CN111187739A - Compound microbial preparation and culture method and application thereof - Google Patents
Compound microbial preparation and culture method and application thereof Download PDFInfo
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- CN111187739A CN111187739A CN202010093435.0A CN202010093435A CN111187739A CN 111187739 A CN111187739 A CN 111187739A CN 202010093435 A CN202010093435 A CN 202010093435A CN 111187739 A CN111187739 A CN 111187739A
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Abstract
The invention relates to the technical field of microbial culture, in particular to a compound microbial preparation and a culture method and application thereof. The invention adopts a mixed culture technology, and lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis are respectively subjected to activation, liquid strain culture, composite strain culture and finished product culture to obtain a composite microbial preparation which can be used for live pig culture and has the effects of promoting growth, helping digestion, resisting diseases, increasing animal weight, reducing feed consumption and the like. The preparation method improves the biomass and physiological metabolism function of microorganism; makes up the deficiency of single culture metabolism, promotes the growth of compound microorganisms by symbiosis, paragenetic symbiosis and the like, and has a plurality of obvious advantages compared with the traditional pure culture technology.
Description
Technical Field
The invention relates to the technical field of microbial culture, in particular to a compound microbial preparation and a culture method and application thereof.
Background
The mixed culture refers to the culture of 2 or more than 2 microorganisms, is a new development of pure culture technology, and is also a novel culture technology which can obtain similar effects without carrying out complicated DNA in vitro recombination. The utilization of the microorganisms by human beings goes through two stages from natural mixed culture to pure culture, the pure culture enables researchers to get rid of the complex situation of coexistence of various microorganisms, and the researchers can study a single target strain without interference, thereby enriching the understanding of morphological structure, physiology and genetic characteristics of the microorganisms. However, in long-term experimental and production practice, it has been found that many important biochemical processes cannot be performed or can be performed only weakly by a single strain of microorganism, and must be performed by co-culturing two or more microorganisms. The mixed culture of various microorganisms becomes an important content in the research of the microbial preparation at present, and the composite microbial preparation prepared by mixed culture of a plurality of microorganisms with different functions and a mutual or symbiotic relationship according to a proper proportion is applied to the aspects of planting, breeding, environmental protection and the like, so that the ecological balance can be adjusted, and the ecological environment can be protected. With the rapid development of modern industrial and agricultural production and the continuous upgrading of industrial structures, the mixed culture of microorganisms has great economic and scientific research values in the aspects of promoting the national economic growth, improving the environment, developing new medicines and the like.
Disclosure of Invention
In order to overcome the defects and shortcomings of the prior art, the invention aims at providing a culture method of a compound microbial preparation, which is characterized in that a plurality of microorganisms are cultured together according to a proper proportion, the combined action advantages of the groups are fully exerted, and the optimal application effect is achieved.
Another object of the present invention is to provide a complex microbial preparation cultured by the above-mentioned culture method.
The invention also aims to provide application of the composite microbial preparation.
The purpose of the invention is realized by the following technical scheme:
a culture method of a compound microbial preparation comprises the following steps:
(1) and (3) strain activation culture: respectively activating the frozen lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis for multiple times to obtain activated strains of lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis;
(2) liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum prepared in the step (1) into a liquid culture medium in a mass ratio, and carrying out anaerobic culture at 37 ℃ for 2-3 days to obtain a mixed lactobacillus seed solution;
② inoculating the activated strain of Bifidobacterium bifidum prepared in step (1), and anaerobically culturing in liquid culture medium at 37 deg.C for 2-3 days to obtain Bifidobacterium bifidum seed solution;
③ inoculating the activated strains of the bacillus subtilis and the bacillus licheniformis prepared in the step (1) into a liquid culture medium in a mass ratio, and culturing for 1-2 days at a shaker rotation speed of 150-200 r/min and a temperature of 30-35 ℃ to obtain a mixed bacillus seed solution;
④ inoculating the activated strain of Rhodopseudomonas palustris prepared in step (1) into a liquid culture medium, and performing anaerobic culture at 60 ℃ and 30-35 ℃ for 5-7 days under the illumination of a tungsten filament lamp to obtain a Rhodopseudomonas palustris seed solution;
⑤ inoculating the beer yeast and the candida utilis activated strain prepared in the step (1) into a liquid culture medium according to the mass ratio, and culturing for 2 days at the rotating speed of a shaking table of 150-200 r/min and the temperature of 28-30 ℃ to obtain mixed yeast seed liquid;
(3) culturing a composite strain: simultaneously inoculating the mixed bacillus seed liquid and the mixed yeast seed liquid prepared in the step (2) into a fermentation culture medium 1, culturing for 1-2 days at the temperature of 30 ℃ at the rotating speed of a shaking table of 150-200 r/min, and then simultaneously inoculating the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid and the rhodopseudomonas palustris seed liquid prepared in the step (2) into the fermentation system, and carrying out anaerobic culture for 3-5 days at the temperature of 30-35 ℃ to obtain a composite microbial preparation strain, wherein the inoculation amount ratio (mass ratio) of the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid, the mixed bacillus seed liquid, the rhodopseudomonas palustris seed liquid and the mixed yeast seed liquid is 5:4:3:3: 5; the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation: inoculating the composite microbial preparation strain prepared in the step (3) into a fermentation culture medium 2 according to the inoculation amount of 5-8% by weight, and performing closed fermentation at 28-30 ℃ for 7-15 days to obtain a finished product of the composite microbial preparation; wherein, the fermentation culture medium in the step is not sterilized;
the strains of lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis in the step (1) are respectively as follows: lactobacillus acidophilus (Lactobacillus acidophilus) CICC 6083, Lactobacillus plantarum (Lactobacillus plantarum) CICC21790, Bifidobacterium bifidum (Bifidobacterium bifidum) CICC 6166, Bacillus subtilis (Bacillus subtilis) CICC24434, Bacillus licheniformis (Bacillus licheniformis) CGMCC1.10257, Rhodopseudomonas palustris (Rhodopseudomonas palustris) CGMCC 1.8929, Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC2.3973, Candida utilis (Candida utilis) CGMCC 2.3047;
the specific operation of the multiple activation in the step (1) is preferably: carrying out three times of slant activation on the frozen and preserved lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, saccharomyces cerevisiae and candida utilis strains, then carrying out flat plate purification culture, and finally carrying out slant culture to obtain lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, saccharomyces cerevisiae and candida utilis strains;
the conditions of slant activation, plate purification culture or slant culture are preferably as follows:
carrying out facultative anaerobic culture on lactobacillus acidophilus or lactobacillus plantarum at 37 ℃ for 2-3 days,
bifidobacterium bifidum is anaerobically cultured for 3-5 days at 37 ℃,
aerobic culture of bacillus subtilis and bacillus licheniformis is carried out for 1-2 days at 30 ℃;
carrying out anaerobic culture on rhodopseudomonas palustris for 5-7 days at the temperature of 30-35 ℃ under the irradiation of 60-watt black-silk lamp light;
carrying out aerobic culture on the beer yeast or the candida utilis at 28-30 ℃ for 2 days;
each liter of the liquid culture medium (lactic acid bacteria) described in the step (2) ① comprises 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 5 g of glucose, 5 g of sodium acetate, 2 g of diamine citrate, 801 mL of tween, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of calcium carbonate, 20mL of tomato juice, 30mL of potato juice, 60mL of carrot juice, 2 g of vitamin C, and 1000mL of weakly alkaline small molecule reducing water, wherein the pH value is 6.8;
each liter of the liquid culture medium (bifidobacterium bifidum) in the step (2) ② comprises 15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5 g of soluble starch, 5.0 g of sodium chloride, 10.0mL of 5% cysteine, 400.0mL of tomato extract, 801.0 mL of tween, 80.0mL of liver extract, and 1000mL of weakly alkaline small molecule reducing water with the pH value of 7.0;
each liter of the liquid culture medium (bacillus) in the step (2) ③ comprises 30 g of glucose, 15 g of yeast extract, 2 g of disodium hydrogen phosphate, 1 g of sodium dihydrogen phosphate and 1000mL of weakly alkaline small molecule reducing water with pH of 7.2;
each liter of the liquid culture medium (rhodopseudomonas palustris) in the step (2) ④ comprises 10 g of yeast powder, 1 g of dipotassium phosphate, 0.5 g of magnesium sulfate and 1000mL of weakly alkaline small molecular reducing water, wherein the pH value is 7.0-7.2;
each liter of the liquid culture medium (yeast) in the step (2) ⑤ comprises 50 g of brown sugar, 20 g of yeast extract, 1 g of ammonium sulfate and 2 g of potassium chloride, the pH value is natural, and the weak alkaline small molecule reducing water is supplemented to 1000 mL;
the fermentation medium 1 in the step (3) comprises the following components in percentage by mass:
5% of industrial waste molasses, 0.5% of yeast powder, 1% of peptone, 1% of banana polysaccharide, 0.1% of ammonium chloride, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weak alkaline small molecular reducing water to 100%;
the fermentation medium 2 in the step (4) comprises the following components in percentage by mass: 5-8% of industrial waste molasses, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weak alkaline small-molecule reducing water to 100%;
the specific operation of the closed fermentation in the step (4) is preferably:
① cleaning culture container and container related to culture medium preparation, and sterilizing with acidic oxidation potential water (acidified disinfectant water for short);
② the components of the fermentation medium (banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, ferrous sulfate, etc.) are weighed according to the proportion and added into the container 1, and then weak alkaline small molecule reducing water is added to make the components fully dissolved;
③ adding industrial waste molasses into the container 2, and adding weakly alkaline small molecule reducing water to dissolve completely;
④ adding the solution prepared in step ② and the solution prepared in step ③ into a culture container, and then adding weak alkaline small molecule reducing water to 80% of the total volume of the culture medium;
⑤, shaking the strains of the compound microbial preparation prepared in the step (3) uniformly, and inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of the culture medium) of 5-8% by weight;
⑥ inoculating, adding weak alkaline micromolecule reducing water into the culture container until the total volume of the culture medium is 100%, uniformly stirring, and carrying out closed fermentation at 28-30 ℃ for 7-15 days to obtain a finished product of the compound microbial preparation;
a compound microbial preparation is prepared by the above preparation method;
the compound microbial preparation is applied to the fields of pig breeding and the like;
compared with the prior art, the invention has the following advantages and effects:
the invention adopts mixed culture technology (figure 1), and various microorganisms mutually provide specific growth nutrients; improving the biomass and physiological metabolism function of the microorganism; make up the deficiency on the metabolism of the single culture, utilize and symbiotic, the growth that favors the compound microorganism of promotion such as symbiosis, etc., compared with traditional pure culture technique, have many obvious advantages, specifically as follows:
(1) the yield is high: many pure culture processes cannot be completed or can only be performed weakly, and good effects are often obtained by using mixed culture.
(2) The growth rate is high: in mixed culture, one microorganism may produce growth factors for another microorganism, or produce carbon or nitrogen sources more readily utilized by another microorganism. Even the metabolites produced will change the pH of the medium making it more suitable for the growth of another microorganism.
(3) Mixed culture can utilize a wider range of substrates: many fermentation substrates are mixtures of carbohydrates, proteins and fats. Since mixed cultures contain different microorganisms and contain correspondingly more enzyme systems than pure cultures, the range of available substrates will also be expanded.
(4) The probability of phage infection is greatly reduced in mixed cultures, and in pure cultures, bacteria often fail fermentation due to phage infection. However, when the hybrid culture is carried out, even if a certain bacterium infects a phage, the fermentation will not fail because of the wide genetic characteristic of the anti-phage, and the other bacterium can carry out the fermentation.
(5) Many fermentation processes which are difficult to achieve in pure culture or have low yields can be achieved in mixed culture.
(6) The fermentation medium can be directly used for culturing the compound microbial preparation without being disinfected, and can also be stirred in the culture process, so that manpower and material resources can be saved, and the economic benefit is improved.
Drawings
FIG. 1 is a process flow diagram of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The strains of lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis related in the embodiment are respectively as follows: lactobacillus acidophilus (Lactobacillus acidophilus) CICC 6083, Lactobacillus plantarum (Lactobacillus plantarum) CICC21790, Bifidobacterium bifidum (Bifidobacterium bifidum) CICC 6166, Bacillus subtilis (Bacillus subtilis) CICC24434, Bacillus licheniformis (Bacillus licheniformis) CGMCC1.10257, Rhodopseudomonas palustris (Rhodopseudomonas palustris) CGMCC 1.8929, Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC2.3973, Candida utilis (Candida utilis) CGMCC 2.3047;
the acidic electrolyzed oxidizing water (called acidified disinfectant water for short) in the embodiment is an aqueous solution which is prepared by adding less than 0.1 percent of salt (sodium chloride) into water, inputting the aqueous solution into an electrolytic cell for electrolysis, generating an anode of the electrolytic cell, wherein the pH value of the aqueous solution is 2-3, the oxidation-reduction potential of the aqueous solution is more than 1100mV, and the active chlorine is 50-70 mg.
In the embodiment, in the strain activation culture (slant activation, plate purification culture or slant culture) process in the step (1), the culture medium of each strain is a conventional culture medium;
in the embodiment, in the liquid strain culture process in the step (2), the culture medium comprises the following components:
the liquid culture medium of lactobacillus acidophilus and lactobacillus plantarum comprises the following components in each liter: 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 5 g of glucose, 5 g of sodium acetate, 2 g of diamine citrate, 801 mL of tween, 2 g of dipotassium phosphate, 0.2 g of magnesium sulfate, 0.05 g of manganese sulfate, 20 g of calcium carbonate, 20mL of tomato juice, 30mL of potato juice, 60mL of carrot juice, 2 g of vitamin C and 1000mL of weakly alkaline small molecular reducing water, wherein the pH value is 6.8;
the liquid culture medium of Bifidobacterium bifidum contains the following components per liter: 15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5 g of soluble starch, 5.0 g of sodium chloride, 10.0mL of 5% cysteine, 400.0mL of tomato extract, 801.0 mL of tween, 80.0mL of liver extract, supplementing weakly alkaline small molecular reducing water to 1000mL, and pH 7.0;
the liquid culture medium of bacillus subtilis and bacillus licheniformis comprises the following components in each liter: 30 g of glucose, 15 g of yeast extract, 2 g of disodium hydrogen phosphate, 1 g of sodium dihydrogen phosphate, 1000mL of weakly alkaline small molecular reducing water and pH 7.2;
the liquid culture medium of rhodopseudomonas palustris comprises the following components per liter: 10 g of yeast powder, 1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate and 1000mL of weakly alkaline small molecular reducing water with the pH value of 7.0-7.2;
the liquid culture medium of beer yeast and candida utilis contains the following components in each liter: 50 g of brown sugar, 20 g of yeast extract, 1 g of ammonium sulfate, 2 g of potassium chloride, natural pH and supplementing to 1000mL of weakly alkaline micromolecular reducing water;
in the process of culturing the composite strain in the step (3), the culture medium comprises the following components:
the fermentation medium 1 comprises the following components in percentage by mass:
5% of industrial waste molasses, 0.5% of yeast powder, 1% of peptone, 1% of banana polysaccharide, 0.1% of ammonium chloride, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weak alkaline small molecular reducing water to 100%;
example 1
(1) And (3) strain activation culture: respectively activating lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis in the frozen storage tube for three times in a test tube inclined plane with the diameter of 18 multiplied by 180mm, then respectively inoculating the activated lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and the candida utilis in a culture dish with the diameter of 90mm for purification culture, and then respectively culturing in a test tube inclined plane with the diameter of 18 multiplied by 180mm to obtain activated strains of the lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and; wherein, the culture conditions are as follows: carrying out facultative anaerobic culture on lactobacillus acidophilus and lactobacillus plantarum at 37 ℃ for 2 days; anaerobic culturing Bifidobacterium bifidum at 37 deg.C for 3 days, and aerobic culturing Bacillus subtilis and Bacillus licheniformis at 30 deg.C for 2 days; culturing Rhodopseudomonas palustris with 60W black-silk lamp at 30 deg.C for 7 days; carrying out aerobic culture on beer yeast and candida utilis for 2 days at 28 ℃.
(2) Liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum prepared in step (1) with inoculating loops respectively, inoculating to sterilized liquid culture medium, and placing at 37 deg.C for anaerobic mixed culture for 2 days to obtain mixed lactobacillus seed solution;
② inoculating the active strain of Bifidobacterium bifidum obtained in step (1) with an inoculating loop to a sterilized liquid culture medium, and placing at 37 deg.C for anaerobic culture for 3 days to obtain a Bifidobacterium bifidum seed solution;
③ respectively inoculating a loop of the activated strains of Bacillus subtilis and Bacillus licheniformis prepared in step (1) into a sterilized liquid culture medium, and performing mixed culture at a shaker rotation speed of 200r/min and a temperature of 35 ℃ in a liquid loading amount of 30mL/250mL for 1 day to obtain a mixed Bacillus subtilis seed solution;
④ inoculating the activated strain of Rhodopseudomonas palustris prepared in step (1) into sterilized liquid culture medium with 90mL/100mL of liquid, and anaerobically culturing at 60 deg.C under tungsten lamp illumination of 35 deg.C for 5 days to obtain Rhodopseudomonas palustris seed liquid;
⑤ respectively inoculating a ring of the activated strains of the beer yeast and the candida utilis prepared in the step (1) into a sterilized liquid culture medium by using an inoculating ring, carrying out mixed culture for 2 days at a shaker rotating speed of 150r/min and a temperature of 28 ℃ and a liquid loading capacity of 25mL/250mL, and obtaining a mixed yeast seed solution;
(3) culturing a composite strain: inoculating the mixed bacillus seed liquid and the mixed yeast seed liquid prepared in the step (2) into a sterilized fermentation culture medium 1 at the same time, culturing for 1 day at the rotating speed of a shaking table of 200r/min at the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then inoculating the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid and the rhodopseudomonas palustris seed liquid prepared in the step (2) into the fermentation system at the same time, and performing anaerobic culture for 5 days at the temperature of 30 ℃ to obtain a composite microbial preparation strain, wherein the inoculation ratio (weight ratio) of the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid, the mixed bacillus seed liquid, the rhodopseudomonas palustris seed liquid and the mixed yeast seed liquid is 5:4:3:3:5, and the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation:
① cleaning culture container and container related to culture medium preparation, and sterilizing with acidic oxidation potential water (acidified disinfectant water for short);
② weighing banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate and ferrous sulfate, adding into container 1, and adding weakly alkaline small molecule reduced water to dissolve completely;
③ adding industrial waste molasses into the container 2, and adding weakly alkaline small molecule reducing water to dissolve completely;
④ adding the solution prepared in step ② and the solution prepared in step ③ into a culture container, washing the container for dissolving the culture medium and molasses with a small amount of weakly alkaline small molecule reducing water for several times to ensure that all the components are added into the culture container, and then adding the weakly alkaline small molecule reducing water to 80% of the total volume of the culture medium;
⑤, shaking the strains of the compound microbial preparation prepared in the step (3), inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of a culture medium) of 5 percent by weight, measuring a small amount of weak alkaline micromolecule reduced water in the strain container, and washing the strain container for several times to ensure that the strains are all added into the culture container;
⑥ inoculating, adding weak alkaline micromolecular reducing water into the culture container to 100% of the total volume of the culture medium, uniformly stirring, covering a cover, sealing and fermenting at 28 ℃ for 15 days to obtain the finished product of the composite microbial preparation, wherein the fermentation culture medium comprises 5 wt% of industrial waste molasses, 0.3 wt% of banana polysaccharide, 0.2 wt% of ammonium chloride, 0.05 wt% of sodium chloride, 0.1 wt% of potassium dihydrogen phosphate, 0.05 wt% of magnesium sulfate, 0.025 wt% of zinc sulfate, 0.025 wt% of ferrous sulfate and the balance of weak alkaline micromolecular reducing water.
Example 2
(1) And (3) strain activation culture: respectively activating lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis in the frozen storage tube for three times in a test tube inclined plane with the diameter of 18 multiplied by 180mm, then respectively inoculating the activated lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and the candida utilis in a culture dish with the diameter of 90mm for purification culture, and then respectively culturing in a test tube inclined plane with the diameter of 18 multiplied by 180mm to obtain activated strains of the lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and; wherein, the culture conditions are as follows: performing facultative anaerobic culture of Lactobacillus acidophilus and Lactobacillus plantarum at 37 deg.C for 3 days; anaerobic culturing Bifidobacterium bifidum at 37 deg.C for 3 days, and aerobic culturing Bacillus subtilis and Bacillus licheniformis at 30 deg.C for 2 days; culturing Rhodopseudomonas palustris under illumination of 60W-black silk lamp at 35 deg.C for 5 days; carrying out aerobic culture on beer yeast and candida utilis for 2 days at 28 ℃.
(2) Liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum prepared in step (1) with inoculating loops respectively, inoculating to sterilized liquid culture medium, and placing at 37 deg.C for 3 days to obtain mixed lactobacillus seed solution;
② inoculating the active strain of Bifidobacterium bifidum obtained in step (1) with an inoculating loop to a sterilized liquid culture medium, and placing at 37 deg.C for anaerobic culture for 2 days to obtain a Bifidobacterium bifidum seed solution;
③ inoculating the activated strains of Bacillus subtilis and Bacillus licheniformis obtained in step (1) with a loop of inoculating loop respectively, and culturing at 30 deg.C and 30mL/250mL for 2 days at a table rotation speed of 150r/min to obtain mixed Bacillus subtilis seed solution;
④ inoculating the activated strain of Rhodopseudomonas palustris prepared in step (1) into sterilized liquid culture medium with 90mL/100mL of liquid, and anaerobically culturing at 60 deg.C under tungsten lamp illumination of 35 deg.C for 5 days to obtain Rhodopseudomonas palustris seed liquid;
⑤ respectively inoculating a ring of the activated strains of the beer yeast and the candida utilis prepared in the step (1) into a sterilized liquid culture medium by using an inoculating ring, carrying out mixed culture for 2 days at a shaker rotating speed of 200r/min and a temperature of 30 ℃ and a liquid loading capacity of 25mL/250mL, and obtaining a mixed yeast seed solution;
(3) culturing a composite strain: inoculating the mixed bacillus seed liquid and the mixed yeast seed liquid prepared in the step (2) into a sterilized fermentation culture medium 1 at the same time, culturing for 2 days at the rotation speed of a shaking table of 150r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then inoculating the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid and the rhodopseudomonas palustris seed liquid prepared in the step (2) into the fermentation system at the same time, and performing anaerobic culture for 3 days at the temperature of 30 ℃ to obtain a composite microbial preparation strain, wherein the inoculation ratio (weight ratio) of the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid, the mixed bacillus seed liquid, the rhodopseudomonas palustris seed liquid and the mixed yeast seed liquid is 5:4:3:3:5, and the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation:
① cleaning culture container and container related to culture medium preparation, and sterilizing with acidic oxidation potential water (acidified disinfectant water for short);
② weighing banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate and ferrous sulfate, adding into container 1, and adding weakly alkaline small molecule reduced water to dissolve completely;
③ adding industrial waste molasses into the container 2, and adding weakly alkaline small molecule reducing water to dissolve completely;
④ adding the solution prepared in step ② and the solution prepared in step ③ into a culture container, washing the container for dissolving the culture medium and molasses with a small amount of weakly alkaline small molecule reducing water for several times to ensure that all the components are added into the culture container, and then adding the weakly alkaline small molecule reducing water to 80% of the total volume of the culture medium;
⑤, shaking the strains of the compound microbial preparation prepared in the step (3), inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of a culture medium) of 6 percent by weight, measuring a small amount of weak alkaline micromolecule reduced water in the strain container, and washing the strain container for several times to ensure that the strains are all added into the culture container;
⑥ inoculating, adding weak alkaline small molecule reducing water into the culture container to 100% of the total volume of the culture medium, stirring uniformly, covering the container, sealing and fermenting at 30 deg.C for 10 days to obtain the final product of compound microorganism preparation, wherein the fermentation medium comprises industrial waste molasses 6%, banana polysaccharide 0.3 wt%, ammonium chloride 0.2 wt%, sodium chloride 0.05 wt%, potassium dihydrogen phosphate 0.1 wt%, magnesium sulfate 0.05 wt%, zinc sulfate 0.025 wt%, ferrous sulfate 0.025 wt%, and weak alkaline small molecule reducing water in balance.
Example 3
(1) And (3) strain activation culture: respectively activating lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis in the frozen storage tube for three times in a test tube inclined plane with the diameter of 18 multiplied by 180mm, then respectively inoculating the activated lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and the candida utilis in a culture dish with the diameter of 90mm for purification culture, and then respectively culturing in a test tube inclined plane with the diameter of 18 multiplied by 180mm to obtain activated strains of the lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and; wherein, the culture conditions are as follows: performing facultative anaerobic culture of Lactobacillus acidophilus and Lactobacillus plantarum at 37 deg.C for 3 days; anaerobic culturing Bifidobacterium bifidum at 37 deg.C for 3 days, and aerobic culturing Bacillus subtilis and Bacillus licheniformis at 30 deg.C for 2 days; culturing Rhodopseudomonas palustris under illumination of 60W-black silk lamp at 35 deg.C for 5 days; carrying out aerobic culture on beer yeast and candida utilis for 2 days at 28 ℃.
(2) Liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum prepared in step (1) with inoculating loops respectively, inoculating to sterilized liquid culture medium, and placing at 37 deg.C for 3 days to obtain mixed lactobacillus seed solution;
② inoculating the active strain of Bifidobacterium bifidum obtained in step (1) with an inoculating loop to a sterilized liquid culture medium, and placing at 37 deg.C for anaerobic culture for 2 days to obtain a Bifidobacterium bifidum seed solution;
③ inoculating the activated strains of Bacillus subtilis and Bacillus licheniformis obtained in step (1) with a loop of inoculating loop respectively, and culturing at 30 deg.C and 30mL/250mL for 1.5 days at a table rotation speed of 150r/min to obtain mixed Bacillus subtilis seed solution;
④ inoculating the activated strain of Rhodopseudomonas palustris prepared in step (1) into sterilized liquid culture medium with 90mL/100mL of liquid, and performing anaerobic culture at 60 deg.C under tungsten lamp illumination at 35 deg.C for 6 days to obtain Rhodopseudomonas palustris seed liquid;
⑤ respectively inoculating a ring of the activated strains of the beer yeast and the candida utilis prepared in the step (1) into a sterilized liquid culture medium by using an inoculating ring, carrying out mixed culture for 2 days at a shaker rotating speed of 200r/min and a temperature of 30 ℃ and a liquid loading capacity of 25mL/250mL, and obtaining a mixed yeast seed solution;
(3) culturing a composite strain: inoculating the mixed bacillus seed liquid and the mixed yeast seed liquid prepared in the step (2) into a sterilized fermentation culture medium 1 at the same time, culturing for 1 day at the rotating speed of a shaking table of 200r/min at the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then inoculating the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid and the rhodopseudomonas palustris seed liquid prepared in the step (2) into the fermentation system at the same time, and performing anaerobic culture for 4 days at the temperature of 30 ℃ to obtain a composite microbial preparation strain, wherein the inoculation ratio (weight ratio) of the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid, the mixed bacillus seed liquid, the rhodopseudomonas palustris seed liquid and the mixed yeast seed liquid is 5:4:3:3:5, and the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation:
① cleaning culture container and container related to culture medium preparation, and sterilizing with acidic oxidation potential water (acidified disinfectant water for short);
② weighing banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate and ferrous sulfate, adding into container 1, and adding weakly alkaline small molecule reduced water to dissolve completely;
③ adding industrial waste molasses into the container 2, and adding weakly alkaline small molecule reducing water to dissolve completely;
④ adding the solution prepared in step ② and the solution prepared in step ③ into a culture container, washing the container for dissolving the culture medium and molasses with a small amount of weakly alkaline small molecule reducing water for several times to ensure that all the components are added into the culture container, and then adding the weakly alkaline small molecule reducing water to 80% of the total volume of the culture medium;
⑤, shaking the strains of the compound microbial preparation prepared in the step (3), inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of a culture medium) of 8 percent of the weight percentage, and measuring a small amount of weak alkaline micromolecule reduced water in the strain container to wash the strain container for several times so as to ensure that the strains are all added into the culture container;
⑥ inoculating, adding weak alkaline small molecule reducing water into the culture container to 100% of the total volume of the culture medium, stirring, covering with a cover, and fermenting at 30 deg.C for 7 days to obtain the final product, wherein the fermentation medium comprises industrial waste molasses 6%, banana polysaccharide 0.3 wt%, ammonium chloride 0.2 wt%, sodium chloride 0.05 wt%, potassium dihydrogen phosphate 0.1 wt%, magnesium sulfate 0.05 wt%, zinc sulfate 0.025 wt%, ferrous sulfate 0.025 wt%, and weak alkaline small molecule reducing water in balance.
Example 4
(1) And (3) strain activation culture: respectively activating lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis in the frozen storage tube for three times in a test tube inclined plane with the diameter of 18 multiplied by 180mm, then respectively inoculating the activated lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and the candida utilis in a culture dish with the diameter of 90mm for purification culture, and then respectively culturing in a test tube inclined plane with the diameter of 18 multiplied by 180mm to obtain activated strains of the lactobacillus acidophilus, the lactobacillus plantarum, the bifidobacterium bifidum, the bacillus subtilis, the bacillus licheniformis, the rhodopseudomonas palustris, the beer yeast and; wherein, the culture conditions are as follows: performing facultative anaerobic culture of Lactobacillus acidophilus and Lactobacillus plantarum at 37 deg.C for 3 days; anaerobic culturing Bifidobacterium bifidum at 37 deg.C for 3 days, and aerobic culturing Bacillus subtilis and Bacillus licheniformis at 30 deg.C for 2 days; culturing Rhodopseudomonas palustris under illumination of 60W-black silk lamp at 35 deg.C for 5 days; carrying out aerobic culture on beer yeast and candida utilis for 2 days at 28 ℃.
(2) Liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum prepared in step (1) with inoculating loops respectively, inoculating to sterilized liquid culture medium, and placing at 37 deg.C for 3 days to obtain mixed lactobacillus seed solution;
② inoculating the active strain of Bifidobacterium bifidum obtained in step (1) with an inoculating loop to a sterilized liquid culture medium, and placing at 37 deg.C for anaerobic culture for 2 days to obtain a Bifidobacterium bifidum seed solution;
③ inoculating the activated strains of Bacillus subtilis and Bacillus licheniformis obtained in step (1) with a loop of inoculating loop respectively, and culturing in a shaking table at a rotation speed of 200r/min and a temperature of 35 deg.C and a liquid loading of 30mL/250mL for 1.5 days to obtain a mixed Bacillus subtilis seed solution;
④ inoculating the activated strain of Rhodopseudomonas palustris prepared in step (1) into a sterilized liquid culture medium by using an inoculating loop, loading the liquid in the culture medium in a volume of 90mL/100mL, and then performing anaerobic culture at 60 ℃ and 35 ℃ under the illumination of a tungsten lamp for 6 days to obtain Rhodopseudomonas palustris seed liquid;
⑤ respectively inoculating a ring of the activated strains of the beer yeast and the candida utilis prepared in the step (1) into a sterilized liquid culture medium by using an inoculating ring, carrying out mixed culture for 2 days at a shaking table rotating speed of 150r/min and a temperature of 30 ℃ and a liquid loading capacity of 25mL/250mL, and obtaining a mixed yeast seed solution;
(3) culturing a composite strain: inoculating the mixed bacillus seed liquid and the mixed yeast seed liquid prepared in the step (2) into a sterilized fermentation culture medium 1 at the same time, culturing for 2 days at the rotating speed of a shaking table of 150r/min at the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, then inoculating the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid and the rhodopseudomonas palustris seed liquid prepared in the step (2) into the fermentation system at the same time, and performing anaerobic culture for 5 days at the temperature of 30 ℃ to obtain a composite microbial preparation strain, wherein the inoculation ratio (weight ratio) of the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid, the mixed bacillus seed liquid, the rhodopseudomonas palustris seed liquid and the mixed yeast seed liquid is 5:4:3:3:5, and the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation:
① cleaning culture container and container related to culture medium preparation, and sterilizing with acidic oxidation potential water (acidified disinfectant water for short);
② weighing banana polysaccharide, ammonium chloride, sodium chloride, potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate and ferrous sulfate, adding into container 1, and adding weakly alkaline small molecule reduced water to dissolve completely;
③ adding industrial waste molasses into the container 2, and adding weakly alkaline small molecule reducing water to dissolve completely;
④ adding the solution prepared in step ② and the solution prepared in step ③ into a culture container, washing the container for dissolving the culture medium and molasses with a small amount of weakly alkaline small molecule reducing water for several times to ensure that all the components are added into the culture container, and then adding the weakly alkaline small molecule reducing water to 80% of the total volume of the culture medium;
⑤, shaking the strains of the compound microbial preparation prepared in the step (3), inoculating the strains into a culture container according to the inoculation amount (relative to the total amount of a culture medium) of 6 percent by weight, measuring a small amount of weak alkaline micromolecule reduced water in the strain container, and washing the strain container for several times to ensure that the strains are all added into the culture container;
⑥ inoculating, adding weak alkaline micromolecular reducing water into the culture container to 100% of the total volume of the culture medium, uniformly stirring, covering a cover, and hermetically fermenting at 30 ℃ for 9 days to obtain the finished product of the composite microbial preparation, wherein the fermentation culture medium comprises 8 wt% of industrial waste molasses, 0.3 wt% of banana polysaccharide, 0.2 wt% of ammonium chloride, 0.05 wt% of sodium chloride, 0.1 wt% of potassium dihydrogen phosphate, 0.05 wt% of magnesium sulfate, 0.025 wt% of zinc sulfate, 0.025 wt% of ferrous sulfate and the balance of weak alkaline micromolecular reducing water.
Comparative example 1
(1) And (3) strain activation culture: the specific procedure is the same as in example 2.
(2) Liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum obtained in step (1) with inoculating loop respectively, inoculating to sterilized liquid culture medium (each strain is cultured separately), and placing at 37 deg.C for 3 days to obtain Lactobacillus acidophilus seed solution and Lactobacillus plantarum seed solution;
② Bifidobacterium bifidum seed liquid, which is prepared by the method described in example 2;
③ respectively inoculating the activated strains of Bacillus subtilis and Bacillus licheniformis prepared in step (1) with an inoculating loop, inoculating into a sterilized liquid culture medium (each strain is cultured independently), and culturing at a table rotation speed of 150r/min and a temperature of 30 deg.C and a liquid loading amount of 30mL/250mL for 2 days to obtain Bacillus subtilis seed liquid and Bacillus licheniformis seed liquid respectively;
④ Rhodopseudomonas palustris seed liquid, the concrete method is the same as example 2;
⑤ respectively inoculating the activated strains of cerevisiae fermentum and Candida utilis prepared in step (1) with an inoculating loop to a sterilized liquid culture medium (each strain is cultured independently), culturing at a table rotation speed of 200r/min and a temperature of 30 deg.C and a liquid loading amount of 25mL/250mL for 2 days to obtain a cerevisiae fermentum seed solution and a Candida utilis seed solution;
(3) culturing a composite strain: simultaneously inoculating the eight seed solutions prepared in the step (2) into a sterilized fermentation culture medium 1, culturing for 2 days at the rotating speed of a shaking table of 200r/min and the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL, and then carrying out anaerobic culture at the temperature of 30 ℃ for 3 days to obtain a composite microbial preparation strain, wherein the inoculation amount ratio of lactobacillus acidophilus, lactobacillus plantarum seed solution, bifidobacterium bifidum seed solution, bacillus subtilis, bacillus licheniformis seed solution, rhodopseudomonas palustris seed solution to beer yeast and candida utilis seed solution is (weight ratio) 5:5:4:3:3:3:5: 5; the total inoculum size was 10% by weight.
Comparative example 2
(1) And (3) strain activation culture: the specific procedure is the same as in example 2.
(2) Liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum obtained in step (1) with inoculating loop respectively, inoculating to sterilized liquid culture medium (each strain is cultured separately), and placing at 37 deg.C for 3 days to obtain Lactobacillus acidophilus seed solution and Lactobacillus plantarum seed solution;
② Bifidobacterium bifidum seed liquid, which is prepared by the method described in example 2;
③ respectively inoculating the activated strains of Bacillus subtilis and Bacillus licheniformis prepared in step (1) with an inoculating loop, inoculating into a sterilized liquid culture medium (each strain is cultured independently), and culturing at a table rotation speed of 150r/min and a temperature of 30 deg.C and a liquid loading amount of 30mL/250mL for 2 days to obtain Bacillus subtilis seed liquid and Bacillus licheniformis seed liquid respectively;
④ Rhodopseudomonas palustris seed liquid, the concrete method is the same as example 2;
⑤ respectively inoculating the activated strains of cerevisiae fermentum and Candida utilis prepared in step (1) with an inoculating loop to a sterilized liquid culture medium (each strain is cultured independently), culturing at a table rotation speed of 200r/min and a temperature of 30 deg.C and a liquid loading amount of 25mL/250mL for 2 days to obtain a cerevisiae fermentum seed solution and a Candida utilis seed solution;
(3) culturing a composite strain: inoculating the lactobacillus acidophilus seed solution, the lactobacillus plantarum seed solution and the rhodopseudomonas palustris seed solution prepared in the step (2) into a sterilized fermentation culture medium 1, carrying out anaerobic culture at the temperature of 30 ℃ for 3 days, then inoculating the bacillus subtilis seed solution, the bacillus licheniformis seed solution, the beer yeast seed solution and the candida utilis seed solution prepared in the step (2) into the fermentation system, the rotating speed of a shaking table is 150r/min, the temperature is 30 ℃, the liquid loading amount is 30mL/250mL, the culture is carried out for 2 days, the composite microbial preparation strain is obtained, wherein the inoculation amount ratio (weight ratio) of lactobacillus acidophilus, lactobacillus plantarum seed liquid, bifidobacterium bifidum seed liquid, bacillus subtilis, bacillus licheniformis seed liquid, rhodopseudomonas palustris seed liquid, beer yeast and candida utilis seed liquid is 5:5:4:3:3:3:5: 5; the total inoculum size was 10% by weight.
Effects of the embodiment
(1) Comparative example 1 seed solutions of 8 strains were simultaneously inoculated into fermentation medium 1, and aerobic culture was performed for 2 days, followed by anaerobic culture for 3 days; comparative example 2 is that lactobacillus acidophilus seed liquid, lactobacillus plantarum seed liquid and rhodopseudomonas palustris seed liquid are inoculated into a fermentation medium 1, anaerobic culture is carried out for 3 days at 30 ℃, and then bacillus subtilis seed liquid, bacillus licheniformis seed liquid, beer yeast seed liquid and candida utilis seed liquid are inoculated into a fermentation system, and aerobic culture is carried out for 2 days. Example 2 the mixed bacillus seed solution and the mixed yeast seed solution were inoculated into the fermentation medium 1 at the same time, aerobically cultured for 2 days, and then the mixed lactobacillus seed solution, bifidobacterium bifidum seed solution and rhodopseudomonas palustris seed solution were inoculated into the above fermentation system at the same time, anaerobically cultured for 3 days. The other parameters are the same.
Table 1 shows the results of counting each strain in the composite microbial preparation strains obtained in example 2 and comparative examples 1 and 2, and it can be seen from Table 1 that the Bacillus in comparative example 1 died largely in the late stage of cultivation; in comparative example 2, both bacillus and yeast had poor growth and died in a large amount in the later period; according to the inoculation sequence of the embodiment 2, the cells all reach higher growth amount; this indicates that the inoculation sequence of example 2 is the optimal inoculation sequence.
TABLE 1 results of different inoculation sequence counts (cfu/ml)
(2) To further explain the problems, the cell growth amount, propagation generation number, growth rate constant, generation time, etc. in the culture of the composite strain in step (3) of example 2 were measured, respectively, and pure culture of each strain (10% by weight of inoculum) was used as a control. The results are shown in tables 2 and 3.
TABLE 2 comparison of pure and mixed cultures
TABLE 3 yield coefficient of metabolites in pure and mixed cultures (Yp/s)
Therefore, the yeast and the bacillus can quickly consume oxygen in the culture medium, an anaerobic environment favorable for survival is created for the lactic acid bacteria, the bifidobacterium bifidum and the rhodopseudomonas palustris, and the growth of the lactic acid bacteria, the bifidobacterium bifidum and the rhodopseudomonas palustris is facilitated; the saccharomycetes can decompose sucrose in the nutrient substances into fructose and glucose to provide carbon sources for lactic acid bacteria, bifidobacterium bifidum and rhodopseudomonas palustris; the lactobacillus and the bifidobacterium bifidum generate lactic acid, acetic acid and other organic acids by taking sugars generated by rhodopseudomonas palustris and saccharomycetes, reduce the pH value, inhibit the proliferation of harmful bacteria and prevent the compound microorganisms from being polluted in the culture process; compared with pure culture, the mixed culture improves the viable count, reduces the link of mixing after pure culture, reduces the pollution rate, effectively ensures the quality of the composite microbial preparation, and improves the utilization rate of raw materials, equipment and energy, thereby reducing the production cost.
The compound microbial preparation prepared by the invention can be used for pig breeding, and has the effects of promoting growth, helping digestion, resisting diseases, increasing animal weight, reducing feed consumption and purifying breeding environment, so that safe, high-quality and residue-free green pork food is produced. The results of the detection of the nutrient content in pork are shown in Table 4, and the results of the detection of the safety of pork are shown in Table 5 (see NY/T2799-2015 for Green food and livestock meat)
TABLE 4 detection results of nutrient content of pork food
| Detecting items | Unit of | The result of the detection | Detection method |
| Aspartic acid | g/100g | 2.06 | GB/T 5009.124-2016 |
| Threonine | g/100g | 0.99 | GB/T 5009.124-2016 |
| Serine | g/100g | 0.87 | GB/T 5009.124-2016 |
| Glutamic acid | g/100g | 3.39 | GB/T 5009.124-2016 |
| Proline | g/100g | 0.90 | GB/T 5009.124-2016 |
| Glycine | g/100g | 1.12 | GB/T 5009.124-2016 |
| Alanine | g/100g | 1.31 | GB/T 5009.124-2016 |
| Valine | g/100g | 1.09 | GB/T 5009.124-2016 |
| Methionine | g/100g | 0.61 | GB/T 5009.124-2016 |
| Isoleucine | g/100g | 1.02 | GB/T 5009.124-2016 |
| Leucine | g/100g | 1.78 | GB/T 5009.124-2016 |
| Tyrosine | g/100g | 0.78 | GB/T 5009.124-2016 |
| Phenylalanine | g/100g | 1.12 | GB/T 5009.124-2016 |
| Histidine | g/100g | 1.91 | GB/T 5009.124-2016 |
| Lysine | g/100g | 0.93 | GB/T 5009.124-2016 |
| Arginine | g/100g | 1.45 | GB/T 5009.124-2016 |
| Total amount of amino acids | g/100g | 21.3 | GB/T 5009.124-2016 |
| Protein | g/100g | 21.0 | GB 5009.5-2016 |
TABLE 5 pork food safety test results
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. A method for culturing a composite microbial preparation, which is characterized by comprising the following steps:
(1) and (3) strain activation culture: respectively activating the frozen lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis for multiple times to obtain activated strains of lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis;
(2) liquid strain culture
① inoculating the activated strains of Lactobacillus acidophilus and Lactobacillus plantarum prepared in the step (1) into a liquid culture medium in a mass ratio, and carrying out anaerobic culture at 37 ℃ for 2-3 days to obtain a mixed lactobacillus seed solution;
② inoculating the activated strain of Bifidobacterium bifidum prepared in step (1) into a liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 2-3 days to obtain a Bifidobacterium bifidum seed solution;
③ inoculating the activated strains of the bacillus subtilis and the bacillus licheniformis prepared in the step (1) into a liquid culture medium in a mass ratio, and culturing for 1-2 days at a shaker rotation speed of 150-200 r/min and a temperature of 30-35 ℃ to obtain a mixed bacillus seed solution;
④ inoculating the activated strain of Rhodopseudomonas palustris prepared in step (1) into a liquid culture medium, and performing anaerobic culture at 60 ℃ and 30-35 ℃ for 5-7 days under the illumination of a tungsten filament lamp to obtain a Rhodopseudomonas palustris seed solution;
⑤ inoculating the beer yeast and the candida utilis activated strain prepared in the step (1) into a liquid culture medium according to the mass ratio, and culturing for 2 days at the rotating speed of a shaking table of 150-200 r/min and the temperature of 28-30 ℃ to obtain mixed yeast seed liquid;
(3) culturing a composite strain: simultaneously inoculating the mixed bacillus seed liquid and the mixed yeast seed liquid prepared in the step (2) into a fermentation culture medium 1, simultaneously inoculating the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid and the rhodopseudomonas palustris seed liquid prepared in the step (2) into the fermentation system at the rotating speed of 150-200 r/min, the temperature of 30 ℃ and the liquid loading amount of 30mL/250mL for 1-2 days, and then carrying out anaerobic culture at the temperature of 30-35 ℃ for 3-5 days to obtain a composite microbial preparation strain, wherein the inoculation amount ratio of the mixed lactobacillus seed liquid, the bifidobacterium bifidum seed liquid, the mixed bacillus seed liquid, the rhodopseudomonas palustris seed liquid and the mixed yeast seed liquid is 5:4:3:3: 5; the total inoculation amount is 10 percent by weight;
(4) and (3) finished product cultivation: inoculating the composite microbial preparation strain prepared in the step (3) into a fermentation culture medium 2 according to the inoculation amount of 5-8% by weight, and performing closed fermentation at 28-30 ℃ for 7-15 days to obtain a finished product of the composite microbial preparation; wherein the fermentation medium is not sterilized in this step.
2. The method for culturing a complex microbial preparation according to claim 1, wherein:
the strains of lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, beer yeast and candida utilis in the step (1) are respectively as follows: lactobacillus acidophilus (Lactobacillus acidophilus) CICC 6083, Lactobacillus plantarum (Lactobacillus plantarum) CICC21790, Bifidobacterium bifidum (Bifidobacterium bifidum) CICC 6166, Bacillus subtilis (Bacillus subtilis) CICC24434, Bacillus licheniformis (Bacillus licheniformis) CGMCC1.10257, Rhodopseudomonas palustris (Rhodopseudomonas palustris) CGMCC 1.8929, Saccharomyces cerevisiae (Saccharomyces cerevisiae) CGMCC2.3973, and Candida utilis (Candida utilis) CGMCC 2.3047.
3. The method for culturing a complex microbial preparation according to claim 1, wherein:
the specific operation of the multiple activation in the step (1) is as follows: carrying out three times of slant activation on the frozen and preserved lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, saccharomyces cerevisiae and candida utilis strains, then carrying out flat plate purification culture, and finally carrying out slant culture to obtain lactobacillus acidophilus, lactobacillus plantarum, bifidobacterium bifidum, bacillus subtilis, bacillus licheniformis, rhodopseudomonas palustris, saccharomyces cerevisiae and candida utilis strains.
4. The method for culturing a complex microbial preparation according to claim 3, wherein:
the conditions of slant activation, plate purification culture or slant culture are as follows:
carrying out facultative anaerobic culture on lactobacillus acidophilus or lactobacillus plantarum at 37 ℃ for 2-3 days,
bifidobacterium bifidum is anaerobically cultured for 3-5 days at 37 ℃,
aerobic culture of bacillus subtilis and bacillus licheniformis is carried out for 1-2 days at 30 ℃;
carrying out anaerobic culture on rhodopseudomonas palustris for 5-7 days at the temperature of 30-35 ℃ under the irradiation of 60-watt black-silk lamp light;
carrying out aerobic culture on the beer yeast or the candida utilis at 28-30 ℃ for 2 days.
5. The method for culturing a complex microbial preparation according to claim 1, wherein:
each liter of the liquid culture medium in the step (2) ① comprises 10 g of peptone, 10 g of beef extract, 5 g of yeast powder, 5 g of glucose, 5 g of sodium acetate, 2 g of diamine citrate, 801 mL of tween, 2 g of dipotassium hydrogen phosphate, 0.2 g of magnesium sulfate, 0.05 of manganese sulfate, 20 g of calcium carbonate, 20mL of tomato juice, 30mL of potato juice, 60mL of carrot juice, 2 g of vitamin C and 1000mL of weakly alkaline small molecular reducing water, wherein the pH value is 6.8;
each liter of the liquid culture medium in the step (2) ② comprises 15.0 g of peptone, 2.0 g of yeast powder, 20.0 g of glucose, 0.5 g of soluble starch, 5.0 g of sodium chloride, 10.0mL of 5% cysteine, 400.0mL of tomato extract, 801.0 mL of tween, 80.0mL of liver extract, and a weakly alkaline small molecule reducing water supplemented to 1000mL, and the pH value is 7.0.
6. The method for culturing a complex microbial preparation according to claim 1, wherein:
each liter of the liquid culture medium in the step (2) ③ comprises 30 g of glucose, 15 g of yeast extract, 2 g of disodium hydrogen phosphate, 1 g of sodium dihydrogen phosphate, 1000mL of weakly alkaline small molecule reduced water and pH 7.2;
each liter of the liquid culture medium in the step (2) ④ comprises 10 g of yeast powder, 1 g of dipotassium hydrogen phosphate, 0.5 g of magnesium sulfate and 1000mL of weakly alkaline small molecular reducing water, wherein the pH value is 7.0-7.2;
each liter of the liquid culture medium in the step (2) ⑤ comprises 50 g of brown sugar, 20 g of yeast extract, 1 g of ammonium sulfate and 2 g of potassium chloride, the pH is natural, and the weak alkaline small molecule reducing water is supplemented to 1000 mL.
7. The method for culturing a complex microbial preparation according to claim 1, wherein:
the fermentation medium 1 in the step (3) comprises the following components in percentage by mass:
5% of industrial waste molasses, 0.5% of yeast powder, 1% of peptone, 1% of banana polysaccharide, 0.1% of ammonium chloride, 0.1% of sodium chloride, 0.05% of potassium dihydrogen phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of weak alkaline small molecular reducing water to 100%.
8. The method for culturing a complex microbial preparation according to claim 1, wherein:
the fermentation medium 2 in the step (4) comprises the following components in percentage by mass: 5-8% of industrial waste molasses, 0.3% of banana polysaccharide, 0.2% of ammonium chloride, 0.05% of sodium chloride, 0.1% of monopotassium phosphate, 0.05% of magnesium sulfate, 0.025% of zinc sulfate, 0.025% of ferrous sulfate and the balance of water to 100%.
9. A complex microbial preparation obtained by the production method according to any one of claims 1 to 8.
10. The use of the complex microbial preparation of claim 9 in the field of pig farming.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115820515A (en) * | 2023-01-03 | 2023-03-21 | 东北农业大学 | Preparation of cheap lactobacillus PTG medium and application of cheap lactobacillus PTG medium in pickled vegetable fermentation |
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Application publication date: 20200522 |