CN114196710A - Application of salinomycin serving as bactericide in alcohol fermentation - Google Patents
Application of salinomycin serving as bactericide in alcohol fermentation Download PDFInfo
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- CN114196710A CN114196710A CN202111438797.XA CN202111438797A CN114196710A CN 114196710 A CN114196710 A CN 114196710A CN 202111438797 A CN202111438797 A CN 202111438797A CN 114196710 A CN114196710 A CN 114196710A
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- salinomycin
- fermentation
- bactericide
- alcohol
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- KQXDHUJYNAXLNZ-XQSDOZFQSA-N Salinomycin Chemical compound O1[C@@H]([C@@H](CC)C(O)=O)CC[C@H](C)[C@@H]1[C@@H](C)[C@H](O)[C@H](C)C(=O)[C@H](CC)[C@@H]1[C@@H](C)C[C@@H](C)[C@@]2(C=C[C@@H](O)[C@@]3(O[C@@](C)(CC3)[C@@H]3O[C@@H](C)[C@@](O)(CC)CC3)O2)O1 KQXDHUJYNAXLNZ-XQSDOZFQSA-N 0.000 title claims abstract description 116
- 239000004189 Salinomycin Substances 0.000 title claims abstract description 115
- 229960001548 salinomycin Drugs 0.000 title claims abstract description 115
- 235000019378 salinomycin Nutrition 0.000 title claims abstract description 115
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 59
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- 229930182555 Penicillin Natural products 0.000 description 6
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 6
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- 206010023076 Isosporiasis Diseases 0.000 description 2
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 2
- 239000001888 Peptone Substances 0.000 description 2
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- PUZPDOWCWNUUKD-UHFFFAOYSA-M sodium fluoride Chemical compound [F-].[Na+] PUZPDOWCWNUUKD-UHFFFAOYSA-M 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
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- 241000219310 Beta vulgaris subsp. vulgaris Species 0.000 description 1
- ZKQDCIXGCQPQNV-UHFFFAOYSA-N Calcium hypochlorite Chemical compound [Ca+2].Cl[O-].Cl[O-] ZKQDCIXGCQPQNV-UHFFFAOYSA-N 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
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- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- KIPLYOUQVMMOHB-MXWBXKMOSA-L [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O Chemical compound [Ca++].CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O.CN(C)[C@H]1[C@@H]2[C@@H](O)[C@H]3C(=C([O-])[C@]2(O)C(=O)C(C(N)=O)=C1O)C(=O)c1c(O)cccc1[C@@]3(C)O KIPLYOUQVMMOHB-MXWBXKMOSA-L 0.000 description 1
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- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 description 1
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- 231100000053 low toxicity Toxicity 0.000 description 1
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 1
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- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
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- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
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- ITCAUAYQCALGGV-UHFFFAOYSA-M sodium;1,4a-dimethyl-7-propan-2-yl-2,3,4,4b,5,6,10,10a-octahydrophenanthrene-1-carboxylate Chemical compound [Na+].C12CCC(C(C)C)=CC2=CCC2C1(C)CCCC2(C)C([O-])=O ITCAUAYQCALGGV-UHFFFAOYSA-M 0.000 description 1
- HCJLVWUMMKIQIM-UHFFFAOYSA-M sodium;2,3,4,5,6-pentachlorophenolate Chemical compound [Na+].[O-]C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl HCJLVWUMMKIQIM-UHFFFAOYSA-M 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
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- 229940063650 terramycin Drugs 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
Images
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-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
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- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Wood Science & Technology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention provides application of salinomycin serving as a bactericide in alcohol fermentation, and belongs to the technical field of biological fermentation. The method comprises the following steps: diluting the saccharic raw material to 20-40 Bx by using water to obtain a saccharic solution; or sequentially gelatinizing, liquefying and saccharifying the starchy raw materials to obtain a saccharified solution; adding 0.1-0.3 wt% of urea into the obtained saccharic solution or saccharic solution, and inoculating 0.4-0.6 wt% of active dry saccharomyces cerevisiae; dissolving salinomycin and/or a salinomycin premix fully by using sterile water at the temperature of 30-35 ℃, adding 8-10 ppm of the salinomycin into fermentation liquor according to the fermentation liquor amount, uniformly mixing, and fermenting for 40-50 hours at the temperature of 30-35 ℃ to obtain the salinomycin premix. In the alcohol fermentation method of the present invention, the bacteria in the fermentation tank are reduced after 4 hours of adding salinomycin, the volatile acid is reduced to below 0.015, the yeast number is increased to more than 1.5 hundred million/mL, and the content of the fermented wine is increased to more than 11% (v/v).
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to application of salinomycin serving as a bactericide in alcoholic fermentation.
Background
Alcohol is used as an industrial basic raw material, is widely applied to many departments of national economy, is always a main product in the fermentation industry of China, in recent years, alcohol is increasingly regarded as renewable clean energy by various countries, and at present, most of alcohol is produced by fermenting starch or sugar raw materials through saccharomyces cerevisiae. Alcohol is relatively extensive in actual production, raw and auxiliary materials for production and water are not strictly sterilized generally, mixed bacteria are inevitably bred in fermentation, and mixed bacteria infection in the fermentation process can influence the alcohol yield of raw materials. The alcohol enterprises generally inhibit the mixed bacteria by adding sulfuric acid, bactericide and the like to avoid bacterial infection. The use of a large amount of concentrated sulfuric acid not only causes great harm to the environment, but also easily causes equipment corrosion and scale deposition, increases the difficulty of alcohol waste liquid treatment, and increases the investment of alcohol environmental protection capital. The sulfuric acid can inhibit mixed bacteria and yeast fermentation, and the wine yield of raw materials is influenced. Commonly used antimicrobial agents typically include antibiotics, antiseptics, or combinations thereof. The use of more preservatives comprises sodium fluoride, bleaching powder, strong chlorine and sodium pentachlorophenate, the inhibition of mixed bacteria can inhibit yeast fermentation, most preservatives have strong toxicity, the use of a large amount of preservatives is easy to harm the health of operators, and the preservative residue in the alcohol waste liquid can pollute the environment. The antibiotics mainly comprise penicillin, streptomycin, terramycin and the like which are common antibacterial agents for human beings, and a large amount of the antibiotics such as penicillin and the like are used, so that bacteria are easy to generate drug resistance, the sterilization effect is lost, and the prevention and control of human diseases are influenced. Therefore, the search for low-toxicity high-efficiency salinomycin bactericide is the research direction of the fermentation alcohol industry at present.
Salinomycin (molecular formula: C)42H69O11Na; CAS: 53003-10-4) is a monocarboxylic acid polyether antibiotic, which is a typical ionophore antibiotic, and salinomycin is often present in the form of a sodium salt. The salinomycin can be used as a metal ion chelating agent, can combine sodium and potassium ions to change the concentration of the sodium and potassium ions inside and outside a cell membrane, and forms an ion gradient to cause cell death. Has strong inhibiting and killing effects on most gram-positive bacteria and various coccidian, and is not easy to generate drug resistance and cross drug resistance. Salinomycin is mainly used as a feed additive in animal feeds, is mainly used for preventing chicken coccidiosis and is not used as a human antibacterial agent. At present, salinomycin sodium is generally used in the market to be prepared into salinomycin premix with soybean meal, rice bran, anhydrous silicic acid or kieselguhr.
Disclosure of Invention
The invention aims to provide the application of salinomycin serving as a salinomycin bactericide in alcohol fermentation according to the problems of strong toxicity, poor sterilization effect and the like of a common bactericide in the existing alcohol fermentation process.
The purpose of the invention is realized by the following technical scheme:
the salinomycin is applied to the preparation of bactericides, wherein the bactericides are acetic acid bacteria and/or lactic acid bacteria.
Further, the minimum inhibitory concentration of salinomycin to acetic acid bacteria is 8 mug/mL, and the minimum inhibitory concentration of salinomycin to acetic acid bacteria is 5 mug/mL.
Further, the bactericide may further comprise one or more pharmaceutically acceptable adjuvants, such as carriers, excipients or diluents.
The salinomycin is used as a bactericide in alcohol fermentation.
Further, the amount of salinomycin added is 1 to 10ppm, preferably 2 to 3ppm, based on the amount of the fermentation broth.
Further, the method for adding salinomycin comprises the following steps: dissolving salinomycin and/or a salinomycin premix fully by using sterile water, and then adding the salinomycin and/or the salinomycin premix into fermentation liquor according to the required addition amount.
An alcohol fermentation method, comprising the following steps:
s1, diluting the saccharine raw material to 20-40 Bx with water to obtain saccharine solution; or sequentially gelatinizing, liquefying and saccharifying the starchy raw materials to obtain a saccharified solution;
s2, adding 0.1-0.3 wt% of urea into the saccharine solution or saccharified solution obtained in the step S1, and inoculating 0.4-0.6 wt% of active dry saccharomyces cerevisiae; dissolving salinomycin and/or a salinomycin premix fully by using sterile water at 30-35 ℃, adding 1-10 ppm of salinomycin into fermentation liquor according to the amount of the fermentation liquor, uniformly mixing, and fermenting at 30-35 ℃ for 40-50 h to obtain a target product.
Further, the sugar raw material in S1 is selected from one or more of sugar cane, sugar beet, sweet sorghum and molasses, and the starchy raw material is selected from one or more of cereals or potato raw materials, preferably from one or more of corn, cassava and sweet potato.
Further, the preparation process of the saccharification liquid in S1 is as follows: dissolving a starchy raw material by using 2-5 times of water, adding 100-150U/g of high-temperature alpha-amylase, uniformly stirring to prepare starch slurry, and slowly heating to 98-102 ℃ for gelatinization for 25-35 min; cooling to 90-95 ℃, keeping the temperature, continuously stirring and liquefying for 60-120 min until the iodine reaction does not show blue color, and preparing a liquefied solution; cooling the liquefied liquid to 60-65 ℃, adjusting the pH value to 4.2-4.5 by using concentrated sulfuric acid, and adding 100-150U/g of saccharifying enzyme for saccharifying for 25-35 min to obtain the saccharifying liquid.
Furthermore, the addition amount of the urea in S2 is 0.2 wt%, and the addition amount of the active dry yeast for brewing is 0.5 wt%.
Further, the fermentation conditions in S2 were 31 ℃ for 48 h.
Further, the salinomycin in S2 may be selected from commercial industrial salt of high-purity (45% to 99%) salinomycin, and the salinomycin premix may be selected from commercial salinomycin premix with 12% or 24% salinomycin content, but 24% salinomycin premix has poor water solubility and poor sterilization effect on fermentation broth. In the practical application process, the salinomycin industrial salt with the purity of 45% and the salinomycin premix with the salinomycin content of 12% are preferably mixed according to the mass ratio of 4:6 for use, so that the cost can be saved to the greatest extent.
The salinomycin industrial salt and the salinomycin premix are preferably purchased from Shandong Zizang pharmaceutical industry Co.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the main raw material salinomycin of the bactericide used for alcohol fermentation has strong antibacterial action on gram-positive bacteria, almost has no medical value to human beings, and is not easy to absorb by oral administration, so that cross drug resistance and drug residue are not easy to generate. Salinomycin is a typical ionophore antibiotic which antagonizes cations in cells, especially K+、Na+、Rb+The affinity of the cell is particularly strong, which can obstruct the transmission of cations inside and outside the cell, influence osmotic pressure, finally disintegrate the cell and play a role in sterilization. K in alcohol production fermentation broth+、Na+The metal ion content is higher, the bacterial cells can be killed more quickly, and the alcohol fermentation liquor can be effectively sterilized, so that the fermentation liquor yield is improved.
(2) The waste liquor of alcohol production is often used for processing feed and fertilizer as resources, the residues of bactericides such as antiseptic and penicillin can influence the resource utilization of the waste liquor of alcohol production, the bactericide raw materials of salinomycin industrial salt and salinomycin premix used for alcohol fermentation are antibiotics which can be used for feed industry, and the waste liquor of alcohol is concentrated to prepare biological feed, so that the premix has high safety for people and animals.
(3) In the alcohol fermentation method of the present invention, bacteria in the fermentation tank are reduced after 4 hours of adding the bactericide, the volatile acid is reduced to below 0.015, the yeast number is increased to more than 1.5 hundred million/mL, and the content of the fermented wine is increased to more than 11% (v/v).
Drawings
FIG. 1 is a graph showing the results of experiments in which salinomycin has an inhibitory effect on lactobacillus by the bacteriostatic circle method; wherein, A is no salinomycin and B is salinomycin.
FIG. 2 is a graph showing the results of an experiment for determining the inhibition effect of salinomycin on acetobacter by a bacteriostatic circle method; wherein, A is no salinomycin and B is salinomycin.
FIG. 3 is a graph comparing the alcohol content of the salinomycin bactericide used in molasses fermentation and the conventional bactericide in example 2.
FIG. 4 is a comparison of the application of salinomycin bactericide in molasses fermentation in comparison with the conventional bactericide volatile acid in example 2.
FIG. 5 is a graph comparing the alcohol content of the salinomycin bactericide used for cassava fermentation in example 3 and the alcohol content of the conventional bactericide.
FIG. 6 is a comparison of the salinomycin bactericide used for cassava fermentation in example 3 and the conventional bactericide volatile acid.
Detailed Description
The present invention is further explained with reference to the following examples, which are not intended to limit the present invention in any way. The alcoholic fermentation feedstock that can be used in the present invention is not particularly limited and can be any starchy and/or saccharine feedstock, especially those commonly used in the current production of ethanol. In a preferred form of the invention, the starchy material is a cereal or potato material, such as corn, tapioca and sweet potato; the sugar raw materials comprise sugarcane, beet, sweet sorghum, molasses and the like.
Example 1
The research on the inhibition effect of salinomycin on common mixed bacteria in alcohol fermentation comprises the following steps:
in the alcohol decomposition process, bacteria are sometimes polluted by fermentation liquor, and the mixed bacteria mainly comprise bacteria, mainly lactic acid bacteria and acetic acid bacteria. The metabolism of the mixed bacteria not only consumes sugar and causes the loss of the sugar, but also can generate metabolic products of lactic acid, acetic acid and other organic acids, and the organic acids can further inhibit the propagation of yeast and reduce the yield of alcohol; acetic acid bacteria also continuously convert alcohol into acetic acid, causing the alcohol content to decrease. The bacteriostatic action of salinomycin on lactobacillus and acetobacter in alcohol fermentation liquor is researched through a bacteriostatic test.
1. Experimental materials: salinomycin (salinomycin content 24%) (Shandong Zizan pharmaceutical Co., Ltd.): lactobacillus ATCC8014 (G)+): acetobacter ATCC15973 (G)-) (preservation of microbiological detection culture Chamber of the institute of food and biotechnology, Guangdong institute of Industrial and technology, light industry and technology, Guangdong). Lactobacillus culture Medium (MRS), MRS agar culture medium, Acetobacter basic culture medium (YG), Acetobacter agar culture medium (YGC)
Lactobacillus culture Medium (MRS): 5.0g of peptone, 10.0g of tryptone, 5.0g of beef extract, 5.0g of yeast powder, 20.0g of glucose, 2.6g of diammonium hydrogen citrate, 2.0g of dipotassium hydrogen phosphate, 5.0g of sodium acetate, 0.58g of magnesium sulfate, 0.25g of manganese sulfate and Tween-801 mL; accurately weighing the reagents according to the formula, fixing the volume in 1000mL of distilled water, uniformly mixing, adjusting the pH value to 5.8, sterilizing at 121 ℃ for 15min, and storing at 4 ℃ for later use.
MRS agar medium: agar accounting for 18 percent of the mass of the MRS culture medium is added into the MRS culture medium.
Acetic acid bacteria basal medium (YG): 1% yeast extract powder, 1% glucose, sterilizing at 0.1MPa and 121 ℃ for 20min, cooling the culture medium to 50-60 ℃, and adding 2% absolute ethyl alcohol.
Acetic acid bacteria plate count medium (YGC): 1% of yeast extract, 1% of glucose and 1.5% of CaCO31.5 percent of agar, sterilizing at the temperature of 121 ℃ under the pressure of 0.1MPa for 20min, cooling the culture medium to 50-60 ℃, and adding 2 percent of absolute ethyl alcohol.
An experimental instrument: biochemical incubator, superclean bench, inoculating loop, alcohol burner, multipoint inoculator and puncher.
2. The experimental method comprises the following steps:
2.1 determination of the Minimum Inhibitory Concentration (MIC)
(1) Lactobacillus (G)+): lactobacillus was inoculated in MRS slant tubes under aseptic conditions for 24h at 37 ℃ activation culture, and then inoculated in MRS liquid tubes (about 15mL per tube) for 24h at 37 ℃. Respectively diluting liquid test tube lactobacillus to 1 × 10 by plate counting method6CFU/mL、1×105CFU/mL、1×104CFU/mL was used.
(2) Acetobacter (G-): acetic acid bacteria in the absence ofInoculating the strain into a YGC slant tube under the condition of strain, performing activation culture at 30 ℃ for 48h, hooking a ring of single strain in the slant tube into a prepared beef extract peptone liquid culture medium, and then placing a shake flask containing the YG liquid culture medium in a 30 ℃ constant temperature shaking table for culturing for 48h to prepare a strain suspension stock solution. Diluting acetic acid bacteria stock solution to 1 × 10 by plate counting method6CFU/mL、1×105CFU/mL、1×104CFU/mL was used.
(3) Accurately weighing 0.2g of salinomycin, diluting with sterile water to a constant volume of 1000mL, and preparing into salinomycin bacteriostatic solution with the concentration of 200 mug/mL for later use. The salinomycin solution is bacteriostatic and diluted by sterile water to be 0, 20, 40, 60, 80, 100 and 120 mu g/mL salinomycin concentration gradient respectively.
(4) And (3) adding the salinomycin solutions with different concentrations prepared in the step (3) into the heated and dissolved MRS agar balanced in a water bath at 45-50 ℃ according to the volume ratio of 1: 9, fully mixing uniformly, pouring into a sterilization plate, and enabling the agar to be 3-4 mm thick. The salinomycin concentrations in the plates were 0, 2, 4, 6, 8, 10, 12. mu.g/mL, respectively. Sucking the lactobacillus suspension (about 1-2 mu L) prepared in the step (1) by using a multi-point inoculator, and respectively inoculating the lactobacillus suspension on the surfaces of agar plates containing salinomycin with different concentrations, wherein the bacterial count of each point is about 104CFU, forming bacterial plaque with the diameter of 5-8 mm. Culturing at 37 deg.C for 48 h. The plate was placed on a dark, non-reflective object surface to determine the end point of the test, with the minimum drug concentration inhibiting bacterial growth being MIC, the minimum inhibitory concentration being given in table 1.
(5) And (3) adding the salinomycin with different concentrations prepared in the step (3) into YGC agar which is heated, dissolved and balanced in a water bath at 45-50 ℃ according to the volume ratio of 1: 9 respectively, fully mixing uniformly, pouring into a sterilization plate, and enabling the agar to be 3-4 mm thick. The salinomycin concentrations in the plates were 0, 2, 4, 6, 8, 10, 12. mu.g/mL, respectively. Sucking the prepared acetic acid bacillus suspension (about 1-2 μ L) in step (2) by using a multi-point inoculator, and respectively inoculating the acetic acid bacillus suspension on the surfaces of agar plates containing salinomycin with different concentrations, wherein the bacterial count of each point is about 104CFU, forming bacterial plaque with the diameter of 5-8 mm. Culturing at 30 deg.C for 48 h. Placing the plate on the surface of dark and non-reflective object to determine the end point of test and minimize the growth of bacteriaThe drug concentration is MIC, and the minimum inhibitory concentration is shown in Table 1.
TABLE 1 determination of minimum inhibitory concentration of salinomycin against lactobacilli and acetobacter
As can be seen from the results shown in Table 1, salinomycin can effectively inhibit bacteria in a certain concentration range, and the MIC of the salinomycin to lactobacillus is more than or equal to 5 mug/mL, and the MIC to acetobacter is more than or equal to 8 mug/mL.
2.2 bacteriostatic loop method
(1) Sterilizing MRS agar culture medium, cooling to 45-50 deg.C, and injecting lactobacillus suspension (concentration of 1 × 10) 10% of culture medium6CFU/mL), mixed well, poured into the plate (about 20 mL/plate), set horizontally and stand for use.
(2) A6.0 mm diameter hole is punched on a culture medium of an MRS agar plate by using a sterilized puncher, 3 holes are punched on each plate, 0.1mL of salinomycin (10 mu g/mL) extract is injected into the hole by using a pipette, the holes are cultured in an incubator at 37 ℃ for 24 hours, then the holes are taken out for observing the growth condition of bacteria, and the diameter of a bacteriostatic circle is measured by using a vernier caliper. And simultaneously carrying out blank experiment comparison. The inhibition zone is shown in figure 1, and the diameter of the inhibition zone is shown in table 2.
(3) Sterilizing YGC agar culture medium, cooling to 45-50 deg.C, and injecting acetic acid bacteria suspension (concentration of 1 × 10) 10% of the culture medium6CFU/mL), mixed well, poured into the plate (about 20 mL/plate), set horizontally and stand for use.
(4) A6.0 mm diameter well was punched out of the culture medium of YGC agar plates using a sterilized punch, 3 wells were punched out of each plate, 0.1mL of a salinomycin (10. mu.g/mL) extract was injected into the well using a pipette, and after incubation at 37 ℃ for 24 hours in an incubator, the well was removed to observe the growth of bacteria, and the diameter of the zone of inhibition was measured using a vernier caliper. And simultaneously carrying out blank experiment comparison. The inhibition zone is shown in figure 2, and the diameter of the inhibition zone is shown in table 2.
TABLE 2 determination of zone of inhibition of salinomycin to lactic acid bacteria and acetic acid bacteria
As can be seen from FIGS. 1 and 2, as compared with the blank sample, salinomycin can significantly inhibit the growth and reproduction of lactobacillus and acetobacter, and as can be seen from Table 2, salinomycin has an average diameter of inhibition zones for lactobacillus of 11.76mm and an average diameter of inhibition zones for acetobacter of 9.36mm, which are both significantly larger than the aperture of the blank sample of 6mm, and has an obvious bacteriostatic effect.
In conclusion, salinomycin can obviously inhibit the growth of gram-positive bacteria (lactobacillus) and gram-negative bacteria (acetobacter) and is not easy to generate drug resistance and cross drug resistance. Salinomycin is mainly used as a feed additive in animal feeds, is mainly used for preventing chicken coccidiosis and is not used as a human antibacterial agent. The above experimental results suggest the possibility of salinomycin as an alcohol fermentation bactericide.
Example 2
(1) Preparing an alcohol fermentation salinomycin bactericide: respectively weighing 4g of salinomycin industrial salt (the salinomycin content is 45%) and 6g of salinomycin premix (the salinomycin content is 12%), weighing 1g of the prepared bactericide, putting the bactericide into a volumetric flask of 1000mL, adding sterile water to fully dissolve the bactericide, and fixing the volume for later use.
(2) Molasses fermentation alcohol: diluting 80Bx cane molasses with tap water to prepare 500mL of 20Bx molasses solution, adding 0.2 wt% of urea, inoculating 0.5 wt% of brewing active dry yeast (Yinglimali), adding 4mL of the bactericide solution prepared in the step (1) to a constant volume, uniformly mixing, fermenting at constant temperature of 31 ℃ for 48h, detecting the alcoholic strength and volatile acid of the fermentation liquid, and repeating the experiment for three times.
(3) The alcohol fermentation bacteriostasis contrast of the traditional bactericide is as follows: diluting 80Bx cane molasses with tap water to prepare 500mL of 20Bx molasses solution, adding 0.2 wt% of urea, inoculating 0.5 wt% of brewing active dry yeast, adjusting the pH value to 3.8 with concentrated sulfuric acid, uniformly mixing, fermenting at constant temperature of 31 ℃ for 48 hours, detecting the alcoholic strength and volatile acid of fermentation liquor, and repeating the experiment for three times.
As a result: as can be seen from the results shown in Table 3, FIG. 3 and FIG. 4, in molasses alcohol fermentation, compared with the traditional sulfuric acid bacteriostasis, the content of alcohol is obviously improved, the volatile acid is obviously reduced, the bacteria producing acid by infection are obviously reduced by adopting the salinomycin bactericide, and the bacteriostasis effect of the salinomycin bactericide is superior to that of concentrated sulfuric acid bacteriostasis.
TABLE 3 comparison of salinomycin disinfectant of the present invention with conventional disinfectant in molasses fermented alcohol
Example 3
(1) Preparing an alcohol fermentation salinomycin bactericide: 0.4g of salinomycin industrial salt (the content of the salinomycin is 45%) and 0.6g of salinomycin premix (the content of the salinomycin is 12%) are respectively weighed and placed into a volumetric flask with the volume of 1000mL, and after the salinomycin industrial salt and the salinomycin premix are fully dissolved by adding sterile water, the volume is fixed for standby.
(2) Fermenting alcohol by cassava: weighing 200g of cassava powder, dissolving with 500mL of tap water, adding 150U/g of high-temperature alpha-amylase (1, 4-alpha-D-glucan hydrolase) and uniformly stirring to prepare starch slurry, slowly heating to 100 ℃ for gelatinization for 30min, cooling to 95 ℃, keeping the temperature and continuously stirring for liquefaction for 60-120 min until iodine reaction does not show blue, thus preparing liquefied liquid. Cooling the liquefied liquid to 60-65 ℃, adjusting the pH value to 4.2-4.5 by using concentrated sulfuric acid, and adding 100U/g of saccharifying enzyme for saccharification for 30min to obtain the saccharifying liquid. Cooling the saccharified liquid to 31 ℃, adding 0.2 wt% of urea, inoculating 0.5 wt% of brewing active dry yeast, adding 7mL of the bactericide solution prepared in the step (1), uniformly mixing, fermenting at constant temperature of 31 ℃ for 48h, and detecting the alcoholic strength and volatile acid of the fermentation liquid. The experiment was repeated three times.
(3) The alcohol fermentation bacteriostasis contrast of the traditional bactericide is as follows: weighing 200g of cassava powder, dissolving with 500mL of tap water, adding 150U/g of high-temperature alpha-amylase, uniformly stirring to prepare starch slurry, slowly heating to 100 ℃ for gelatinization for 30min, cooling to 95 ℃, keeping the temperature, continuously stirring and liquefying for 60-120 min until iodine reaction does not show blue, and thus preparing liquefied liquid. Cooling the liquefied liquid to 60-65 ℃, adjusting the pH value to 4.2-4.5 by using concentrated sulfuric acid, and adding 100U/g of saccharifying enzyme for saccharification for 30min to obtain the liquefied liquid. Cooling the saccharified liquid to 31 ℃, adding 0.2 wt% of urea, inoculating 0.5 wt% of brewing active dry yeast, adding 5ppm of penicillin potassium, uniformly mixing, fermenting at constant temperature of 31 ℃ for 48 hours, detecting the alcoholic strength and volatile acid of the fermentation liquid, and repeating the experiment for three times. Data pairs such as table 5.
As a result: as can be seen from the results shown in table 4, fig. 5 and fig. 6, in the cassava alcohol fermentation, compared with the traditional sulfuric acid and penicillin bacteriostasis, the alcohol content is obviously improved, the volatile acid is obviously reduced, the bacteria producing acid by infection are obviously reduced by adopting the salinomycin bactericide, and the bacteriostasis effect of the salinomycin bactericide is better than that of concentrated sulfuric acid, penicillin and the like.
TABLE 4 comparison of salinomycin disinfectant of the present invention with conventional disinfectant in cassava fermented alcohol
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The application of salinomycin in preparing bactericides is characterized in that: the bacteria are acetic acid bacteria and/or lactic acid bacteria.
2. The use of salinomycin according to claim 1 in the preparation of a bactericide, wherein:
the minimum inhibitory concentration of salinomycin to acetic acid bacteria is 8 mug/mL, and the minimum inhibitory concentration of salinomycin to acetic acid bacteria is 5 mug/mL.
3. Use of salinomycin according to claim 1 or 2 for the preparation of a fungicide, characterized in that:
the bactericide further comprises one or more pharmaceutically acceptable carriers, excipients or diluents.
4. The salinomycin is used as a bactericide in alcohol fermentation.
5. The use of salinomycin as a bactericidal agent in alcoholic fermentation according to claim 4, wherein:
the addition amount of salinomycin is 1-10 ppm, and further 2-3 ppm of the fermentation liquor amount.
6. The use of salinomycin as a bactericidal agent in alcoholic fermentation according to claim 5, wherein:
the method for adding salinomycin comprises the following steps: dissolving salinomycin and/or a salinomycin premix fully by using sterile water, and then adding the salinomycin and/or the salinomycin premix into fermentation liquor according to the required addition amount.
7. An alcohol fermentation method is characterized in that: the method comprises the following steps:
s1, diluting the saccharine raw material to 20-40 Bx with water to obtain saccharine solution; or sequentially gelatinizing, liquefying and saccharifying the starchy raw materials to obtain a saccharified solution;
s2, adding 0.1-0.3 wt% of urea into the saccharine solution or saccharified solution obtained in the step S1, and inoculating 0.4-0.6 wt% of active dry saccharomyces cerevisiae; dissolving salinomycin and/or a salinomycin premix fully by using sterile water at 30-35 ℃, adding 1-10 ppm of salinomycin into fermentation liquor according to the amount of the fermentation liquor, uniformly mixing, and fermenting at 30-35 ℃ for 40-50 h to obtain a target product.
8. The alcohol fermentation method according to claim 7, wherein:
the sugar raw material in S1 is selected from one or more of sugarcane, beet, sweet sorghum and molasses, and the starchy raw material is selected from one or more of corn, cassava and sweet potato.
9. The alcohol fermentation method according to claim 7 or 8, wherein:
the preparation process of the saccharified solution in S1 is as follows: dissolving a starchy raw material by using 2-5 times of water, adding 100-150U/g of high-temperature alpha-amylase, uniformly stirring to prepare starch slurry, and slowly heating to 98-102 ℃ for gelatinization for 25-35 min; cooling to 90-95 ℃, keeping the temperature, continuously stirring and liquefying for 60-120 min until the iodine reaction does not show blue color, and preparing a liquefied solution; cooling the liquefied liquid to 60-65 ℃, adjusting the pH value to 4.2-4.5 by using concentrated sulfuric acid, and adding 100-150U/g of saccharifying enzyme for saccharifying for 25-35 min to obtain the saccharifying liquid.
10. The alcohol fermentation method according to claim 7, wherein:
the addition amount of the urea in the S2 is 0.2 wt%, and the addition amount of the active dry yeast for brewing is 0.5 wt%;
the fermentation conditions in S2 were 31 ℃ for 48 h.
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