CN112741090B - Method for inhibiting hybrid bacteria propagation in spirulina culture process by using di-primary/secondary alkyl thiosulfinate - Google Patents
Method for inhibiting hybrid bacteria propagation in spirulina culture process by using di-primary/secondary alkyl thiosulfinate Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N41/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a sulfur atom bound to a hetero atom
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention discloses a method for inhibiting the reproduction of mixed bacteria in the process of spirulina culture by using di-primary/secondary alkyl thiosulfinate, belonging to the technical field of aquatic food culture production. The invention realizes the effects of inhibiting the growth of microorganisms and killing mixed bacteria by matching the specific substituted primary and secondary alkyls in the di-primary/secondary alkyl thiosulfinate with a disulfide bond, and has obvious inhibiting effect on the growth and reproduction of the mixed bacteria in the production of spirulina; meanwhile, the di-primary/secondary alkyl sulfenesulfonate used in the method is easy to naturally degrade, can be decomposed into harmless degradation products after staying in a water body for 1-2 days, has no influence on a water body ecological system, and provides a new means for solving the problem of mixed bacteria pollution in the spirulina culture production process.
Description
Technical Field
The invention belongs to the technical field of aquaculture production, and particularly relates to a method for inhibiting the propagation of mixed bacteria in the process of spirulina culture by using di-primary/secondary alkyl thiosulfinate.
Background
Spirulina is an ancient photoautotrophic microalgae, named because it is spiral under a microscope. The spirulina is an alkaline nutrient food, contains various nutrient components necessary for human body, is rich in various vitamins, trace elements, algal polysaccharides, phycocyanin, linolenic acid, insulin-like substances and other bioactive substances, and has various medicinal and health-care functions of reducing cholesterol, removing kidney toxicity, improving the immune function of human body, promoting prostaglandin synthesis, inhibiting and preventing cancers, accelerating wound healing and the like under certain conditions. The spirulina is completely certified by the national ministry of health and the Food and Drug Administration (FDA), Germany, Japan and the like to have no toxic or side effect, and is the most safe health care product. In China, the annual total national output of spirulina reaches 10000 tons. One third of the output of spirulina is used for direct export, and other spirulina is mostly used as health care products in the form of algae powder, microalgae tablets and perfusion capsules.
The production of the spirulina is carried out in a semi-closed runway type circulating culture pond, various mixed bacteria brought by air flow and in a water body can be propagated in a large quantity by utilizing nutrient components in the culture pond, basic nutrition and dissolved oxygen required by the growth of the spirulina are consumed, the activity of the spirulina is reduced, the production period is prolonged, and the pond can be scrapped due to sudden pollution in extreme cases. It has been shown that certain bacteria can promote the aging and settlement of microalgae cells, thereby limiting the growth and propagation of microalgae. Therefore, prevention of contamination by other microorganisms is an important research area for microalgae culture. Patent techniques for inhibiting or killing bacteria during the production of spirulina have been reported, for example: in CN201621426240.9, the collected algae mud is soaked and sterilized by high-concentration ozone water, and the strong oxidation inevitably damages various nutritional components in the spirulina, so that the feasibility is not high; CN201910884771.4 adopts ultraviolet rays to sterilize the culture solution, the ultraviolet rays have weak penetrating power and poor effect, and the rapid propagation of mixed bacteria in the culture period is hardly influenced, so the method is only suitable for the culture of spirulina in a laboratory scale and is not suitable for the large-scale culture and production of the spirulina.
Disclosure of Invention
In order to solve the problems, the invention utilizes di-primary/secondary alkyl sulfenate to control the hybrid bacteria reproduction in the spirulina culture process. The invention makes the mercaptan specificity oxidative stress reaction caused by disulfide bond and sulfur stress cause the sensitive protein damage of the microorganism such as bacteria and the like through the matching of characteristic substitution primary and secondary alkyls in the di-primary/secondary alkyl thiosulfinate, thereby inhibiting the growth of the microorganism and killing the mixed bacteria. Meanwhile, the di-primary/secondary alkyl sulphosulfinate used in the method is easy to naturally degrade, can be decomposed into harmless degradation products after staying in water for 1-2 days, and has no influence on a water ecological system. In the production process of spirulina, the diprimary/secondary alkyl sulphosulfinate with the concentration of 10 mug/L-10 mg/L is added to obviously inhibit the growth and the propagation of mixed bacteria.
The first purpose of the invention is to provide the application of di-primary/secondary alkyl sulphosulfinate in inhibiting the propagation of mixed bacteria in the culture process of spirulina; the structure of the di-primary/secondary alkyl thiosulfinic acid is shown as a formula (1):
wherein R is1、R2Independently is selected from C4-C8Primary and secondary alkyl groups.
In one embodiment of the invention, R1、R2Preferably C5Primary and secondary alkyl groups.
The invention also provides a spirulina culture method, which is characterized in that di-primary/secondary alkyl sulphosulfinate with the structure shown in the formula (1) is added into the water body of the spirulina culture pond as a bacteriostatic agent.
In one embodiment of the invention, the method is to add di-primary/secondary alkyl sulphosulfinate with the structure shown in formula (1) into the water body of the spirulina culture pond in an amount of 10 mg-10 g per ton of culture water body per day until the spirulina is cultured and harvested.
In one embodiment of the present invention, a process for the preparation of a diprimary/secondary alkyl thiosulfinic acid comprises:
taking a mixed system of dichloromethane and methanol as a solvent, adding diprimary/secondary alkyl disulfide and dimethyl selenate, and uniformly mixing; and then adding hydrogen peroxide, reacting at 20-50 ℃, extracting, washing and drying after the reaction is finished, and obtaining the target product di-primary/secondary alkyl sulphosulfinate.
In one embodiment of the invention, the structure of the diprimary/secondary alkyl disulfide is as follows:
wherein R is1、R2Independently is selected from C4-C8Primary and secondary alkyl groups.
In one embodiment of the present invention, the concentration of the diprimary/secondary alkyl disulfide relative to the solvent is 10 to 50 mol/L.
In one embodiment of the present invention, the concentration of the dimethyl selenate relative to the solvent is 1 to 5 mol/L.
In one embodiment of the invention, the volume ratio of dichloromethane to methanol in the solvent is 100: 0-80: 20.
in one embodiment of the present invention, the concentration of hydrogen peroxide to the solvent is 10 to 50 mol/L.
In one embodiment of the present invention, the reaction time is 3 to 10 hours.
In one embodiment of the invention, the method further comprises: after the reaction was complete, the reaction mixture was washed three times with three volumes of saturated sodium chloride solution. And drying the reaction mixture at 20-55 ℃ in vacuum to obtain a product. The content of the di-primary/secondary alkyl sulphosulfinate in the product is 75-90%.
The invention has the beneficial effects that:
1. the invention realizes the effects of inhibiting the growth of microorganisms and killing mixed bacteria by matching the specific substituted primary and secondary alkyls in the di-primary/secondary alkyl thiosulfinate with a disulfide bond, and has obvious inhibiting effect on the growth and reproduction of the mixed bacteria in the production of spirulina; meanwhile, the di-primary/secondary alkyl sulfenesulfonate used in the method is easy to naturally degrade, can be decomposed into harmless degradation products after staying in a water body for 1-2 days, has no influence on a water body ecological system, and provides a new means for solving the problem of mixed bacteria pollution in the spirulina culture production process.
2. The invention utilizes the di-primary/secondary alkyl thiosulfinate to obviously inhibit the growth and the propagation of the mixed bacteria, reduces the total number of the mixed bacteria in each milliliter of water by 6 to 7 orders of magnitude, has no influence on the growth of the spirulina, and is suitable for the industrial production requirements of different scales and fields. The production period of the spirulina is reduced from 7 days to 4.5 days, the yield of each 500 tons of aquaculture water reaches 2.0-2.5 tons, and the economic benefit is obviously increased.
Detailed Description
The invention relates to methylene dichloride which is purchased from salt city cloud peak chemical company Limited, methanol is purchased from national medicine group chemical reagent company Limited, di-n-amyl disulfide, di-isoamyl disulfide, di-n-octyl disulfide and di-tert-butyl disulfide are purchased from Shanghai Vast chemical engineering company Limited, dimethyl selenate is purchased from national medicine group chemical reagent company Limited, hydrogen peroxide is purchased from national medicine group chemical reagent company Limited, and saturated sodium chloride is purchased from national medicine group chemical reagent company Limited.
Example 1
(1) Preparation of di-n-pentylthiosulfinate:
adding di-n-amyl disulfide according to a molar concentration of 50mol/L into a dichloromethane-methanol (the volume ratio of dichloromethane to methanol is 90: 10), and adding dimethyl selenate according to a molar concentration of 5 mol/L; then, hydrogen peroxide was added at 20mol/L and the reaction was stirred at 25 ℃ for 5 hours. After the reaction was complete, the reaction mixture was washed three times with three volumes of saturated sodium chloride solution. The reaction mixture was dried under vacuum at 35 ℃ to obtain the product. The di-n-pentylthiosulfinate content of the product was 90%.
And (3) substance characterization: m/z 222.11;1HNMR (deuterated DMSO): δ 0.8ppm (triplet, 6H, -CH)3) 1.28 to 1.50ppm (multiplet, 12H, -CH)2) 2.4ppm (triplet, 2H, -CH)2-S-),2.5ppm (triplet, 2H, -CH)2-S＝O)。
(2) Inhibition of growth of infectious microbes in spirulina culture ponds using di-n-pentylthiosulfinate:
100g of the obtained di-n-pentylthiosulfinate was added to a water body of a spirulina culture pond of 100 tons of water body. During the cultivation of spirulina, 100g of spirulina is added every day.
As a result, it was found that: and collecting the spirulina after 5 days, wherein the yield of the spirulina in each 500 tons of aquaculture water reaches 2.5 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.5X 10 per mL2one/mL.
Example 2
(1) Preparation of di-n-octyl thiosulfinate:
adding di-n-octyl disulfide (purchased from Shanghai Hansi chemical Co., Ltd.) with a molar concentration of 35mol/L into a dichloromethane-methanol (the volume ratio of dichloromethane to methanol is 80: 20) solution, and adding dimethyl selenate with a molar concentration of 3 mol/L; then, after hydrogen peroxide was added at 40mol/L, the reaction was stirred at 50 ℃ for 10 hours. After the reaction was complete, the reaction mixture was washed three times with three volumes of saturated sodium chloride solution. The reaction mixture was dried under vacuum at 55 ℃ to obtain the product. The di-n-octylthiosulfinate content in the product was 75%.
And (3) substance characterization: m/z 306.21;1HNMR (deuterated DMSO): δ 0.88ppm (triplet, 6H, -CH)3) 1.21 to 1.50ppm (multiplet, 24H, -CH)2) 2.4ppm (triplet, 2H, -CH)2-S-),2.5ppm (triplet, 2H, -CH)2-S＝O)。
(2) The use of di-n-octyl thiosulfinate to inhibit the growth of infectious microbes in spirulina culture ponds:
1000g of the obtained di-n-octyl thiosulfinate is added into the water body of the spirulina culture pond with 100 tons of water body. During the cultivation of spirulina, 1000g of spirulina is added every day.
As a result, it was found that: and collecting the spirulina after 6 days, wherein the yield of the spirulina in each 500 tons of aquaculture water reaches 2.0 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.5X 10 per mL3one/mL.
Example 3
(1) Preparation of diisoamyl thiosulfinate:
adding diisoamyl disulfide according to a molar concentration of 45mol/L and adding dimethyl selenate according to a molar concentration of 4mol/L into a dichloromethane-methanol (the volume ratio of dichloromethane to methanol is 100: 0); then, hydrogen peroxide was added at 15mol/L, and the reaction was stirred at 45 ℃ for 3 hours. After the reaction was completed, the reaction mixture was washed three times with three times the volume of a saturated sodium chloride solution (available from national institute of chemical Co., Ltd.). The reaction mixture was dried under vacuum at 35 ℃ to obtain the product. The content of diisoamyl thiosulfinate in the product was 80%.
And (3) substance characterization: m/z 222.11;1HNMR (deuterated DMSO): delta is 0.93-0.99 ppm (multiplet, 12H, -CH)3) 1.55ppm (multiplet, 4H, -CH)2) 1.77ppm (multiplet, 2H, -CH), 2.36ppm (2H, -CH)2-S-),2.62ppm(2H，-CH2-S＝O)。
(2) Inhibition of mixed bacterial growth in spirulina culture ponds using diisoamyl thiosulfinate:
500g of the obtained diisoamyl thiosulfinate was added to a water body of a spirulina culture pond of 100 tons of water body. During the cultivation of spirulina, 500g is required to be added every day.
As a result, it was found that: and collecting the spirulina after 6 days, wherein the yield of the spirulina in each 500 tons of aquaculture water reaches 2.2 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 2X 10 per mL3one/mL.
Comparative example 1:
the absence of di-primary/secondary alkyl thiosulfinate during spirulina culture inhibits the growth of infectious microbes in spirulina culture ponds.
As a result, it was found that: after 5 days, the spirulina is harvested, and the yield of the spirulina in each 500 tons of aquaculture water is 0.9 ton. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.7X 10 per mL9～1010one/mL.
The spirulina is collected after 6 days, and the yield of the spirulina per 500 tons of aquaculture water is 1.1 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.5X 10 per mL9～1010one/mL.
After 7 days, the spirulina is harvested, and the yield of the spirulina in each 500 tons of aquaculture water is 1.5 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.2X 10 per mL9～1010one/mL.
Comparative example 2:
(1) preparation of di-tert-butylthiosulfinate:
adding di-tert-butyl disulfide into dichloromethane according to a molar concentration of 60mol/L, and adding dimethyl selenate according to a molar concentration of 5 mol/L; then, hydrogen peroxide was added at 20mol/L, and the reaction was stirred at 35 ℃ for 12 hours. After the reaction was completed, the reaction mixture was washed three times with three times the volume of a saturated sodium chloride solution (available from national institute of chemical Co., Ltd.). The reaction mixture was dried under vacuum at 35 ℃ to obtain the product. The content of di-tert-butylthiosulfinate in the product was 90%.
And (3) substance characterization: m/z 194.08;1HNMR (deuterated DMSO): δ 1.32ppm (singlet, 9H, -CH)3) 1.44ppm (singlet, 9H, -CH)3)。
(2) The use of di-tert-butylthiosulfinate to inhibit the growth of mixed bacteria in spirulina culture ponds:
700g of the obtained di-tert-butylthiosulfinate was added to a water body of a spirulina culture pond of 100 tons of water body. During the cultivation of spirulina, 600g is required to be added every day.
As a result, it was found that: after 5 days, the spirulina is harvested, and the yield of the spirulina in each 500 tons of aquaculture water is 0.9 ton. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.5X 10 per mL9～1010one/mL.
The spirulina is collected after 6 days, and the yield of the spirulina per 500 tons of aquaculture water is 1.2 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.7X 10 per mL9～1010one/mL.
After 7 days, the spirulina is harvested, and the yield of the spirulina in each 500 tons of aquaculture water is 1.3 tons. The concentration of the algae filament is 2 x 105The total number of colonies (determined according to national Standard GB 4789.2) was 1.4X 10 per mL9～1010one/mL.
Claims (8)
1. A spirulina cultivation method is characterized in that di-primary/secondary alkyl thiosulfinate with a structure shown as a formula (1) is added into a water body of a spirulina cultivation pond as a bacteriostatic agent for cultivation;
wherein R is1、R2Is C5A primary alkyl group.
2. The method of claim 1, wherein the di-primary/secondary alkyl thiosulfinate with the structure shown in formula (1) is added into the water body of the spirulina culture pond in an amount of 10 mg-10 g per ton of aquaculture water body per day until the spirulina is cultured and harvested.
3. The process according to any one of claims 1-2, characterized in that the process for the preparation of di-primary/secondary alkyl thiosulfinic acids comprises:
taking a mixed system of dichloromethane and methanol as a solvent, adding diprimary/secondary alkyl disulfide and dimethyl selenate, and uniformly mixing; and then adding hydrogen peroxide, reacting at 20-50 ℃, extracting, washing and drying after the reaction is finished, and obtaining the target product di-primary/secondary alkyl sulphosulfinate.
4. The method according to claim 3, wherein the concentration of the di-primary/secondary alkyl disulfide relative to the solvent is 10 to 50 mol/L.
5. The method of claim 3, wherein the concentration of the dimethyl selenate relative to the solvent is 1-5 mol/L.
6. The process of claim 3, wherein the volume ratio of dichloromethane to methanol in the solvent is 100: 0-80: 20.
7. the method according to claim 3, wherein the concentration of hydrogen peroxide to the solvent is 10 to 50 mol/L.
8. The method according to claim 3, wherein the reaction time is 3 to 10 hours.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5064419A (en) * | 1973-10-17 | 1975-05-31 | ||
| CN110484476A (en) * | 2019-09-19 | 2019-11-22 | 南昌绿锦茶疗食品有限公司 | A kind of cultural method of spirulina |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS5064419A (en) * | 1973-10-17 | 1975-05-31 | ||
| CN110484476A (en) * | 2019-09-19 | 2019-11-22 | 南昌绿锦茶疗食品有限公司 | A kind of cultural method of spirulina |
Non-Patent Citations (3)
| Title |
|---|
| Alkyl thiolsulfinates;LA VERNE D.SMALL et al;《Journal of the American Chemical Society》;19470731;第69卷;第1710-1713页 * |
| Nematicidal and Antimicrobial Constituents from Allium Grayi Regel and Allium fistulosum L.var. caespitosum;Masahiro Tada et al;《Agric.Biol.Chem.》;20160106;第52卷(第9期);第2383-2385页 * |
| Oxidation of Disulfides to Thiolsulfinates with Hydrogen Peroxide and a Cyclic Seleninate Ester Catalyst;Nicole M.R.McNeil et al;《Molecules》;20151231;第20卷;第10748-10762页 * |
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