CN109735461B - Lactobacillus plantarum and application thereof in reducing biogenic amine content of fish tea - Google Patents
Lactobacillus plantarum and application thereof in reducing biogenic amine content of fish tea Download PDFInfo
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Abstract
The invention discloses a Lactobacillus plantarum (Lactobacillus plantarum) Yc-2 with the preservation number CGMCC NO.16614; the method for reducing the biogenic amine content of fish tea by using the lactobacillus plantarum Yc-2 comprises the following steps: to a concentration of 10 6 ～10 8 The bacterial liquid of the Lactobacillus plantarum Yc-2 of CFU/mL is used as a leaven, and is inoculated into a fermentation system according to 1-4% of the weight of the raw material fish for fermentation. By inoculating the lactobacillus plantarum Yc-2, biogenic amine in a fish tea system is well inhibited, the degradation rate of the biogenic amine content in the fish tea can reach more than 95%, and particularly the degradation rate of histamine can reach more than 91%.
Description
Technical Field
The invention relates to the technical field of microbial leavening agents, in particular to lactobacillus plantarum capable of degrading biogenic amine and application of lactobacillus plantarum in reducing biogenic amine content of fish tea.
Background
The fish tea, also called as fish acid, is a flavor fermented food generally made by the Li Miao nationality. The fish tea is prepared from fresh fish and cooked carbohydrate, and is fermented by microorganism to obtain fish processed product with strong taste, rich nutrition and stable quality. In the natural fermentation process, the microbial population is complex, and partial microorganisms can catalyze decarboxylation of free amino acid to generate biogenic amine harmful to human bodies, so that potential safety hazards are brought to the foods. The human health is not influenced by a small amount of biogenic amine, but when the biogenic amine is externally taken by a human body, the biogenic amine can cause severe reactions such as headache, blood pressure rise, damage to the nervous system and the cardiovascular system, diarrhea, vomiting and the like. Histamine is the most toxic of biogenic amines, tyramine; putrescine and cadaverine are less toxic by themselves, but can potentiate the toxicity of histamine and tyramine. The US FDA regulates that histamine levels in aquatic products not exceed 50mg/kg and recommends that tyramine levels not exceed 100mg/kg and that the total biogenic amine levels not exceed 1000mg/kg. Biogenic amines are thermally stable and, once produced, are difficult to remove during cooking; therefore, controlling the accumulation of biogenic amines during fermentation is very important for food safety.
Due to the particularity of the food production process, it is generally difficult to control biogenic amines by changing physical parameters of the production process; the treatment of the product by physical or chemical addition may result in loss of nutrients, change of flavor, and doubt of safety. The reasonable selection of the leaven, especially the microorganism which has the ability of degrading biogenic amine and does not have potential safety hazard, can effectively control biogenic amine accumulation in food.
Lactic acid bacteria, which are a general term for a group of bacteria that ferment carbohydrates and produce lactic acid, are important leavening agents in the food industry. The lactobacillus has the advantages of not damaging food nutrition and improving product flavor; meanwhile, the antibacterial agent has an antibacterial effect, can acidify the environment and secrete antibacterial substances, and further effectively inhibits some pathogenic bacteria and putrefying bacteria. In addition, most lactic acid bacteria are safe, healthy and non-toxic.
Disclosure of Invention
The invention aims to provide a method for reducing biogenic amine in a fish tea product by using lactobacillus plantarum.
The invention is realized by the following technical scheme:
in order to achieve the aim, the invention provides a Lactobacillus plantarum Yc-2 with the preservation number of CGMCC NO.16614, which is preserved in China general microbiological culture Collection center (CGMCC) in 2018, 10 months and 24 days, and the address is as follows: the microbial research institute of western road 1, 3, national academy of sciences, north-south, morning-yang, beijing, zip code: 10010. the lactobacillus plantarum is separated from fermented food, 16S rDNA sequence identification is carried out by adopting a bacterial universal primer 27F/1492R, and the lactobacillus plantarum is lactobacillus plantarum.
The invention also provides a method for reducing the biogenic amine content of fish tea by using the lactobacillus plantarum, which comprises the following steps: to a concentration of 10 6 ～10 8 Taking the Lactobacillus plantarum bacterial liquid of CFU/mL as a leaven, inoculating the Lactobacillus plantarum bacterial liquid into a fermentation system according to 1-4% of the weight of the raw material fish, and performing synchronous fermentation; wherein the lactobacillus plantarum is lactobacillus plantarum Yc-2 with the preservation number of CGMCC NO.16614.
In a preferred mode, the method for reducing the content of biogenic amine in fish tea by using lactobacillus plantarum comprises the following steps:
s1, raw material pretreatment: taking raw material fish, cutting into fish blocks, placing the fish blocks in saturated salt solution, soaking the fish blocks for 1 to 3 hours at the temperature of between 4 and 15 ℃, and draining the fish blocks for later use;
s2, making fish tea: mixing the fish blocks, rice and 10 obtained in step S1 6 ～10 8 Uniformly mixing the bacterial liquid of the Lactobacillus plantarum Yc-2 with the CFU/mL, placing the mixture in a container, and hermetically placing the container for 32 to 37 days at the temperature of between 20 and 25 ℃ to obtain the fish tea; the collection number of the lactobacillus plantarum Yc-2 is CGMCC NO.16614;
wherein the adding weight ratio of the fish blocks, the rice and the lactobacillus plantarum Yc-2 bacterial liquid is 1 (1-4) to 0.01-0.04.
Preferably, the fish block size in step S1 is: 2cm × 2cm × 2cm; the raw material fish is grass carp, carp or bighead carp.
Preferably, the container in the step S2 is used after being sterilized at 121 ℃ for 20min, and the rice is obtained by mixing rice and water according to a weight ratio of 1; the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into an MRS broth culture medium, and carrying out oxygen consumption and static culture at 37 ℃ for 12 hours; centrifugally collecting thalli, and diluting to 10% with 0.85% normal saline 6 ～10 8 CFU/mL；
Wherein the MRS agar medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14.0g/L of agar and 1.0g/L of Tween 80, wherein the pH value at 25 ℃ is 6.5 +/-0.2;
the MRS broth culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 1.0g/L of Tween-80, and the pH value at 5 ℃ is 5.7 +/-0.2.
In a preferred mode, the method for reducing the biogenic amine content of the fish tea by using the lactobacillus plantarum comprises the following steps:
s1, raw material pretreatment:
removing head, tail, fish bones and viscera from grass carp, and cutting fish into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt water at 4 ℃ for 2h, and draining for later use;
s2, making fish tea:
cleaning the container, and sterilizing at 121 ℃ for 20min; collecting 240g cooked rice, 60g fish fillet obtained in step S1 and 0.6g 10 8 Uniformly mixing the CFU/mL lactobacillus plantarum Yc-2 bacterial liquid, placing the mixture in the container, sealing and fermenting for 35 days at the temperature of 20 ℃ to obtain fish tea;
wherein the rice is prepared by heating rice and water at a weight ratio of 1.5 for 30min at 100 ℃; the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into 3ml of MRS broth culture medium, and carrying out oxygen consumption and static culture at 37 ℃ for 12 hours; centrifuging at 10000r/min for 10min, collecting thallus, and chlorinatingDiluting with 0.85% sodium water solution to obtain 10 8 CFU/mL of bacterial suspension.
The invention has the beneficial effects that:
the lactobacillus plantarum Yc-2 provided by the invention can be used for reducing the content of biogenic amine in fermented fish products, especially fish tea products, and has a very wide application prospect.
Detailed Description
The invention is further illustrated by the following specific examples.
The invention provides a Lactobacillus plantarum (Lactobacillus plantarum) Yc-2 with the preservation number of CGMCC NO.16614, which is preserved in China general microbiological culture Collection center (CGMCC) in 24 months and 10 months in 2018, and the address is as follows: the microbial research institute of western road 1, 3, national academy of sciences, north-south, morning-yang, beijing, zip code: 10010.
the lactobacillus plantarum is separated from fermented food, 16SrDNA sequence identification is carried out by adopting a bacterial universal primer 27F/1492R, and the lactobacillus plantarum is obtained.
The lactobacillus plantarum preservation method comprises the following steps: well-grown single colonies were selected from MRS agar medium plates, inoculated into 3mL of MRS broth medium, cultured at 37 ℃ under oxygen-consuming and static conditions for 24 hours, and 1mL of the culture medium was added to a cryopreservation tube containing 1mL of 30% glycerol and stored in a refrigerator at-80 ℃.
The culture method of the lactobacillus plantarum comprises the following steps: the preserved lactobacillus plantarum is streaked on an MRS agar culture medium plate, subjected to oxygen consumption and static culture at 37 ℃ for 24 hours, and a single colony is picked and inoculated into an MRS broth culture medium, and subjected to oxygen consumption and static culture at 37 ℃ for 12 hours.
The MRS agar culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14.0g/L of agar and 1.0g/L of Tween 80, and the pH value at 25 ℃ is 6.5 +/-0.2.
The MRS broth culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef extract powder, 4.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate and 1.0g/L of Tween-80, and the pH value at 25 ℃ is 5.7 +/-0.2.
Example 1:
the method for reducing the biogenic amine content of fish tea by using lactobacillus plantarum Yc-2 (CGMCC No. 16614) comprises the following steps:
s1, raw material pretreatment:
removing head, tail, fish bones and viscera from fresh grass carp, and cutting fish into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt water at 4 ℃ for 2h, and draining for later use;
washing rice, wherein the weight ratio of the rice to water is 1.5;
s2, making fish tea:
cleaning the container, and sterilizing at 121 deg.C for 20min; collecting 240g cooked rice, 60g fish fillet obtained in step S1 and 0.6g 10 8 Uniformly mixing the CFU/mL lactobacillus plantarum Yc-2 bacterial liquid, placing the mixture in the container, sealing and fermenting for 35 days at the temperature of 20 ℃ to obtain fish tea;
the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into 3ml of MRS broth culture medium, and carrying out oxygen consumption and static culture at 37 ℃ for 12 hours; centrifuging at 10000r/min for 10min, collecting thallus, and preparing thallus with sterile normal saline (sodium chloride 0.85% water solution, w/v, g/mL) to 10 8 CFU/mL of bacterial suspension.
The fish tea obtained in the example was taken, and the contents of histamine, tyramine, putrescine, cadaverine, tryptamine and phenethylamine in the fish meat were detected by dansyl chloride pre-column derivatization-high performance liquid chromatography, and the detection results are shown in table 1.
Example 2:
the method for reducing the biogenic amine content of the fish tea by using the lactobacillus plantarum Yc-2 (CGMCC No. 16614) comprises the following steps:
s1, raw material pretreatment:
removing head, tail, fish bone and viscera from fresh Cyprinus Carpio, and cutting into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt water at 10 ℃ for 3h, and draining for later use;
washing rice, wherein the weight ratio of the rice to water is 1.5;
s2, making fish tea:
cleaning the container, and sterilizing at 121 deg.C for 20min; 120g of rice, 120g of fish blocks obtained in the step S1 and 2.4g of 10 are taken 7 Uniformly mixing the bacterial liquid of the Lactobacillus plantarum Yc-2 in a ratio of CFU/mL, placing the mixture in the container, sealing the container at 25 ℃, and fermenting for 37 days to obtain the fish tea;
the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture for 24 hours at 37 ℃, selecting a single colony, inoculating the single colony into 3ml of MRS broth culture medium, and carrying out oxygen consumption and static culture for 12 hours at 37 ℃; centrifuging at 10000r/min for 10min, collecting thallus, and preparing thallus with 2mL sterile normal saline (sodium chloride 0.85% water solution, w/v, g/mL) to 10 7 CFU/mL of bacterial fluid.
The fish tea obtained in the example was taken, and the contents of histamine, tyramine, putrescine, cadaverine, tryptamine and phenethylamine in the fish meat were detected by dansyl chloride pre-column derivatization-high performance liquid chromatography, and the detection results are shown in table 1.
Example 3:
the method for reducing the biogenic amine content of fish tea by using lactobacillus plantarum Yc-2 (CGMCC No. 16614) comprises the following steps:
s1, raw material pretreatment:
removing head, tail, fish bone and viscera from fresh Aristichthys nobilis, and cutting into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt solution at 15 deg.C for 1h, and draining;
washing rice, wherein the weight ratio of the rice to water is 1.5, heating at 100 ℃ for 30min to obtain rice, and cooling for later use;
s2, making fish tea:
cleaning the container, and sterilizing at 121 deg.C for 20min; 200g of rice, 100g of fish blocks obtained in the step S1 and 4g of 10 are taken 6 Uniformly mixing the bacterial liquid of the Lactobacillus plantarum Yc-2 of CFU/mL, and placing the mixture in the containerSealing and fermenting at 22 deg.C for 32 days to obtain fish tea;
the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into 3ml of MRS broth culture medium, and carrying out oxygen consumption and static culture at 37 ℃ for 12 hours; centrifuging at 10000r/min for 10min, collecting thallus, and preparing thallus into 10 with 2mL sterile physiological saline (sodium chloride 0.85% water solution, w/v, g/mL) 6 CFU/mL of bacterial fluid.
The fish tea obtained in the example was taken, and the contents of histamine, tyramine, putrescine, cadaverine, tryptamine and phenethylamine in the fish meat were detected by dansyl chloride pre-column derivatization-high performance liquid chromatography, and the detection results are shown in table 1.
Comparative example 1
The preparation of the fish tea product by natural fermentation comprises the following steps:
s1, raw material pretreatment:
removing head, tail, fish bone and viscera from fresh grass carp, and cutting fish meat into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt solution at 4 deg.C for 2 hr, and draining;
washing rice, wherein the weight ratio of the rice to water is 1.5, heating at 100 ℃ for 30min to obtain rice, and cooling for later use;
s2, making fish tea:
cleaning the container, and sterilizing at 121 deg.C for 20min; and (3) uniformly mixing 240g of rice and 60g of the fish blocks obtained in the step (S1), placing the mixture in the container, sealing the container at 22 ℃, and fermenting for 35 days to obtain the fish tea.
Taking the fish tea obtained in the comparative example, detecting the contents of histamine, tyramine, putrescine, cadaverine, tryptamine and phenethylamine in the fish meat by dansyl chloride pre-column derivatization-high performance liquid chromatography, wherein the detection results are shown in table 1.
Comparative example 2
A method for reducing the biogenic amine content of fish tea by using Lactobacillus plantarum PL-2 (CGMCC No. 13919) comprises the following steps:
the lactobacillus plantarum PL-2 is separated from pickle and purchased from 'China general microbiological culture Collection center', and has the preservation number as follows: CGMCC 13919;
s1, raw material pretreatment:
removing head, tail, fish bone and viscera from fresh grass carp, and cutting fish meat into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt solution at 4 deg.C for 2 hr, and draining;
washing rice, wherein the weight ratio of the rice to water is 1.5;
s2, making fish tea:
cleaning the container, and sterilizing at 121 deg.C for 20min; collecting 240g cooked rice, 60g fish fillet obtained in step S1 and 0.6g 10 8 Uniformly mixing the CFU/mL lactobacillus plantarum PL-2 bacterial liquid, placing the mixture in the container, sealing and fermenting for 35 days at 20 ℃ to obtain fish tea;
the preparation method of the lactobacillus plantarum PL-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum PL-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into 3ml of MRS broth culture medium, and carrying out oxygen consumption and static culture at 37 ℃ for 12 hours; centrifuging at 10000r/min for 10min, collecting thallus, and preparing thallus into 10 with 2mL sterile physiological saline (sodium chloride 0.85% water solution, w/v, g/mL) 8 CFU/mL of bacterial suspension.
Taking the fish tea obtained in the comparative example, detecting the contents of histamine, tyramine, putrescine, cadaverine, tryptamine and phenethylamine in the fish meat by dansyl chloride pre-column derivatization-high performance liquid chromatography, wherein the detection results are shown in table 1.
The influence of Lactobacillus plantarum Yc-2 (CGMCC No. 16614) on the biogenic amine content in fish tea is shown in Table 1:
TABLE 1 influence of Lactobacillus plantarum Yc-2 (CGMCC No. 16614) on biogenic amine content in fish tea
Experimental results show that biogenic amine in a fish tea system is well inhibited by inoculating lactobacillus plantarum Yc-2 (CGMCC No. 16614), the degradation rates of the total contents of six biogenic amines in examples 1, 2 and 3 are respectively 96.94%, 96.15% and 95.66%, and particularly the degradation rates of histamine are respectively 95.25%, 94.09% and 91.23%; compared with the comparative example 2, the lactobacillus plantarum Yc-2 (CGMCC No. 16614) has better degradation efficiency on biogenic amine, so the strain can be used as a leavening agent of a low biogenic amine fish tea product.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (9)
1. A Lactobacillus plantarum (Lactobacillus plantarum) Yc-2 is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC NO.16614.
2. A method for reducing the biogenic amine content of fish tea by using lactobacillus plantarum is characterized by comprising the following steps: to a concentration of 10 6 ～10 8 The bacterial liquid of the Lactobacillus plantarum Yc-2 with CFU/mL is used as a leaven, and is inoculated into a fermentation system according to 1-4% of the weight of the raw material fish for fermentation; wherein the collection number of the lactobacillus plantarum Yc-2 is CGMCC NO.16614.
3. The method for reducing the biogenic amine content of fish tea by using lactobacillus plantarum as claimed in claim 2, which comprises the steps of:
s1, raw material pretreatment: taking raw material fish, cutting into fish blocks, placing the fish blocks in saturated salt solution, soaking the fish blocks for 1 to 3 hours at the temperature of between 4 and 15 ℃, and draining the fish blocks for later use;
s2, making fish tea: mixing the fish blocks, rice and 10 obtained in step S1 6 ～10 8 Mixing the bacterial liquid of the Lactobacillus plantarum Yc-2 with the CFU/mL bacterial liquid, placing the mixture in a container, and sealing and placing the container for 32-37 days at the temperature of 20-25 ℃ to obtain the fish tea; the collection number of the lactobacillus plantarum Yc-2 is CGMCC NO.16614;
wherein the adding weight ratio of the fish blocks, the rice and the lactobacillus plantarum Yc-2 bacterial liquid is 1 (1-4) to 0.01-0.04.
4. The method for reducing the biogenic amine content of fish tea by using lactobacillus plantarum as claimed in claim 3, wherein the fish fillet size in step S1 is: 2cm by 2cm.
5. The method for reducing the content of biogenic amine in fish tea by using lactobacillus plantarum as claimed in claim 3, wherein the raw material fish in step S1 is grass carp, carp or bighead carp.
6. The method for reducing the biogenic amine content in fish tea by using lactobacillus plantarum as claimed in claim 3, wherein the container in step S2 is used after being sterilized at 121 ℃ for 20 min.
7. The method for reducing biogenic amine content in fish tea by using lactobacillus plantarum as claimed in claim 3, wherein the cooked rice in step S2 is prepared by mixing rice and water in a weight ratio of 1.5, and heating at 100 ℃ for 30 min.
8. The method for reducing the content of biogenic amine in fish tea by using lactobacillus plantarum according to claim 3, wherein the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid in the step S2 comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, consuming oxygen at 37 ℃, and performing static culture for 24 hours; selecting single colony, inoculating to MRS broth culture medium, culturing at 37 deg.C with oxygen consumption and standing for 12 hr; centrifugally collecting thalli, diluting the thalli to 10 percent by using normal saline with the mass fraction of 0.85 percent 6 ～10 8 CFU/mL。
9. The method for reducing the biogenic amine content of fish tea by using lactobacillus plantarum as claimed in claim 3, comprising the steps of:
s1, raw material pretreatment: taking grass carp, removing head, tail, fish bones and viscera, and cutting fish meat into small pieces of 2cm × 2cm × 2cm; soaking the fish blocks in saturated salt water at 4 ℃ for 2h, and draining for later use;
s2, making fish tea: cleaning the container, and sterilizing at 121 deg.C for 20min; collecting 240g cooked rice, 60g fish fillet obtained in step S1 and 0.6g 10 8 Uniformly mixing the bacterial liquid of the Lactobacillus plantarum Yc-2 in a ratio of CFU/mL, placing the mixture in the container, sealing and fermenting the mixture for 35 days at the temperature of 20 ℃ to obtain the fish tea;
wherein the rice is prepared by heating rice and water at a weight ratio of 1.5 for 30min at 100 ℃; the preparation method of the lactobacillus plantarum Yc-2 bacterial liquid comprises the following steps: streaking lactobacillus plantarum Yc-2 on an MRS agar culture medium plate, carrying out oxygen consumption and static culture at 37 ℃ for 24 hours, selecting a single colony, inoculating the single colony into 3ml of MRS broth culture medium, and carrying out oxygen consumption and static culture at 37 ℃ for 12 hours; centrifuging at 10000r/min for 10min, collecting thallus, diluting with 0.85% sodium chloride water solution, and making into 10 8 CFU/mL of bacterial suspension.
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