CN111924981B - Application of amylolytic halococcus HL-6 for efficiently removing nitrite - Google Patents
Application of amylolytic halococcus HL-6 for efficiently removing nitrite Download PDFInfo
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Abstract
The invention belongs to the technical field of biological and aquatic product processing, and particularly relates to an application of a strain of deinococcus amyloliquefaciens HL-6 for efficiently removing nitrite, wherein the preservation number of the deinococcus amyloliquefaciens HL-6 is GDMCC No: 60715. the invention screens out a strain of amylolytic halococcus (a) for removing nitrite for the first timeSalinicoccus amylolyticus) The strain has salt tolerance, has good removal effect on nitrite with high concentration and low concentration, and particularly can still remove more than 90% of nitrite under the condition of high salt.
Description
Technical Field
The invention belongs to the technical field of biological and aquatic product processing, and particularly relates to application of a strain of amylolytic halococcus HL-6 for efficiently removing nitrite.
Background
Nitrite is a precursor substance for nitrosamine synthesis. Numerous animal experiments have confirmed that nitrosamines are strong carcinogens and can trigger subsequent tumors via placenta and breast milk. Nitrosamines also cause deformities and mutations. Epidemiological investigations have shown that certain cancers in humans, such as gastric, esophageal, liver, colon and bladder cancers, are associated with the intake of nitrosamines. Thus, the nitrite in the food can pose a great safety hazard.
The fish sauce, also called fish sauce, has rich nutrition and delicious taste, and is a traditional aquatic product fermentation condiment which is deeply loved by residents in various countries in south east Asia and coastal areas of China. The fish sauce is usually prepared by immediately salting the small sea fish after being caught or adding salt for salting after being kept on the shore, so that nitrite is inevitably brought into raw materials and sea water salt which are not cleaned, and a part of the nitrite is also generated in the open fermentation process. Nitrite exists in fish sauce produced by traditional fermentation, but the content is different, and the limit of the nitrite is not specified in the current Chinese fish sauce product standard (SB/T10324-1999). River body fluid and the like detect 16 commercial fish gravy sold in Guangzhou and fish gravy fermentation liquid of certain enterprises, wherein the production places of the fish gravy are Vietnam, hong Kong, Thailand, Shantou and Fujian respectively, and the nitrite content is 0.72-18.97 mg/kg and is less than the nitrite standard limit value (20mg/kg) of pickled vegetables. However, for people taking fish sauce as a seasoning in coastal areas of China, the fish sauce has a high eating proportion in daily diet, and the potential safety hazard caused by nitrous acid is not negligible. It has been reported that most people suffer from nasopharyngeal carcinoma and gastric cancer in southern coastal areas because fish gravy is eaten for a long time. Therefore, for the fish gravy, the removal of the nitrite is of great significance.
At present, various methods for reducing the content of nitrite have been developed at home and abroad, wherein the microbial fermentation is the most researched method with the best effect and the safest method for reducing the nitrite. At present, most of nitrite removal methods reported are lactic acid bacteria. However, the existing lactic acid bacteria have poor salt tolerance, and the nitrite is difficult to remove from the high-salt food such as fish gravy.
Disclosure of Invention
The invention aims to provide application of enterococcus amyloliquefaciens (Salinicoccus amylolyticus) HL-6 in removing nitrite, wherein the deposited number of the enterococcus amyloliquefaciens is as follows: GDMCC NO: 60715.
in order to achieve the purpose, the invention adopts the following technical measures:
the applicant selects a bacterium which can efficiently degrade nitrite from fish sauce, the bacterium is identified as a deinococcus amyloliquefaciens through biological morphology and molecular identification, the bacterium is delivered to Guangdong province microbial strain preservation center for preservation in 7-5.7.2019, and the preservation numbers are as follows: GDMCC NO: 60715, sort name: salinicoccus amylolyticus HL-6, address: the institute of microorganisms, Guangdong province, Guangzhou, China.
The colony morphology of the strain Salinicoccus amylolyticus HL-6 cultured in a nutrient agar culture medium with the sodium chloride content of 4% at 25 ℃ for 72 hours is shown in figure 1, the colony is circular, pink to red, the edge is neat, and the individual morphology is spherical.
The application of the enterococcus amyloliquefaciens HL-6 in removing nitrite comprises preparing the bacterial agent into a nitrite remover of a water-produced fermentation product, or directly using the bacterial agent in the treatment of the nitrite in a water body, particularly the nitrite treatment in a high-salt environment;
in the above application, preferably, the aquatic product is fish sauce, shrimp paste, pickled fish or crab sauce;
in the above-described application, it is preferable that the content of sodium chloride to be removed in the application is not more than 35%.
Compared with the prior art, the invention has the following advantages:
the invention screens out a strain of starch degrading brine coccus (Salinicoccus amylolyticus) for nitrite removal for the first time, the strain has salt tolerance, has good removal effect on nitrite with high concentration and low concentration, and particularly can still achieve more than 90% of nitrite removal under the high-salt condition.
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FIG. 1 is a diagram showing colony morphology and individual morphology of enterococcus amyloliquefaciens (Salinicoccus amylolyticus) HL-6.
FIG. 2 is a graph showing the effect of sodium chloride content and temperature on the growth of enterococcus faecium HL-6 for starch decomposition;
wherein A is the influence of the sodium chloride content on the growth of the amylolytic halococcus HL-6;
and B is the influence of temperature on the growth of the starch-decomposing halococcus HL-6.
Detailed Description
The technical solutions of the present invention, if not specifically mentioned, are conventional in the art, and the reagents or materials, if not specifically mentioned, are commercially available.
Example 1:
isolation and characterization of enterococcus amyloliquefaciens (Salinicoccus amylolyticus) HL-6:
the enterococcus faecalis (Salinicoccus amylolyticus) HL-6 is screened and separated from fish gravy, the colony morphology of the bacterial strain HL-6 is shown in figure 1, the colony is circular, pink to red, the edge is neat, and the individual morphology is spherical.
Identification of isolated Strain HL-6 bacterial colony Polymerase chain reaction (Polymerase chain reaction PCR) amplification was performed using a Mighty Amp DNA Polymerase using 27F and 1492R bacterial universal primers as forward and reverse primers. PCR amplification System: 27F 1.5. mu.L, 1492R 1.5. mu.L, MightyAmp DNA Polymerase 1.5. mu.L, 2 XMightyAmp Buffer 30. mu.L, ddH2O25.5 μ L, reaction conditions: the process cycles from 98 ℃ (pre-denaturation) 2min, 98 ℃ (denaturation) 10S, 55 ℃ (renaturation) 15S, 68 ℃ (extension) 90S, 72 ℃ for 10min, and from denaturation to first extension 40 times, the PCR products are sent to the Shanghai industries for sequencing, the sequencing results are subjected to homology retrieval from GenBank by using BLAST search program, and 16S rDNA gene sequences of model strains with high similarity are downloaded for analysis.According to the sequencing result of the strain HL-6, 16S rDNA database BLAST is carried out through NCBI, and the similarity of the strain HL-6 and a standard strain Salinicoccus amylolytic JC304 is the highest and is as high as 99.58%. Selecting partial strains with the sequence similarity of 99%, manufacturing an phylogenetic tree by adopting a software Mega5.05 and a Maximum likelihood method, enabling the strain HL-6 and the Salinicoccus amylolyticus JC304 to belong to the same branch system on the phylogenetic tree, and identifying the strain HL-6 as the deinococcus amyloliquefaciens. The strain is sent to Guangdong province microbial strain collection center for preservation in 2019, 7 and 5 days, and is classified and named: salinicoccus amylolyticus HL-6 with the preservation number: GDMCC NO: 60715, address: the institute of microorganisms, Guangdong province, Guangzhou, China.
Example 2:
effect of sodium chloride content and temperature on growth of enterococcus faecium for starch decomposition:
1) inoculating amylolytic halococcus HL-6 into nutrient broth, and culturing at 25 deg.C and 120r/min for 24h to obtain amylolytic halococcus seed solution. The seed culture solution was inoculated into liquid nutrient broth culture media of different salinity at an inoculum size of 5%, respectively, with sodium chloride content set at a gradient of 4% (in 4% for example, 1L of the medium containing 40g of sodium chloride), 6%, 9%, 12%, 15%, 18%, 21%, 24% and 27%, and cultured in a 96-well plate at 25 ℃ for 48 hours, and absorbance was measured at a wavelength of 600nm every 4 hours. As shown in FIG. 2A, it can be seen that the enterococcus amyloliquefaciens has good salt tolerance, can tolerate 27% of sodium chloride, and can grow slowly when the sodium chloride content is as high as 27%.
2) Inoculating 5% inoculum size culture solution of amylolytic enterococcus salina HL-6 seed into liquid culture medium containing 4% sodium chloride in nutrient broth, culturing with 96-well plate, respectively culturing at different temperatures for 48h, measuring absorbance at wavelength of 600nm every 4h, and setting temperature gradient at 20 deg.C, 25 deg.C, 30 deg.C and 37 deg.C. As shown in FIG. 2B, it is understood from the results shown in FIG. 2 that the number of viable bacteria in the amylolytic enterococcus strain is smaller than that in the case of culturing at 37 ℃ for 16 hours in the stationary phase, but is smaller than that in the case of culturing at 25 ℃ in the stationary phase, indicating that the growth of the amylolytic enterococcus strain is fast at a high temperature, but the number of viable bacteria in the case of culturing at the stationary phase is low; when the culture temperature is 20 ℃, a stationary phase appears in 36h, but the viable count is still less than that of the viable count cultured at 25 ℃, which indicates that when the temperature is too low, the growth of the amylolytic halococcus is not facilitated.
Example 3:
the application of amylolytic halococcus HL-6 in degrading nitrite in different sodium chloride contents is as follows:
inoculating amylolytic enterococcus salina HL-6 into 4% sodium chloride nutrient broth, culturing at 25 deg.C and 120r/min for 32h, centrifuging at 5000r/min for 10min after fermentation is finished, and collecting thallus. Preparing nutrient broth culture medium containing sodium chloride 15%, 18%, 21%, 24%, 27%, 30% and 35%, adding sodium nitrite to make sodium nitrite concentration be 20mg/L, and inoculating thallus of collected amylolytic halococcus HL-6 to make amylolytic halococcus final concentration be 107CFU/mL, and placing the culture medium after inoculation at 37 ℃ for culturing for 16h at 120 r/min. And (4) measuring the content of nitrite in the culture medium after the fermentation is finished. Nitrite was measured according to the naphthyl ethylenediamine hydrochloride method described in GB/T5009.33-2010 for nitrite and nitrate determination in foods, and the results are shown in the following table. As can be seen from the results in the following table, the concentration of the enterococcus faecalis was 107In CFU/mL, the amylolytic enterococcus salina HL-6 still has a good nitrite removal effect under high-concentration salinity, and the nitrite removal effect is not greatly influenced along with the increase of the sodium chloride content.
TABLE 1 Effect of different sodium chloride contents on nitrite removal by Amylolysis of Hydrococcus halophilus
Example 4:
application of amylolytic enterococcus salina HL-6 in nitrate reduction of fish gravy
Inoculating amylolytic enterococcus salina HL-6 into 4% sodium chloride nutrient broth, culturing at 25 deg.C and 120r/min for 32h, centrifuging at 5000r/min for 10min after fermentation is finished, and collecting thallus. Taking commercially available fish sauce (sodium chloride content is 27%, nitrite content is5.82mg/L) are divided into 5 parts, sodium nitrite is respectively added to ensure that the final concentration of the exogenously added sodium nitrite is respectively 0, 20, 40, 60 and 80mg/L, and then the collected thalli of the deinocstarch-dissolving halococcus HL-6 are inoculated to ensure that the final concentration of the deinocstarch-dissolving halococcus HL-6 is 107CFU/mL, and culturing the fish sauce inoculated with the deinococcus amyloliquefaciens at 37 ℃ at 120r/min for 16 h.
The nitrite content of the fish gravy was measured after the end of the fermentation and the results are shown in the table below. As can be seen from the results in the following table, the amylolytic enterococcus hirae HL-6 has good capability of removing nitrite in the fish sauce, and the content of nitrite is reduced from 5.82mg/L to 0.042mg/L through the fermentation of microorganisms in the fish sauce without exogenous nitrite, and the nitrite removal rate reaches 99.3%; even when the nitrite concentration in the fish gravy reaches 85.82mg/L by adding the exogenous nitrite, the nitrite removal rate can still reach 92.4%.
TABLE 2 removal of sodium nitrite in fish gravy by amylolytic enterococcus sp
Claims (4)
1. Amylolytic enterococcus salina (Salinicoccus amylolyticus) The application of the salt water coccus amyloliquefaciens in removing nitrite is disclosed in the specification, wherein the code of the salt water coccus amyloliquefaciens is GDMCC No: 60715.
2. salmonella amylovora(Salinicoccus amylolyticus)The application of the nitrite remover in preparing aquatic fermentation products, wherein the preservation number of the deinococcus amyloliquefaciens is GDMCC No: 60715.
3. amylolytic enterococcus salina (Salinicoccus amylolyticus) The application of the strain in treating nitrite in water body, wherein the preservation number of the deinococcus amyloliquefaciens is GDMCC No: 60715, the content of sodium chloride in the water body is less than or equal to 35%.
4. The use according to claim 2, wherein the marine fermentation product is one of fish sauce, shrimp paste, pickled fish or crab sauce.
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| CN109439602A (en) * | 2018-12-29 | 2019-03-08 | 中蓝连海设计研究院有限公司 | Halophilic vibrio YL5-2 and its microbial inoculum are degraded the application in conversion pollutant under high salt conditions |
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| CN109439602A (en) * | 2018-12-29 | 2019-03-08 | 中蓝连海设计研究院有限公司 | Halophilic vibrio YL5-2 and its microbial inoculum are degraded the application in conversion pollutant under high salt conditions |
Non-Patent Citations (6)
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| "Culturable halophilic bacteria inhabiting Algerian saline ecosystems: A source of promising features and potentialities";Taha Menasria1 et al.;《World Journal of Microbiology and Biotechnology 》;20190820;第132页 * |
| "Isolation and initial characterization of the tellurite reducing moderately halophilic bacterium, Salinicoccus sp. strain QW6";Mohammad AliAmoozegar et al.;《Microbiological Research》;20080715;第163卷(第4期);第456-465页 * |
| "Salinicoccus halodurans sp nov., a moderate halophile from saline soil in China";Wang, XW et al.;《International Journal of Systematic and Evolutionary Microbiology》;20080731;第58卷;第1537-1541页 * |
| "Salinicoccus kunmingensis sp nov., a moderately halophilic bacterium isolated from a salt mine in Yunnan, south-west China";Chen, YG et al.;《International Journal of Systematic and Evolutionary Microbiology》;20071031;第57卷;第2327-2332页 * |
| "蜢虾酱中细菌的分离鉴定及其耐盐性研究";李可心等;《中国食品科学技术学会第十五届年会论文摘要集》;20181107;第138-139页 * |
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