CN109456922B - Moderately halophilic bacteria strain Zhan beach bacillus for improving fermentation quality of fish paste - Google Patents
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Abstract
The invention discloses a moderate halophilic bacteria strain Zymobacter floribundum for improving fermentation quality of fish paste, and relates to the field of application of food microbial technology. The strain is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 9 and 27 months, and the serial number of the strain is CGMCC NO.16542, and the strain can be called Bacillus hwajinoensis CGMCC 16542. The strain is screened from traditional fermented shrimp paste by using a Gibbons culture medium, has strong protease production capacity, can be applied to fermentation of animal aquatic products, and can accelerate fermentation of the animal aquatic products and improvement of flavor.
Description
Technical Field
The invention relates to the field of application of food microbial technology, in particular to a moderately halophilic bacteria strain Huajin beach bacillus for improving the fermentation quality of fish paste, and a new strain capable of effectively promoting the fermentation of the fish paste and improving the quality of the fish paste.
Background
The freshwater aquaculture development of China is rapid, but the processing industry development is relatively lagged. Therefore, the development of freshwater fish products suitable for domestic consumption market demands has important practical significance for promoting the healthy development of freshwater fish industry. At present, fermented aquatic products are rich in amino acid content and high in nutritive value, are popular with consumers, but have the problems of long fermentation time, high salt content, unstable fermentation and the like in the fermented products. The moderately halophilic bacteria are extreme microorganisms that grow optimally at NaCl concentrations of between 0.5mol/L and 2.5 mol/L. Compared with mild halophilic bacteria, moderate halophilic bacteria can grow in high-salinity environment, so that the mixed bacteria pollution in the fermentation process is reduced. Compared with extreme halophilic archaea, the moderate halophilic archaea has a wider halophilic range than halophilic archaea, is wider in distribution and source, has lower requirements on nutrient substances, is easier to adapt to living environment, and is easier to be applied in actual production. Some moderate halophilic bacteria have strong property of producing extracellular protease. The invention utilizes a new moderately halophilic bacteria strain to ferment the fish paste to obtain the fish paste product with short fermentation time, lower salinity and higher flavor and nutritive value. Provides a theoretical basis and a method for improving the fermentation quality of aquatic products.
Disclosure of Invention
In order to accelerate the fermentation of animal aquatic products and improve the quality of fermented products, the invention screens out a new strain for improving the quality of fermented aquatic products from the traditional fermented shrimp paste. The strain can effectively improve the flavor and the nutritive value of the fermented aquatic product, and provides guidance for improving the quality of the fermented product.
The technical scheme adopted by the invention is as follows:
the moderately halophilic bacteria JD (Bacillus hwajinpoiensis JD) provided by the invention is a new strain capable of being applied to improving the quality of fermented fish paste. The moderately halophilic bacteria strain is preserved in China general microbiological culture Collection center (CGMCC) of the institute of microbiology, China academy of sciences, institute of microbiology, No.1, Sai Lu, No. 3, located in the area of Chaoyang, Beijing for 27 months and 2018, the serial number is CGMCC NO.16542, and the suggested classification name is as follows: bacillus sphaericus Bacillus hwajinpoensis.
In one aspect of the invention, the application of Bacillus hwajinpoiensis CGMCC16542 is provided for improving the flavor and quality of fish sauce fermentation, and the method for fermenting the fish sauce by using the moderately halophilic bacteria strain Bacillus tsumadai comprises the following steps:
(1) treatment of raw materials: chopping the minced fish meat by using a chopper mixer, adding 5-20% (w/w) of pickled sea salt, and placing in a refrigerator at 4 ℃ for 5-24 hours before fermentation.
(2) Preparation of moderately halophilic bacteria strain: the moderately halophilic bacteria Bacillus hwajinpoensis CGMCC16542 is inoculated on a culture medium for activated culture, and the operation is repeated three times. Centrifuging the cultured strain liquid at 4 deg.C and 10000r/min for 10min, washing with sterile normal saline twice, and suspending in sterile normal saline. Finally, adjusting the concentration of the bacterial liquid to 105～106CFU/mL, the bacterial solution was stored at 4 ℃ and used within 24 h.
(3) Addition of a starter: adding the intermediate halophilic bacteria strain Bacillus hwajinpoensis CGMCC16542 according to the final adding amount (10)4～107CFU/g) is mixed into the pretreated raw fish to ensure that the final bacterial count meets the addition requirement
(4) Fermenting the fish paste: fermenting the mixed material obtained in the step (3) at the temperature of 28-40 ℃ for 5-30 d in a heat preservation manner; placing the fermented fish meat paste in a-20 refrigerator for later use.
(5) Stirring and ventilating in the fish meat sauce fermentation process: and (3) stirring the fermented fish paste clockwise for 1-5 min every 1-5 days within 5-30 days of fish paste fermentation, and standing for ventilation for 10-120 s after stirring.
(6) Cooking the fish meat paste: and (4) steaming the fish paste obtained in the step (4) in a steaming drawer for 20-40 min by using water vapor until the fish paste is well cooked.
(7) Fish sauce sterilization: and (4) filling and sealing the fish paste cooked in the step (6), and performing secondary sterilization at 120 ℃ for 15-20 min to obtain a fish paste finished product.
(8) And (3) evaluating the physical and chemical indexes of the product fermentation: the content of amino acid nitrogen is evaluated according to the acidity meter method of GB 5009.235-2016. The content of volatile basic nitrogen is evaluated by the method according to the GB5009.228-2016 microdiffusion method. The method for measuring the histamine content refers to GB5009.208-2016 spectrophotometry.
(8) Sensory evaluation: a sensory panel of 10 persons was evaluated and flavor training was performed on panellists before evaluation. The scores comprise 0-9 scores, and the overall flavor of the fish paste is comprehensively scored according to the fish flavor, fishy smell, fermented taste, ammonia taste, delicate flavor, salty taste, sweet taste and bitter taste.
The invention has the advantages of
The fish paste fermented by the moderately halophilic bacteria strain Zhang Jintan bacillus has short fermentation time, low salinity, high content of amino acid nitrogen, delicious taste, rich nutritive value, low content of volatile basic nitrogen which is lower than European Union standard, and low content of histamine which is far lower than national standard, and is a fermented fish paste product with high quality and safety.
In conclusion, the invention utilizes the uniqueness, the outstanding substantive features and the innovativeness and practicability of remarkable progress of the invention patent, and produces beneficial effects.
Drawings
FIG. 1 shows the colony morphology of a moderately halophilic bacterium Bacillus hwajinpoensis JD;
FIG. 2 shows a phylogenetic tree constructed from the 16SrRNA sequence of the moderately halophilic bacterium Bacillus hwajinpoensis JD. Note: zzy9 is halophilic Bacillus hwajinpoensis JD.
Detailed Description
1. Separating and screening strains:
the method is characterized in that the traditional fermented fresh shrimp paste is used as a raw material, the shrimp paste is prepared by fishermen in black-border son town in Hanshan city of Hebei province, and moderately halophilic bacteria are separated and screened from the shrimp paste. And (3) separating and purifying the moderately halophilic bacteria by using a Gibbons culture medium added with 1% skimmed milk powder, coating the shrimp paste diluted by sterile water according to a certain multiple on a solid Gibbons culture medium, culturing at 30 ℃ for 48h, picking a bacterial colony with typical characteristics, and continuously performing multiple separation and purification on the solid Gibbons culture medium added with 1% skimmed milk powder to obtain the halophilic bacteria strain which is purely cultured and produces the protease. Inoculating the separated and purified strain to a Gibbons slant, culturing at 30 ℃ for 48h, and storing in a refrigerator at 4 ℃ for short-term storage.
2. Identification of strains
2.1 morphological characteristics of the Strain
The bacterial colony is orange yellow, has smooth surface, neat edge, non-transparency, compact bacterial colony, positive gram stain and rod-shaped thallus. After 24h incubation in liquid Gibbons medium, the broth was cloudy.
2.2 physiological characteristics of the Strain
The strain can grow when the NaCl concentration is 0-20%; when the pH value is 7-9, the growth is fast; can grow well at the temperature ranging from 25 ℃ to 43 ℃;
2.3 molecular biological identification
Sequencing the separated and screened moderately halophilic bacteria by 16SrRNA, retrieving a gene sequence in Ezbiocloud after operation arrangement, and determining the gene sequence as (Bacillus hwajinpoiensis). Based on the retrieved homologous strains, a tree of biological evolutionary relationships was constructed using the Mege5.1 program of DNAStar software as shown in FIG. 2.
The moderately halophilic bacteria strain is preserved in China general microbiological culture Collection center (CGMCC) of the institute of microbiology, China academy of sciences, institute of microbiology, No.1, Sai Lu, No. 3, located in the area of Chaoyang, Beijing for 27 months and 2018, the serial number is CGMCC NO.16542, and the suggested classification name is as follows: bacillus sphaericus Bacillus hwajinpoensis.
Example 1:
the method for fermenting the fish paste by using the moderately halophilic bacteria strain Bacillus tsukubaensis is carried out according to the following steps:
(1) treatment of raw materials: chopping the minced fish meat with a chopper mixer, adding 5% (w/w) of pickled sea salt, and placing in a refrigerator at 4 deg.C for 24h before fermentation.
(2) Preparation of moderately halophilic bacteria strain: the moderately halophilic bacteria strain Bacillus hwajinpoensis CGMCC16542 is inoculated on a culture medium for activated culture, and the operation is repeated three times. Centrifuging the cultured strain liquid at 4 deg.C and 10000r/min for 10min, washing with sterile normal saline twice, and suspending in sterile normal saline. Finally, adjusting the concentration of the bacterial liquid to 105～106CFU/mL, the bacterial solution was stored at 4 ℃ and used within 24 h.
(3) Addition of a starter: the final addition amount of the moderate halophilic bacteria strain Bacillus hwajinpoensis CGMCC16542 is 104CFU/g) is mixed into the pretreated raw fish to ensure that the final bacterial count meets the addition requirement
(4) Fermenting the fish paste: fermenting the mixed material obtained in the step (3) at the temperature of 28 ℃ for 30d in a heat preservation way; placing the fermented fish meat paste in a-20 refrigerator for later use.
(5) Stirring and ventilating in the fish meat sauce fermentation process: stirring the fermented fish paste clockwise every 5 days for 1min within 30 days of fish paste fermentation, and standing for ventilation for 10s after stirring.
(6) Cooking the fish meat paste: and (4) steaming the fish paste obtained in the step (4) in a steaming drawer for 20min by using water vapor until the fish paste is well cooked.
(7) Fish sauce sterilization: and (4) filling and sealing the fish paste cooked in the step (6), and performing secondary sterilization at 120 ℃ for 15min to obtain a finished fish paste product.
(8) And (3) evaluating the physical and chemical indexes of the product fermentation: the content of amino acid nitrogen is evaluated according to the acidity meter method of GB 5009.235-2016. The content of volatile basic nitrogen is evaluated by the method according to the GB5009.228-2016 microdiffusion method. The method for measuring the histamine content refers to GB5009.208-2016 spectrophotometry.
(9) Sensory evaluation: a sensory panel of 10 persons was evaluated and flavor training was performed on panellists before evaluation. The scores comprise 0-9 scores, and the overall flavor of the fish paste is comprehensively scored according to the fish flavor, fishy smell, fermented taste, ammonia taste, delicate flavor, salty taste, sweet taste and bitter taste.
(10) Physicochemical index and sensory evaluation of fish meat paste
The content of amino acid state in the fish paste fermented by the moderate halophilic bacteria strain is 0.639g/100 g; the content of volatile basic nitrogen is 24.87mg/100g, which is lower than 35mg/100g of European Union standard; the histamine content is 3.82mg/100g, which is far lower than the histamine content of 20mg/100g in the fish of national standard of food safety; the overall score for sensory evaluation was 6.33. The sensory score of the fish paste without added strain was 5.67. The raw materials of the fish meat paste have greatly reduced native fishy smell and obvious fresh taste, and compared with the fish paste without the added bacterial strain, the fish meat paste has obviously improved flavor.
Example 2:
the method for fermenting the fish paste by using the moderately halophilic bacteria strain Bacillus tsukubaensis is carried out according to the following steps:
(1) treatment of raw materials: chopping the minced fish meat with a chopper mixer, adding 20% (w/w) of pickled sea salt, and placing in a refrigerator at 4 deg.C for 24h before fermentation.
(2) Preparation of moderately halophilic bacteria strain: the moderately halophilic bacteria strain Bacillus hwajinpoensis CGMCC16542 is inoculated on a culture medium for activated culture, and the operation is repeated three times. Centrifuging the cultured strain liquid at 4 deg.C and 10000r/min for 10min, washing with sterile normal saline twice, and suspending in sterile normal saline. Finally, adjusting the concentration of the bacterial liquid to 105～106CFU/mL, the bacterial solution was stored at 4 ℃ and used within 24 h.
(3) Addition of a starter: the final addition amount of the moderate halophilic bacteria strain Bacillus hwajinpoensis CGMCC16542 is 107CFU/g) is mixed into the pretreated raw fish to ensure that the final bacterial count meets the addition requirement
(4) Fermenting the fish paste: fermenting the mixed material obtained in the step (3) at the temperature of 40 ℃ for 5d in a heat preservation way; placing the fermented fish meat paste in a-20 refrigerator for later use.
(5) Stirring and ventilating in the fish meat sauce fermentation process: and (4) stirring the fermented fish paste clockwise every 1d for 5min within 5d of fish paste fermentation, and standing for ventilation for 120s after stirring.
(6) Cooking the fish meat paste: and (4) steaming the fish paste obtained in the step (4) in a steaming drawer for 40min by using water vapor until the fish paste is well cooked.
(7) Fish sauce sterilization: and (4) filling and sealing the fish paste cooked in the step (6), and performing secondary sterilization at 120 ℃ for 25min to obtain a finished fish paste product.
(8) And (3) evaluating the physical and chemical indexes of the product fermentation: the content of amino acid nitrogen is evaluated according to the acidity meter method of GB 5009.235-2016. The content of volatile basic nitrogen is evaluated by the method according to the GB5009.228-2016 microdiffusion method. The method for measuring the histamine content refers to GB5009.208-2016 spectrophotometry.
(9) Sensory evaluation: a sensory panel of 10 persons was evaluated and flavor training was performed on panellists before evaluation. The scores comprise 0-9 scores, and the overall flavor of the fish paste is comprehensively scored according to the fish flavor, fishy smell, fermented taste, ammonia taste, delicate flavor, salty taste, sweet taste and bitter taste.
(10) Physicochemical index and sensory evaluation of fish meat paste
The content of amino acid state in the fish paste fermented by the moderate halophilic bacteria strain is 0.312g/100 g; the content of volatile basic nitrogen is 7.80mg/100g, which is lower than 35mg/100g of European Union standard; the histamine content is 1.98mg/100g, which is far lower than the histamine content of 20mg/100g in the fish of national standard of food safety; the overall score for sensory evaluation was 5.93. The sensory score of the fish paste without the added strain was 5.48. The raw materials of the fish meat paste have greatly reduced native fishy smell and obvious fresh taste, and compared with the fish paste without the added bacterial strain, the fish meat paste has obviously improved flavor.
Claims (2)
1. Moderate halophilic strain Zhanjintan bacillus (B.tsugae) (B.tsugae)Bacillus hwajinpoensis) It is characterized in that the preservation number is CGMCC NO. 16542.
2. A method for fermenting fish meat paste using the bacillus tsunobilis of claim 1, comprising the steps of:
(1) pretreatment of raw fish: chopping the minced fish meat by using a chopper mixer, adding 5-20% (w/w) of pickled sea salt, and placing in a refrigerator at 4 ℃ for 5-24 hours before fermentation;
(2) preparation of moderately halophilic bacteria strain: subjecting moderately halophilic bacteriaBacillus hwajinpoensisCGMCC No.16542 is inoculated on a culture medium for activated culture, and the operation is repeated for three times; centrifuging the cultured strain liquid at 4 deg.C and 10000r/min for 10min, washing with sterile normal saline twice, and suspending in sterile normal saline; finally, adjusting the concentration of the bacterial liquid to 105～106CFU/mL, storing the bacterial liquid at 4 ℃ and using the bacterial liquid within 24 h;
(3) addition of a starter: subjecting moderately halophilic bacteria strainBacillus hwajinpoensisMixing CGMCC number 16542 into pretreated raw fish, and adding strain at final addition of 104～107The CFU/g is measured, so that the final bacteria number can meet the addition requirement;
(4) fermenting the fish paste: fermenting the mixed material obtained in the step (3) at the temperature of 28-40 ℃ for 5-30 d in a heat preservation manner; placing the fermented fish meat paste in a-20 refrigerator for later use;
(5) stirring and ventilating in the fish meat sauce fermentation process: stirring the fermented fish paste clockwise for 1-5 min every 1-5 days within 5-30 days of fish paste fermentation, and standing for ventilation for 10-120 s after stirring is finished;
(6) cooking the fish meat paste: steaming the fish paste obtained in the step (4) in a steaming drawer for 20-40 min by using water vapor until the fish paste is well cooked;
(7) fish sauce sterilization: and (4) filling and sealing the fish paste cooked in the step (6), and performing secondary sterilization at 120 ℃ for 15-20 min to obtain a fish paste finished product.
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| CN201811452816.2A CN109456922B (en) | 2018-11-30 | 2018-11-30 | Moderately halophilic bacteria strain Zhan beach bacillus for improving fermentation quality of fish paste |
| PCT/CN2019/080282 WO2020107760A1 (en) | 2018-11-30 | 2019-03-29 | Moderately halophilic bacteria and method for fermenting fish meat sauce by using same |
| US16/761,132 US11974590B2 (en) | 2018-11-30 | 2019-03-29 | Method of fermentation of fish paste by medium halophilic bacteria |
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