score for the overall flavor of the fish flavor, fishy flavor, fermentation flavor, ammonia flavor, umami flavor, salty flavor, sweet flavor and bitterness.
  • the amino acid content of the fermented fish paste in this process is 0.549g/100g; the volatile base nitrogen content is 21.90mg/100g, which is lower than the EU standard 35mg/100g; the histamine content is 1.97mg/100g, which is far lower than food safety
  • the content of histamine in national standard fish is 20mg/100g; the comprehensive score of sensory evaluation is 6.34.
  • the fermentation taste is obvious, the umami taste is sufficient, the amino acid nitrogen content is high, the nutritional value is high, and the safety is high.

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Abstract

本发明公开了中度嗜盐菌及利用其发酵鱼肉酱的方法，属于利用菌株发酵水产品固态发酵技术领域。利用中度嗜盐菌发酵剂作为生产发酵剂生产鱼肉酱，通过发酵鱼肉酱的理化指标评价和感官评价，研究混合发酵剂比例、接种量、发酵温度，发酵时间和盐度对鱼肉酱的影响，使得鱼肉酱具有发酵时间短、盐度较低、安全性高和风味与营养价值较高的特性。利用此混合发酵剂能够得到风味较好的鱼肉酱发酵制品。

Description

中度嗜盐菌及利用其发酵鱼肉酱的方法 技术领域
本发明涉及中度嗜盐菌及利用其发酵鱼肉酱的方法，属于利用菌株发酵水产品固态发酵技术领域。
背景技术
我国淡水养殖业发展迅速，但加工业发展中相对滞后。因此开发适合国内消费市场需求的淡水鱼产品，对推动淡水鱼产业健康发展具有重要的现实意义。目前，发酵水产品中氨基酸含量丰富，营养价值高，深受消费者喜爱，但对于发酵产品又存在着发酵时间长、含盐量高和发酵不稳定等问题。中度嗜盐菌是一种在NaCl浓度为0.5mol/L到2.5mol/L之间生长最佳的极端微生物。与轻度嗜盐菌相比，中度嗜盐菌能够在高盐环境中生长，从而减少发酵过程中杂菌污染。与极端嗜盐古菌相比，中度嗜盐菌比嗜盐古菌的嗜盐范围宽，分布及来源更广泛、对营养物质要求也更低、更容易适应生存环境，在实际生产中更容易被应用。有些中度嗜盐菌又有较强的产胞外蛋白酶特性。本发明将利用中度嗜盐菌混合菌株发酵鱼肉酱，得到发酵时间短，盐度较低和风味和营养价值较高的鱼肉酱产品。为水产品发酵品质的提升提供了理论基础和方法。
发明内容
本发明利用中度嗜盐菌，结合菌株的优良发酵特性，采用菌株发酵法生产鱼肉风味酱。该方法可有效提高的风味和营养价值。
本发明提供的中度嗜盐菌JD(Bacillus aquimaris JD)，已经于2018年9月27日保藏在位于北京市朝阳区北辰西路1号院3号的中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，编号为CGMCC NO.16541，建议的分类命名：海水芽孢杆菌Bacillus aquimaris。
一种利用中度嗜盐菌菌株海水芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌Bacillus aquimaris CGMCC 16541接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Bacillus aquimaris CGMCC 16541按最终添加量(10 4～10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔1～5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
本发明提供的中度嗜盐菌JD(Bacillus hwajinpoensis JD)，已经于2018年9月27日保藏在位于北京市朝阳区北辰西路1号院3号的中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，编号为CGMCC NO.16542，建议的分类命名：花津滩芽孢杆菌Bacillus hwajinpoensis。
一种利用中度嗜盐菌菌株花津滩芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌Bacillus hwajinpoensis CGMCC 16542接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Bacillus hwajinpoensis CGMCC 16542按最终添加量(10 4～10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔1～5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
本发明提供的中度嗜盐菌JD(Halobacillus faecis JD)，该中度嗜盐菌菌株已经于2018年9月27日保藏在位于北京市朝阳区北辰西路1号院3号的中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，编号为CGMCC NO.16543，建议的分类命名：沉泥喜盐芽孢杆菌Halobacillus faecis。
一种利用中度嗜盐菌菌株沉泥喜盐芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌Halobacillus faecis CGMCC 16543接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Halobacillus faecis CGMCC 16543按最终添加量(10 4～10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔1～5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
本发明提供如下技术方案：一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法。本发明包括原料的处理、混合菌株的制备与添加、鱼肉酱的发酵、氨基酸态氮的测定、感官评定等程序，具体生产工艺如下：
一种利用中度嗜盐菌混合菌株发酵的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例(1～3):(1～3):(1～3)进行混合制备成混合发酵剂，再按最终添加量(10 4～10 7CFU/g)将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例(1～3):(1～3):(1～3)和最终添加量(10 4～10 7CFU/g)亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好的置于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔2～30d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
本发明的优点：(1)利用该中度嗜盐菌菌株发酵的鱼肉酱的发酵时间短，盐度较低，氨基酸态氮含量较高，滋味鲜美，营养价值丰富，挥发性盐基氮的含量低于欧盟标准，组胺的含量远低于国家标准，是一种品质和安全性高的的发酵鱼肉酱产品。
(2)将上述混合发酵剂在发酵中的接种比例、接种量、发酵时间、发酵温度、加盐量等发酵工艺进行了优化，综合发酵鱼酱的理化指标评价和感官评价对最终鱼肉酱品质进行了综合评价。此方法获得的鱼肉酱发酵时间短，盐度较低，氨基酸态氮含量较高，滋味鲜美，营养价值丰富，挥发性盐基氮的含量低于欧盟标准，组胺的含量远低于国家标准，是一种品质和安全性高的的发酵鱼肉酱产品。
附图说明
图1为中度嗜盐菌Bacillus aquimaris JD菌落形态；
图2为中度嗜盐菌Bacillus aquimaris JD的16SrRNA序列构建的系统发育树。注：zzy8为本发明中度嗜盐菌Bacillus aquimaris JD。
图3为中度嗜盐菌Bacillus hwajinpoensis JD菌落形态；
图4为中度嗜盐菌Bacillus hwajinpoensis JD的16SrRNA序列构建的系统发育树。注：zzy9为本发明中度嗜盐菌Bacillus hwajinpoensis JD。
图5为中度嗜盐菌Halobacillus faecis JD菌落形态；
图6为中度嗜盐菌Halobacillus faecis JD的16SrRNA序列构建的系统发育树。注：zzy5为本发明中度嗜盐菌Halobacillus faecis。
具体实施方式
(一)菌株海水芽孢杆菌Bacillus aquimaris。
1、菌株的分离筛选：
以传统发酵的新鲜虾酱为原材料，虾酱来源于河北省唐山市丰南区黑沿子镇渔民自制虾酱，从虾酱中分离筛选中度嗜盐菌。中度嗜盐菌的分离和纯化使用添加1％脱脂乳粉的Gibbons 培养基，将无菌水按一定倍数稀释后的虾酱涂布于固体Gibbons培养基上，30℃培养48h，挑取具有典型特征的菌落，继续在添加1％脱脂乳粉的固体Gibbons培养基上进行多次分离纯化，获得纯培养产蛋白酶的嗜盐菌菌株。将分离并纯化后的菌株接种到Gibbons斜面上，30℃培养48h，置于4℃冰箱短期保藏备用。
2、菌株的鉴定
2.1菌株的形态特征
菌落淡黄色、表面光滑、边缘整齐、不透明、菌落致密，革兰氏染色为阴性，菌体呈杆状。在液体Gibbons培养基中培养24h后，菌液浑浊。
2.2菌株的生理特征
该菌株在NaCl浓度为0～15％时均可进行生长；在pH为7～9时，生长较快；在温度范围为25℃～43℃都能进行良好生长；
2.3分子生物学鉴定
对分离筛选出的中度嗜盐菌经16SrRNA测序，基因序列经操作整理后，在Ezbiocloud中检索，确定为(Bacillus aquimaris)。根据检索到的同源菌株，应用DNAStar软件的Mege5.1程序，构建生物进化关系树如图2。
该中度嗜盐菌菌株已经于2018年9月27日保藏在位于北京市朝阳区北辰西路1号院3号的中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，编号为CGMCC NO.16541，建议的分类命名：海水芽孢杆菌Bacillus aquimaris。
(二)菌株花津滩芽孢杆菌Bacillus hwajinpoensis。
1、菌株的分离筛选：
以传统发酵的新鲜虾酱为原材料，虾酱来源于河北省唐山市丰南区黑沿子镇渔民自制虾酱，从虾酱中分离筛选中度嗜盐菌。中度嗜盐菌的分离和纯化使用添加1％脱脂乳粉的Gibbons培养基，将无菌水按一定倍数稀释后的虾酱涂布于固体Gibbons培养基上，30℃培养48h，挑取具有典型特征的菌落，继续在添加1％脱脂乳粉的固体Gibbons培养基上进行多次分离纯化，获得纯培养产蛋白酶的嗜盐菌菌株。将分离并纯化后的菌株接种到Gibbons斜面上，30℃培养48h，置于4℃冰箱短期保藏备用。
2、菌株的鉴定
2.1菌株的形态特征
菌落呈橘黄色、表面光滑、边缘整齐、不透明、菌落致密，革兰氏染色为阳性，菌体呈杆状。在液体Gibbons培养基中培养24h后，菌液浑浊。
2.2菌株的生理特征
该菌株在NaCl浓度为0～20％时均可进行生长；在pH为7～9时，生长较快；在温度范围为25℃～43℃都能进行良好生长；
2.3分子生物学鉴定
对分离筛选出的中度嗜盐菌经16SrRNA测序，基因序列经操作整理后，在Ezbiocloud中检索，确定为(Bacillus hwajinpoensis)。根据检索到的同源菌株，应用DNAStar软件的Mege5.1程序，构建生物进化关系树如图2。
该中度嗜盐菌菌株已经于2018年9月27日保藏在位于北京市朝阳区北辰西路1号院3号的中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，编号为CGMCC NO.16542，建议的分类命名：花津滩芽孢杆菌Bacillus hwajinpoensis。
(三)菌株沉泥喜盐芽孢杆菌Halobacillus faecis。
1、菌株的分离筛选：
以传统发酵的新鲜虾酱为菌株采样源，虾酱来源于河北省唐山市丰南区黑沿子镇渔民传统法自制虾酱，从虾酱中分离筛选中度嗜盐菌。中度嗜盐菌的分离和纯化使用添加1％脱脂乳粉的Gibbons培养基，将无菌水按一定倍数稀释后的虾酱涂布于固体Gibbons培养基上，30℃培养48h，挑取具有典型特征的菌落，继续在添加1％脱脂乳粉的固体Gibbons培养基上进行多次分离纯化，获得纯培养产蛋白酶的嗜盐菌菌株。将分离并纯化后的菌株接种到Gibbons斜面上，30℃培养48h，置于4℃冰箱短期保藏备用。
2、菌株的鉴定
2.1菌株的形态特征
菌落呈浅黄色、表面光滑、边缘整齐、不透明、菌落致密，革兰氏染色为阳性，菌体呈杆状。在液体Gibbons培养基中培养24h后，菌液浑浊。
2.2菌株的生理特征
该菌株在NaCl浓度为0～15％时均可进行生长；在pH为7～9时，生长较快；在温度范围为25℃～43℃都能进行良好生长；
2.3分子生物学鉴定
对分离筛选出的中度嗜盐菌经16SrRNA测序，基因序列经操作整理后，在Ezbiocloud中检索，确定为(Halobacillus faecis)。根据检索到的同源菌株，应用DNAStar软件的Mege5.1程序，构建生物进化关系树如图2。
该中度嗜盐菌菌株已经于2018年9月27日保藏在位于北京市朝阳区北辰西路1号院3号的中国科学院微生物研究所的中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，编号为CGMCC NO.16543，建议的分类命名：沉泥喜盐芽孢杆菌Halobacillus faecis。
实施例1：
一种利用中度嗜盐菌菌株海水芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5％(w/w)，在发酵前放置在4℃冰箱内24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌菌种Bacillus aquimaris CGMCC 16541接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Bacillus aquimaris CGMCC 16541按最终添加量10 4CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28℃下保温发酵30d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1min，搅拌结束后静置透气10s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
利用该株中度嗜盐菌菌株发酵鱼肉酱的氨基酸态的含量为0.643g/100g；挥发性盐基氮含量为25.32mg/100g，低于欧盟标准35mg/100g；组胺含量为3.77mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.27。未添加菌株鱼酱的感官评分为5.69。本鱼肉酱中原料中固有的土腥味大大减少，鲜味明显，与未添加菌株鱼酱相比，风味有显著提升。
实施例2：
一种利用中度嗜盐菌菌株海水芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐20％(w/w)，在发酵前放置在4℃冰箱内24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌菌种Bacillus aquimaris CGMCC 16541接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Bacillus aquimaris CGMCC 16541按最终添加量10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度40℃下保温发酵5d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5d内，每隔1d在无菌条件下对发酵鱼酱进行顺时针的搅拌5min，搅拌结束后静置透气120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌25min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
利用该株中度嗜盐菌菌株发酵鱼肉酱的氨基酸态的含量为0.307g/100g；挥发性盐基氮含量为7.57mg/100g，低于欧盟标准35mg/100g；组胺含量为2.01mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为5.88。未添加菌株鱼酱的感官评分为5.49。本鱼肉酱中原料中固有的土腥味大大减少，鲜味明显，与未添加菌株鱼酱相比，风味有显著提升。
实施例3：
一株中度嗜盐菌菌株花津滩芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5％(w/w)，在发酵前放置在4℃冰箱内24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌菌种Bacillus hwajinpoensis CGMCC 16542接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Bacillus hwajinpoensis CGMCC 16542按最终添加量10 4CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28℃下保温发酵30d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1min，搅拌结束后静置透气10s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
利用该株中度嗜盐菌菌株发酵鱼肉酱的氨基酸态的含量为0.639g/100g；挥发性盐基氮含量为24.87mg/100g，低于欧盟标准35mg/100g；组胺含量为3.82mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.33。未添加菌株鱼酱的感官评分为5.67。本鱼肉酱中原料中固有的土腥味大大减少，鲜味明显，与未添加菌株鱼酱相比，风味有显著提升。
实施例4：
一株中度嗜盐菌菌株花津滩芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐20％(w/w)，在发酵前放置在4℃冰箱内24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌菌种Bacillus hwajinpoensis CGMCC 16542接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min， 然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Bacillus hwajinpoensis CGMCC 16542按最终添加量10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度40℃下保温发酵5d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5d内，每隔1d在无菌条件下对发酵鱼酱进行顺时针的搅拌5min，搅拌结束后静置透气120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌25min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
利用该株中度嗜盐菌菌株发酵鱼肉酱的氨基酸态的含量为0.312g/100g；挥发性盐基氮含量为7.80mg/100g，低于欧盟标准35mg/100g；组胺含量为1.98mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为5.93。未添加菌株鱼酱的感官评分为5.48。本鱼肉酱中原料中固有的土腥味大大减少，鲜味明显，与未添加菌株鱼酱相比，风味有显著提升。
实施例5：
一种利用中度嗜盐菌菌株沉泥喜盐芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5％(w/w)，在发酵前放置在4℃冰箱内24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌菌种Halobacillus faecis CGMCC 16543接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Halobacillus faecis CGMCC 16543按最终添加量10 4CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28℃下保温发酵30d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1min，搅拌结束后静置透气10s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
利用该株中度嗜盐菌菌株发酵鱼肉酱的氨基酸态的含量为0.647g/100g；挥发性盐基氮含量为24.82mg/100g，低于欧盟标准35mg/100g；组胺含量为3.81mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.32。未添加菌株鱼酱的感官评分为5.66。本鱼肉酱中原料中固有的土腥味大大减少，鲜味明显，与未添加菌株鱼酱相比，风味有显著提升。
实施例6：
一种利用中度嗜盐菌菌株沉泥喜盐芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐20％(w/w)，在发酵前放置在4℃冰箱内24h。
(2)中度嗜盐菌菌株的制备：将中度嗜盐菌菌种Halobacillus faecis CGMCC 16543接种在培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌菌种Halobacillus faecis CGMCC 16543按最终添加量10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度40℃下保温发酵5d；将发酵好鱼肉酱置于-20冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5d内，每隔1d在无菌条件下对发酵鱼酱进行顺时针的搅拌5min，搅拌结束后静置透气120s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸40min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌25min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
利用该株中度嗜盐菌菌株发酵鱼肉酱的氨基酸态的含量为0.314g/100g；挥发性盐基氮含量为7.63mg/100g，低于欧盟标准35mg/100g；组胺含量为2.11mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为5.94。未添加菌株鱼酱的感官评分为5.53。本鱼肉酱中原料中固有的土腥味大大减少，鲜味明显，与未添加菌株鱼酱相比，风味有显著提升。
实施例7：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐12％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例2:1:1进行混合制备成混合发酵剂，再按最终添加量10 4CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例2:1:1和最终添加量10 4CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度40℃下保温发酵5d；将发酵好的置于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.311g/100g；挥发性盐基氮含量为7.54mg/100g，低于欧盟标准35mg/100g；组胺含量为1.69mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为5.79。本鱼肉酱中原料中固有的土腥味大大减少，略有腥味，鲜味略显不足，与未发酵原料相比，风味有显著提升。
实施例8：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐12％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例1:1:1进行混合制备成混合发酵剂，再按最终添加量10 6CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例1:1:1和最终添加量10 6CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度37℃下保温发酵15d；将发酵好的置于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.519g/100g；挥发性盐基氮含量为23.86mg/100g，低于欧盟标准35mg/100g；组胺含量为2.17mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为7.20。本鱼肉酱中原料中固有的土腥味几乎没有，无不良气味，滋味鲜美，氨基酸态氮含量较高，营养价值较为丰富，安全性也较高。
实施例9：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐14％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例1:1:1进行混合制备成混合发酵剂，再按最终添加量10 5CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例1:1:1和最终添加量10 5CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度31℃下保温发酵15d；将发酵好的置于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.476g/100g；挥发性盐基氮含量为20.11mg/100g，低于欧盟标准35mg/100g；组胺含量为1.92mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.62。本鱼肉酱中鱼原料中固有的土腥味几乎没有，无不良气味，滋味鲜美，氨基酸态氮含量较高，营养价值较为丰富，安全性较高。
实施例10：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐20％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例3:1:1进行混合制备成混合发酵剂，再按最终添加量10 7CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例3:1:1和最终添加量10 7CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度40℃下保温发酵30d；将发酵好的置于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.621g/100g；挥发性盐基氮含量为39.81mg/100g，高于欧盟标准35mg/100g；组胺含量为6.98mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为5.87。本鱼肉酱中几乎没有原料中固有的土腥味几乎没有，有不良气味产生，鱼酱有明显的氨味。
实施例11：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例2:2:1进行混合制备成混合发酵剂，再按最终添加量10 6CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例2:2:1和最终添加量10 6CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28℃下保温发酵5d；将发酵好的置于 -20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.362g/100g；挥发性盐基氮含量为6.49mg/100g，低于欧盟标准35mg/100g；组胺含量为1.13mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.11。本鱼肉酱中原料中固有的土腥味较少，略有不良风味。
实施例12：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐16％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6菌株，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例2:1:1进行混合制备成混合发酵剂，再按最终添加量10 6CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例2:1:1和最终添加量10 6CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度31℃下保温发酵20d；将发酵好的置 于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.501g/100g；挥发性盐基氮含量为23.17mg/100g，低于欧盟标准35mg/100g；组胺含量为2.48mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.94。本鱼肉酱中原料中固有的土腥味几乎没有，鲜味突出，滋味鲜美，偏咸，氨基酸态氮含量高，营养价值丰富，安全性高。
实施例13：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐20％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例2:2:1进行混合制备成混合发酵剂，再按最终添加量10 5CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例2:2:1和最终添加量10 5CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度34℃下保温发酵10d；将发酵好的置 于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.287g/100g；挥发性盐基氮含量为16.23mg/100g，低于欧盟标准35mg/100g；组胺含量为1.88mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为5.97。本鱼肉酱中原料中固有的土腥味减少，腥味相对明显但安全性较高。
实施例14：
一种利用中度嗜盐菌混合菌株发酵鱼肉酱的方法，按照下述步骤进行：
(1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐10％(m/m)，在发酵前放置在4℃冰箱内24h。
(2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次。将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中。最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用。
(3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例1:1:3进行混合制备成混合发酵剂，再按最终添加量10 4CFU/g将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例1:1:3和最终添加量10 6CFU/g亦可；
(4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度34℃下保温发酵15d；将发酵好的置 于-20℃冰箱中备用。
(5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔5d在无菌条件下对发酵鱼酱进行顺时针的搅拌2min，搅拌结束后静置透气30s。
(6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20min至鱼肉酱熟透。
(7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15min，即得鱼肉酱成品。
(8)产品发酵理化指标的评价：氨基酸态氮含量评价，方法参照GB5009.235-2016酸度计法。挥发性盐基氮含量评价，方法参照GB5009.228-2016微量扩散法。组胺含量的测定，方法参照GB5009.208-2016分光光度法。
(9)感官评定：由10人组成感官评定小组，评定前需对小组成员进行风味培训。评分包括0～9十个分数，对鱼肉酱的鱼味、腥味、发酵味、氨味、鲜味、咸味、甜味和苦涩味进行整体风味的综合评分。
(10)鱼肉酱的理化指标和感官评价
本工艺发酵鱼肉酱的氨基酸态的含量为0.549g/100g；挥发性盐基氮含量为21.90mg/100g，低于欧盟标准35mg/100g；组胺含量为1.97mg/100g，远低于食品安全国家标准鱼类中组胺的含量20mg/100g；感官评价的综合得分为6.34。本鱼肉酱中几乎没有原料中固有的土腥味，发酵味明显，鲜味较足，氨基酸态氮含量多，营养价值较高，安全性高。

Claims (7)

  1. 中度嗜盐菌菌株海水芽孢杆菌，其保藏编号为CGMCC NO.16541，建议的分类命名：海水芽孢杆菌Bacillus aquimaris。
  2. 利用权利要求1所述的一种利用中度嗜盐菌菌株海水芽孢杆菌发酵鱼肉酱的方法，按照下述步骤进行：
    (1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h；
    (2)中度嗜盐菌菌株的制备：将中度嗜盐菌Bacillus aquimaris CGMCC 16541接种在培养基上活化培养，重复操作三次；将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中；最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用；
    (3)发酵剂的添加：将中度嗜盐菌菌种Bacillus aquimaris CGMCC 16541按最终添加量(10 4～10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
    (4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好鱼肉酱置于-20冰箱中备用；
    (5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔1～5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s；
    (6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透；
    (7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
  3. 中度嗜盐菌菌株，其保藏编号为CGMCC NO.16542，建议的分类命名：花津滩芽孢杆菌Bacillus hwajinpoensis。
  4. 一种利用权利要求3所述的一种利用中度嗜盐菌菌株花津滩芽孢杆菌发酵鱼肉酱的方法，其特征在于按照下述步骤进行：
    (1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h；
    (2)中度嗜盐菌菌株的制备：将中度嗜盐菌Bacillus hwajinpoensis CGMCC 16542 接种在培养基上活化培养，重复操作三次；将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中；最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用；
    (3)发酵剂的添加：将中度嗜盐菌菌种Bacillus hwajinpoensis CGMCC 16542按最终添加量(10 4～10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
    (4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好鱼肉酱置于-20冰箱中备用；
    (5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔1～5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s；
    (6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透；
    (7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
  5. 中度嗜盐菌菌株，其保藏编号为CGMCC NO.16543，建议的分类命名：沉泥喜盐芽孢杆菌Halobacillus faecis。
  6. 一种利用权利要求5所述的中度嗜盐菌菌株沉泥喜盐芽孢杆菌发酵鱼肉酱的方法，其特征在于按照下述步骤进行：
    (1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h；
    (2)中度嗜盐菌菌株的制备：将中度嗜盐菌Halobacillus faecis CGMCC 16543接种在培养基上活化培养，重复操作三次；将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中；最后将菌液浓度调整到10 5～10 6CFU/mL，将菌液于4℃保存，并于24h内使用；
    (3)发酵剂的添加：将中度嗜盐菌菌种Halobacillus faecis CGMCC 16543按最终添加量(10 4～10 7CFU/g)混入到预处理过的原料鱼中，使最终菌数达到添加要求即可
    (4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30 d；将发酵好鱼肉酱置于-20冰箱中备用；
    (5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔1～5d在无菌条件下对发酵鱼酱进行顺时针的搅拌1～5min，搅拌结束后静置透气10～120s；
    (6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20～40min至鱼肉酱熟透；
    (7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15～20min，即得鱼肉酱成品。
  7. 一种利用中度嗜盐菌混合菌株发酵的方法，其特征在于按照下述步骤进行：
    (1)原料的处理：碎鱼肉利用斩拌机斩断，加入腌制海盐5～20％(w/w)，在发酵前放置在4℃冰箱内5～24h；
    (2)混合菌株的制备：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis分别接种在不同的培养基上活化培养，重复操作三次；将培养的菌种菌液于4℃、10000r/min离心10min，然后用无菌生理盐水洗涤两次，再悬浮于无菌生理盐水中；最后将菌液浓度调整到10 5-10 6CFU/mL，将菌液于4℃保存，并于24h内使用；
    (3)发酵剂的添加：将中度嗜盐菌混合菌种Halobacillus faecis、Bacillus aquimaris和Bacillus hwajinpoensis菌株按菌数比例(1～3):(1～3):(1～3)进行混合制备成混合发酵剂，再按最终添加量(10 4～10 7CFU/g)将混合发酵剂混入到预处理过的原料鱼中，使最终菌数达到添加要求即可；或3株菌株分别加入到原料鱼中，最终菌数符合添加比例(1～3):(1～3):(1～3)和最终添加量(10 4～10 7CFU/g)亦可；
    (4)鱼肉酱发酵：把步骤(3)得到的混合物料在温度28～40℃下保温发酵5～30d；将发酵好的置于-20冰箱中备用；
    (5)鱼肉酱发酵过程中的搅拌与透气：在鱼酱发酵的5～30d内，每隔2～30d在无菌条件下对发酵鱼酱进行顺时针的搅拌1-5min，搅拌结束后静置透气10-120s；
    (6)鱼肉酱熟制：将步骤(4)得到的鱼肉酱利用水蒸气在蒸屉中蒸20-40min 至鱼肉酱熟透；
    (7)鱼肉酱灭菌：将步骤(6)熟制的鱼肉酱灌装密封，在120℃下二次灭菌15-20min，即得鱼肉酱成品。
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