CN1687408A - Method for producing nitrite reductase by ferment of lactic acid bacteria - Google Patents
Method for producing nitrite reductase by ferment of lactic acid bacteria Download PDFInfo
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- CN1687408A CN1687408A CN 200510073389 CN200510073389A CN1687408A CN 1687408 A CN1687408 A CN 1687408A CN 200510073389 CN200510073389 CN 200510073389 CN 200510073389 A CN200510073389 A CN 200510073389A CN 1687408 A CN1687408 A CN 1687408A
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Abstract
The invention provide a method for producing nitrite reductase by ferment of lactic acid bacteria, activating lactic acid bacteria, enlarge breeding gradually, preparation liquid seeds, according to ferment liquid 3%-8% put it into the liquid zymogenic culture medium, in the temperature of 26-32 Deg. C breeding 36-72h, adding 40-150mg/L nitrite, continue breeding 12-24h.
Description
Technical Field
The invention relates to the fields of microbiology, enzyme engineering, fermentation engineering, biochemistry, food chemistry, food science, food safety and the like, in particular to a method for producing nitrite reductase by fermenting lactic acid bacteria. The nitrite reductase produced by the invention is mainly applied to eliminating nitrite residues in agricultural products, foods, feeds and the environment, improving the food safety and the food quality and reducing the incidence of food poisoning and cancer caused by nitrite.
Background
The agricultural production nitrogen fertilizer is applied in large quantity, and the N, P, K proportion is maladjusted, so that the nitrate and nitrite in the environment are enriched in large quantity, and a large amount of nitrite residues in agricultural products, food and feed are caused. Nitrite is a substance harmful to human body, and not only causes direct poisoning and death of people, livestock and poultry; but also precursors of the carcinogen nitrosamines. According to the analysis of nitrite in vegetables, grains, fresh fishes, fresh meats, fresh eggs, salt, pickled vegetables, milk and dairy products in Beijing, Henan, Qingdao and Jilin areas by the food health supervision and inspection institute of the Ministry of health in China, investigation results show that nitrite is detected in all eight normal types of food supplied in the market, wherein the detection rate of the pickled vegetables is as high as 94.7%, and the highest value is 85.6 mg/kg. According to the food poisoning data of food health supervision and inspection institute of Shandong province, the following are shown: food poisoning caused by nitrite is commonly caused in the whole provinces from 1983 to 1997 for 82, 1089 and 39 deaths are caused. Nitrite poisoning occurs in a latency period of 10min at the shortest time and 4h at the longest time, and the average time is about 30 min. According to incomplete statistics, the current national morbidity is increased by dozens of times or even dozens of times. Therefore, the effective measures for eliminating and reducing the nitrite residue in agricultural products, foods, feeds and the environment are necessary conditions for safe production of foods, improvement of food quality and reduction of the incidencerate of food poisoning and cancer caused by nitrite. The new look-up shows that: no scientific, convenient, effective and rapid method for eliminating the nitrite residues in agricultural products, feeds, foods and environments is available at home and abroad.
Nitrite reductase is the most efficient pathway for degrading nitrite. Nitrite is converted to ammonia by nitrite reductase and can be expressed as: therefore, nitrite reductase can be added into agricultural products, food, feed and environment to rapidly degrade nitrite, so that the harm and pathogenicity to human are eliminated. Nitrite reductase produced by fermenting lactic acid bacteria beneficial to human bodies is added with 0.01-0.5 thousandth of nitrite reductase during the pretreatment period of raw materials, and the temperature is kept for 5-60 min at 20-60 ℃, so that nitrite remained in agricultural products, food, feed and the environment can be completely degraded. The application solves the harm and pathogenicity of the nitrite residue to human, livestock and poultry.
Disclosure of Invention
The invention aims to provide a method for producing nitrite reductase by fermenting lactic acid bacteria, which is a method for producing nitrite reductase by fermenting lactic acid bacteria under the induction of nitrite at 26-32 ℃, wherein the activity of a crude enzyme liquid of the nitrite reductase obtained by the production method can reach 2600U/ml, and enzyme preparations with different concentrations and purities can be obtained by separating and purifying. When the activity of the enzyme is 1000U/ml or 1000U/g, the usage amount of 0.01-0.5 per mill is applied to agricultural products, food, feed and environment, and the residual nitrite can be completely degraded by keeping thetemperature at 20-60 ℃ for 5-60 min. The nitrite reductase is used for eliminating the nitrite residue, and the operation is simple, convenient, fast, low in cost, safe and reliable. Can fundamentally eliminate the harm of direct poisoning, death and carcinogenesis of people, livestock and poultry caused by nitrite residue.
The method for producing nitrite reductase by fermenting lactic acid bacteria, disclosed by the invention, comprises the following steps of:
(1) activating lactic acid bacteria generating nitrite reductase, and performing step-by-step amplification culture according to a conventional method to prepare liquid first-stage seeds and second-stage seeds;
(2) inoculating the liquid primary seeds or secondary seeds into a liquid enzyme production culture medium according to the inoculation amount of 3-8% of the volume of fermentation liquid, adding 40-150 mg/L nitrite when culturing for 36-72 h at 26-32 ℃, and culturing for 12-24 h, namely finishing the production of nitrite reductase by fermenting lactic acid bacteria;
(3) centrifuging the fermentation liquor obtained in the step (2) at 8,000-10,000 rpm to collect lactobacillus thalli;
(4) cleaning with a buffer solution with the pH value of 7.0 which is 2-3 times of the volume of the collected thallus in the step (3), centrifugally collecting the lactobacillus thallus at 8,000-10,000 rpm after the thallus is completely suspended, repeating the operation of cleaning the thallus for 2-3 times, and finally preparing the thallus into a lactobacillus suspension;
(5) decompressing and crushing the lactobacillus suspension prepared in the step (4);
(6) and (4) centrifuging the bacterial cell crushing suspension obtained in the step (5) at 12,000-140,000 rpm and 4 ℃, and collecting supernate, namely the crude enzyme solution.
(7) According to different requirements and different using objects, the crude enzyme liquid obtained in the step (6) can be further concentrated, separated and purified to prepare enzyme preparations with different activities, purities and formulations.
The strain used in the invention is derived from China general microbiological culture Collection center (CGMCC), can be obtained commercially, and the activation and growth conditions are carried out according to the instructions provided by a strain preservation unit. Nitrite reductase-producing lactic acid bacteria (e.g., CGMCC strain No.: 1.11 or 1.19 or 1.557 or 1.2029 or 1.1480 or 1.1878) are cultured under enzyme-producing conditions in the case of producing nitrite reductase by fermentation after activation of the strain, and the strain can be stored at 4 ℃ for 4 months.
The specific implementation mode is as follows:
the first embodiment is as follows:
(1) preparation of the culture Medium
① culture medium for activating bacteria strain, including casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 5.0g, sodium acetate 5.0g, diamine citrate 2.0g, Tween 801.0 g, and K2HPO41.0g，MgSO4·7H2O 0.2g，MnSO4·H2O0.05g，CaCO320.0g, agar 15.0g, distilled water 1.0L, pH 6.8, and autoclave sterilization at 121 deg.C for 30 min.
② liquid seed culture medium containing casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 5.0g, sodium acetate 5.0g, citric acid diamine 2.0g, Tween 801.0 g, and K2HPO41.0g，MgSO4·7H2O 0.2g，MnSO4·H2O0.05g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
③ enzyme-producing culture medium comprising 3.0-7.0 g of casein peptone, 2.0-8.0 g of beef extract, 3.0-9.0 g of yeast extract, 3.0-10.0 g of corn starch sugar, 0.5-2.0 g of sodium acetate, 0.6-2.0 g of citric acid diamine, 800.3-1.4 g of Tween and K2HPO40.4～1.5g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
(2) Activating lactobacillus for generating nitrite reductase according to strain specification provided by China general microbiological culture Collection center (CGMCC);
(3) inoculating the activated lactobacillus in the step (2) into a liquid seed test tube with 3 needles to 5ml, culturing for 48-72 h at 26-28 ℃, and then performing stepwise amplification culture according to 5% of inoculation amount to prepare liquid first-stage seeds and liquid second-stage seeds;
(4) inoculating the secondary seeds prepared in the step (3) into 10L of enzyme production culture medium according to the inoculation amount of 3-5% of the volume of the fermentation liquid, adding 40-80 mg/L of nitrite when culturing for 60-72 h at 26-28 ℃, and culturing for 12-24 h, namely finishing the production of nitrite reductase by fermenting lactic acid bacteria.
(5) Centrifuging the fermentation liquor obtained in the step (4) at 8,000-10,000 rpm to collect lactobacillus thalli;
(6) washing with phosphate buffer solution with pH value of 7.0 which is 2-3 times of the volume of the collected thallus in the step (5), centrifugally collecting lactobacillus thallus at 8,000-10,000 rpm after the thallus is completely suspended, repeating the operation of washing the thallus for 2-3 times, and finally preparing the thallus into lactobacillus suspension;
(7) decompressing and crushing the lactobacillus suspension prepared in the step (6);
(8) and (4) centrifuging the bacterial cell crushing suspension obtained in the step (7) at 12,000-140,000 rpm and 4 ℃, and collecting supernate, namely the crude enzyme solution.
(9) According to different requirements and different using objects, the crude enzyme liquid obtained in the step (8) can be further concentrated, separated and purified to prepare enzyme preparations with different activities, purities and formulations.
Example two:
(1) preparation of the culture Medium
① culture medium for activating bacteria strain, including casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 5.0g, sodium acetate 5.0g, diamine citrate 2.0g, Tween 801.0 g, and K2HPO41.0g，MgSO4·7H2O 0.2g，MnSO4·H2O0.05g，CaCO320.0g, agar 15.0g, distilled water 1.0L, pH 6.8, and autoclave sterilization at 121 deg.C for 30 min.
② liquid seed culture medium including casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g,5.0g of glucose, 5.0g of sodium acetate, 2.0g of citric acid diamine, 801.0 g of Tween and K2HPO41.0g，MgSO4·7H2O 0.2g，MnSO4·H2O0.05g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
③ enzyme-producing culture medium comprising 3.0-7.0 g of casein peptone, 2.0-8.0 g of beef extract, 3.0-9.0 g of yeast extract, 3.0-10.0 g of corn starch sugar, 0.5-2.0 g of sodium acetate, 0.6-2.0 g of citric acid diamine, 800.3-1.4 g of Tween and K2HPO40.4～1.5g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
(2) Activating lactobacillus for generating nitrite reductase according to strain specification provided by China general microbiological culture Collection center (CGMCC);
(3) inoculating the activated lactobacillus in the step (2) into a liquid seed test tube with 3 needles to 5ml, culturing for 48-60 h at 28-30 ℃, and then performing stepwise amplification culture according to 5% of inoculation amount to prepare liquid first-stage seeds and liquid second-stage seeds;
(4) inoculating the secondary seeds prepared in the step (3) into 20L of enzyme production culture medium according to the inoculation amount of 6-8% of the volume of the fermentation liquid, adding 81-120 mg/L of nitrite when culturing for 36-48 h at 28-30 ℃, and culturing for 12-24 h, namely finishing the production of nitrite reductase by fermenting lactic acid bacteria.
(5) Centrifuging the fermentation liquor obtained in the step (4) at 8,000-10,000 rpm to collect lactobacillus thalli;
(6) washing with phosphate buffer solution with pH value of 7.0 which is 2-3 times of the volume of the collected thallus in the step (5), centrifugally collecting lactobacillus thallus at 8,000-10,000 rpm after the thallus is completely suspended, repeating the operation of washing the thallus for 2-3 times, and finally preparing the thallus into lactobacillus suspension;
(7) decompressing and crushing the lactobacillus suspension prepared in the step (6);
(8) and (4) centrifuging the bacterial cell crushing suspension obtained in the step (7) at 12,000-140,000 rpm and 4 ℃, and collecting supernate, namely the crude enzyme solution.
(9) According to different requirements and different using objects, the crude enzymeliquid obtained in the step (8) can be further concentrated, separated and purified to prepare enzyme preparations with different activities, purities and formulations.
Example three:
(1) preparation of the culture Medium
① culture medium for activating bacteria strain, including casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 5.0g, sodium acetate 5.0g, diamine citrate 2.0g, Tween 801.0 g, and K2HPO41.0g，MgS04·7H2O0.2g，MnSO4·H2O0.05g，CaCO320.0g, agar 15.0g, distilled water 1.0L, pH 6.8, and autoclave sterilization at 121 deg.C for 30 min.
② liquid seed culture medium containing casein peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, glucose 5.0g, sodium acetate 5.0g, citric acid diamine 2.0g, Tween 801.0 g, and K2HPO41.0g，MgSO4·7H2O 0.2g，MnSO4·H2O0.05g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
③ enzyme-producing culture medium comprising 3.0-7.0 g of casein peptone, 2.0-8.0 g of beef extract, 3.0-9.0 g of yeast extract, 3.0-10.0 g of corn starch sugar, 0.5-2.0 g of sodium acetate, 0.6-2.0 g of citric acid diamine, 800.3-1.4 g of Tween and K2HPO40.4～1.5g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
(2) Activating lactobacillus for generating nitrite reductase according to strain specification provided by China general microbiological culture Collection center (CGMCC);
(3) inoculating the activated lactobacillus in the step (2) into a liquid seed test tube with 3 needles to 5ml, culturing for 36-48 h at 30-32 ℃, and then performing stepwise amplification culture according to 5% of inoculation amount to prepare liquid first-stage seeds and liquid second-stage seeds;
(4) inoculating the secondary seeds prepared in the step (3) into 50L of enzyme production culture medium according to the inoculation amount of 3-5% of the volume of the fermentation liquid, adding 121-150 mg/L of nitrite when culturing for 36-48 h at 30-32 ℃, and culturing for 12-24 h, namely finishing the production of nitrite reductase by fermenting lactic acid bacteria.
(5) Centrifuging the fermentation liquor obtained in the step (4) at 8,000-10,000 rpm to collect lactobacillus thalli;
(6) washing with phosphate buffer solution with pH value of 7.0 which is 2-3 times of the volume of the collected thallus in the step (5), centrifugally collecting lactobacillus thallus at 8,000-10,000 rpm after the thallus is completely suspended, repeating the operation of washing the thallus for 2-3 times, and finally preparing the thallus into lactobacillus suspension;
(7) decompressing and crushing the lactobacillus suspension prepared in the step (6);
(8) and (4) centrifuging the bacterial cell crushing suspension obtained in the step (7) at 12,000-140,000 rpm and 4 ℃, and collecting supernate, namely the crude enzyme solution.
(9) According to different requirements and different using objects, the crude enzyme liquid obtained in the step (8) can be further concentrated, separated and purified to prepare enzyme preparations with different activities, purities and formulations.
Example four:
the lactic acid bacteria nitrite reductase produced according to the first, second and third methods of the example was determined to have an average enzyme activity of 2600U/ml in crude enzymesolution, and if separated and purified, enzyme preparations of different activities, purities and dosage forms could be obtained. Nitrite reductase with the activity of 1000U/ml or 1000U/g is added into agricultural products, food, feed and environment according to the weight of 0.01-0.5 per mill of the raw material, and is processed for 5-60 min at 20-60 ℃ for nitrite detection, and the detection result is shown in the following table.
| Sample name | Treatment ofFront side Nitrite content (mg/Kg) | After treatment Nitrite content (mg/Kg) | Remarks for note |
| Wheat flour | 0.007 | Is not picked up | |
| Soybean flour | 0.009 | Is not picked up | |
| Vegetable for protected area | |||
| Potato | 0.078 | Is not picked up | |
| Tomato | 0.046 | Is not picked up | |
| Chinese cabbage | 0.064 | Is not picked up | |
| Rape seed | 0.082 | Is not picked up | |
| Cucumber (Cucumis sativus) | 0.045 | Is not picked up | |
| Bean | 0.038 | Is not picked up | |
| Radish (radish) | 0.029 | Is not picked up | |
| Vegetables for field | |||
| Potato | 0.006 | Is not picked up | |
| Tomato | 0.037 | Is not picked up | |
| Chinese cabbage | 0.029 | Is not picked up | |
| Rape seed | 0.032 | Is not picked up | |
| Cucumber (Cucumis sativus) | 0.023 | Is not picked up | |
| Bean | 0.016 | Is prepared fromPick up out | |
| Radish (radish) | 0.014 | Is not picked up | |
| Feed stuff | |||
| Pig mixed feed | 0.058 | Is not picked up | |
| Chicken mixed feed | 0.047 | Is not picked up |
The detection result shows that: nitrite remained in agricultural products, food, feed and environment is treated by nitrite reductase preparation, and is completely degraded; meanwhile, the nitrite reductase is the simplest, convenient, rapid and effective method for eliminating the nitrite residue.
Claims (3)
1. A method for producing nitrite reductase by fermenting lactic acid bacteria comprises the following steps:
(1) activating lactic acid bacteria generating nitrite reductase by a conventional method, and performing step-by-step amplification culture to prepare liquid first-stage seeds and liquid second-stage seeds;
(2) inoculating the liquid primary seeds or secondary seeds into a liquid enzyme production culture medium according to the inoculation amount of 3-8% of the volume of fermentation liquid, adding 40-150 mg/L nitrite when culturing for 36-72 h at 26-32 ℃, and culturing for 12-24 h;
(3) centrifuging the fermentation liquor obtained in the step (2) at 8,000-10,000 rpm to collect lactobacillus thalli;
(4) cleaning with a buffer solution with the pH value of 7.0 which is 2-3 times the volume of the collected bacteria in the step (3), after the bacteria are completely suspended, centrifugally collecting the lactobacillus bacteria at 8,000-10,000 rpm, repeating the operation of cleaning the bacteria for 2-3 times, and finally preparing the bacteria into a lactobacillus suspension;
(5) decompressing and crushing the lactobacillus suspension prepared in the step (4);
(6) and (4) centrifuging the bacterial cell crushing suspension obtained in the step (5) at 12,000-140,000 rpm and 4 ℃, and collecting supernate, namely the crude enzyme solution.
2. The method of claim 1, comprising, after step (6): and (4) further concentrating, separating and purifying the crude enzyme liquid obtained in the step (6) to prepare enzyme preparationswith different activities, purities and dosage forms.
3. The method of claim 1 or 2, wherein the liquid enzyme production medium is: 3.0-7.0 g of casein peptone, 2.0-8.0 g of beef extract, 3.0-9.0 g of yeast extract, 3.0-10.0 g of corn starch sugar, 0.5-2.0 g of sodium acetate, 0.6-2.0 g of citric acid diamine, 800.3-1.4 g of tween, and K2HPO40.4～1.5g，CaCO320.0g, 1.0L tap water, pH 7.0, and autoclave sterilization at 121 deg.C for 30 min.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807610B (en) * | 2006-01-23 | 2011-01-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN101228928B (en) * | 2008-02-28 | 2011-05-18 | 中国农业大学 | Nitrite reductase compound enzyme preparation and method of using the same to degrade nitrite in sausage |
| CN102286438A (en) * | 2011-07-25 | 2011-12-21 | 上海应用技术学院 | Method for producing nitrite reductase from lactobacillus |
| CN104068410A (en) * | 2007-06-11 | 2014-10-01 | 杰伯里国际有限公司 | Method and composition for preparing cured meat products |
| CN109679930A (en) * | 2019-02-15 | 2019-04-26 | 大连大学 | A kind of fermentation process producing nitrite reductase |
-
2005
- 2005-06-03 CN CN 200510073389 patent/CN1687408A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1807610B (en) * | 2006-01-23 | 2011-01-26 | 迟乃玉 | Method for producing low temperature cellulase using microbe fermentation |
| CN104068410A (en) * | 2007-06-11 | 2014-10-01 | 杰伯里国际有限公司 | Method and composition for preparing cured meat products |
| CN101228928B (en) * | 2008-02-28 | 2011-05-18 | 中国农业大学 | Nitrite reductase compound enzyme preparation and method of using the same to degrade nitrite in sausage |
| CN102286438A (en) * | 2011-07-25 | 2011-12-21 | 上海应用技术学院 | Method for producing nitrite reductase from lactobacillus |
| CN109679930A (en) * | 2019-02-15 | 2019-04-26 | 大连大学 | A kind of fermentation process producing nitrite reductase |
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Open date: 20051026 |