Preparation method and application of a liquid compound microecological preparation and its protective agent

Abstract

The present invention discloses a preparation method and application of a liquid compound microecological preparation and its protective agent. The components of the protective agent include glucose, soybean meal, peptone, sodium sulfite, disodium hydrogen phosphate, citric acid, calcium carbonate, glycerol, sodium butyrate, sodium lactate, D-sodium propionate, and nucleoside phosphorylase. By adding a certain proportion of the protective agent to the liquid compound microecological preparation, the present invention can enable the bacteria in the liquid compound microecological preparation to have a longer preservation time, with a slow decline in the bacterial count during long-term preservation, ensuring a higher viable bacteria count and stronger bacterial vitality, and greatly promoting production activities.

Description

Technical Field

[0001] The present invention relates to the technical field of microecological agents, and more particularly, to a preparation method and application of a liquid compound microecological agent and its protective agent. Background Art

[0002] With the development of the livestock industry and the increase in breeding density, many breeders use antibiotics during the feeding process to prevent and control diseases. However, people have gradually realized that the long-term use of antibiotics will pose a hazard to human health through the food chain. At present, a complete ban on antibiotics has been initiated, and among the alternatives to antibiotics, microecological agents have attracted people's attention due to their diverse effects, long onset time, non-toxicity, non-antibiotic resistance, no residue, and no side effects, and have broad application prospects.

[0003] Microecological agents can be classified into pure bacterial agents and compound bacterial agents according to their contents. Because the intestinal microecosystem is complex, existing microecological bacterial agents are mainly compound bacterial agents. In terms of form, microecological agents can be further divided into solid preparations and liquid preparations. Compared with solid microecological agents, liquid microecological agents have better retention of metabolites, higher viable bacteria counts, and stronger strain vitality. However, due to the high water content of liquid microecological agents, how to improve the storage time of viable bacteria therein is a major difficulty. At the same time, in the case of compound bacterial agents, the storage conditions of different bacteria are different, so a preservation method that can comprehensively improve the survival time of different bacterial species in the liquid state is needed.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] The purpose of the present invention is to provide a preparation method and application of a liquid compound microecological agent and its protective agent. By adding a certain proportion of protective agent to the liquid compound microecological agent, the storage time of the bacteria in the liquid compound microecological agent can be prolonged, the decline of the bacteria amount is slow during long-term storage, ensuring a higher viable bacteria count and stronger bacterial vitality, and greatly promoting production activities.

[0006] Since the liquid compound microecological agent contains lactic acid bacteria, yeast, Bacillus coagulans, and Clostridium butyricum, and the preservation methods of different bacterial species are different, which leads to obstacles in the preservation of viable bacteria in the liquid microecological agent. The inventor has found through research that Bacillus coagulans and Clostridium butyricum can form spores, and their stress resistance is extremely strong in the spore state and they are not easily killed; while lactic acid bacteria and yeast cannot form spores, so while inhibiting their metabolism, it is also necessary to ensure that there is a certain amount of nutrients for their metabolism. However, in the presence of nutrients, the spores will gradually germinate, and once the spores germinate, they will lose their resistance to external stress.

[0007] After a large amount of creative work, the inventor obtained a protective agent for liquid compound microecological agents. On the one hand, this protective agent can ensure the survival rate of lactic acid bacteria and yeasts, and on the other hand, it can prevent the spore germination of Bacillus coagulans and Clostridium butyricum.

[0008] The present invention is implemented as follows:

[0009] In a first aspect, the present invention provides a protective agent for liquid compound microecological agents, which includes glucose, soybean meal, peptone, sodium sulfite, disodium hydrogen phosphate, citric acid, calcium carbonate, glycerol, sodium butyrate, sodium lactate, D-sodium propionate, and nucleoside phosphorylase.

[0010] Among them, glucose, peptone, and soybean meal all serve as basic nutrient sources. Glucose, as a rapid-acting carbon source, and peptone, as a rapid-acting carbon source, can both provide certain nutrients for lactic acid bacteria and yeasts. At the same time, soybean meal, as a slow-acting carbon source and nitrogen source, can be slowly decomposed under the action of bacterial enzymes to provide nutrients.

[0011] Sodium sulfite plays a role in reducing the dissolved oxygen in the liquid bacterial agent. On the one hand, it can inhibit the metabolism of lactic acid bacteria and yeasts, and on the other hand, the low dissolved oxygen state can also prevent the resuscitation of Bacillus coagulans spores. At the same time, excessive dissolved oxygen may cause the death of Clostridium butyricum.

[0012] Disodium hydrogen phosphate and citric acid are pH buffer components, which play a role in stabilizing the pH.

[0013] Calcium in calcium carbonate exists as calcium ions under acidic conditions, which can reduce the cell membrane permeability.

[0014] Glycerol can increase the surface tension of the liquid. When used together, they can effectively reduce the material exchange between cells and the outside world. On the one hand, it can inhibit the metabolism of lactic acid bacteria and yeasts, and on the other hand, it can prevent factors promoting spore germination from entering the spore and binding to the germination-promoting receptor protein on the spore inner membrane. At the same time, it maintains the amount of substances inside the spore and inhibits spore germination.

[0015] Sodium butyrate and sodium lactate, as substances similar to the main metabolites of Clostridium butyricum and Bacillus coagulans respectively, can play a role in inhibiting the spore germination of Clostridium butyricum and Bacillus coagulans respectively. At the same time, sodium lactate, as the main metabolite of lactic acid bacteria, can also reduce the activity of lactic acid bacteria to a certain extent and inhibit its metabolism.

[0016] D-sodium propionate can antagonize L-sodium propionate, and the binding of L-sodium propionate to the corresponding receptor on the spore inner membrane can induce germination. By antagonizing L-sodium propionate that has already bound to the nutrient receptor, D-sodium propionate can effectively inhibit spore germination.

[0017] The role of nucleoside phosphorylase is to decompose inosine. And inosine is also the main factor inducing spore germination.

[0018] Specifically, by weight parts, the above-mentioned protective agent includes: 0.9 - 1 part of glucose, 1.8 - 2 parts of soybean meal, 0.4 - 0.5 part of peptone, 0.5 - 0.6 part of sodium sulfite, 1.6 - 1.65 parts of disodium hydrogen phosphate, 1.15 - 1.16 parts of citric acid, 0.5 - 0.6 part of calcium carbonate, 0.5 - 0.6 part of glycerol, 1 - 1.2 parts of sodium butyrate, 1.8 - 2 parts of sodium lactate, 0.5 - 0.6 part of D-sodium propionate, and 0.18 - 0.2 part of nucleoside phosphorylase.

[0019] Further, the above-mentioned protective agent includes: 0.95 - 1 part of glucose, 1.9 - 2 parts of soybean meal, 0.45 - 0.5 part of peptone, 0.5 - 0.55 part of sodium sulfite, 1.6 - 1.62 parts of disodium hydrogen phosphate, 1.15 - 1.155 parts of citric acid, 0.5 - 0.55 part of calcium carbonate, 0.5 - 0.55 part of glycerol, 1.1 - 1.2 parts of sodium butyrate, 1.9 - 2 parts of sodium lactate, 0.5 - 0.55 part of D-sodium propionate, and 0.19 - 0.2 part of nucleoside phosphorylase.

[0020] In a second aspect, the present invention also provides the use of the above-mentioned protective agent for the liquid compound microecological preparation in the preparation of a liquid compound microecological preparation.

[0021] In a third aspect, the present invention also provides a liquid compound microecological preparation, which includes the protective agent for the liquid compound microecological preparation.

[0022] In some embodiments, the above-mentioned liquid compound microecological preparation further includes a compound bacterial suspension, and the compound bacterial suspension contains lactic acid bacteria, Bacillus coagulans, Clostridium butyricum, and yeast.

[0023] In some embodiments, the above-mentioned lactic acid bacteria are selected from any one or more of Enterococcus faecalis, Lactobacillus plantarum, and Pediococcus acidilactici. In the present invention, the lactic acid bacteria can be any one of Enterococcus faecalis, Lactobacillus plantarum, and Pediococcus acidilactici, or any combination of any two or three of them. Any method can achieve the purpose, and the present invention does not make further limitations in this regard.

[0024] In some embodiments, the above-mentioned yeast includes Saccharomyces boulardii.

[0025] In some embodiments, the viable cell number ratio of lactic acid bacteria, Bacillus coagulans, Clostridium butyricum, and yeast in the above-mentioned liquid compound microecological preparation is 9.2 - 13:3.7 - 6.5:3.0 - 3.8:3.7 - 6.5.

[0026] In some embodiments, the added mass of the above-mentioned protective agent is 10.83% - 12.11% of the mass of the compound bacterial suspension.

[0027] In some embodiments, the mass ratios of the components of the protective agent to the compound bacterial suspension include: glucose 0.9%-1%, soybean meal 1.8%-2%, peptone 0.4%-0.5%, sodium sulfite 0.5%-0.6%, disodium hydrogen phosphate 1.6%-1.65%, citric acid 1.15%-1.16%, calcium carbonate 0.5%-0.6%, glycerol 0.5%-0.6%, sodium butyrate 1%-1.2%, sodium lactate 1.8%-2%, D-sodium propionate 0.5%-0.6%, nucleoside phosphorylase 0.18%-0.2%.

[0028] Fourthly, the present invention also provides a preparation method of the liquid compound microecological preparation, which includes adding a protective agent to the compound bacterial suspension in proportion and adjusting the pH value to obtain the liquid compound microecological preparation.

[0029] In some embodiments, the pH value of the above liquid compound microecological preparation is 4-4.5.

[0030] In the present invention, the pH is adjusted to 4.4-4.5 because for Clostridium butyricum and Bacillus coagulans, within the range of pH 4-7, as the pH increases, the spore germination rate increases, while if the pH is too low, Lactobacillus and yeast are likely to die. Therefore, pH 4.4-4.5 is the appropriate acidity and alkalinity.

[0031] In some embodiments, the preparation steps of the compound bacterial suspension include separately activating and expanding the cultures of the strains of Lactobacillus, yeast, Bacillus coagulans and Clostridium butyricum to obtain a Lactobacillus suspension, a yeast suspension, a Bacillus coagulans suspension and a Clostridium butyricum suspension, and then mixing them in proportion to obtain the compound bacterial suspension.

[0032] In some embodiments, the viable count in the Lactobacillus suspension is 9.2×10 8 -1.3×10 9 cfu / ml, the viable count in the Bacillus coagulans suspension is 3.7×10 8 -6.5×10 8 cfu / ml, the viable count in the Clostridium butyricum suspension is 3.0×10 8 -3.8×10 8 cfu / ml, and the viable count in the yeast suspension is 3.7×10 8 -6.5×10 8 cfu / ml.

[0033] The present invention has the following beneficial effects:

[0034] The protectant of the liquid compound microecological preparation of the present invention contains a small amount of nutrients for the metabolism of the bacteria and components that inhibit the metabolism rate of the bacteria and the germination of spores. Adding it to the liquid compound microecological preparation results in a high total bacterial count of the prepared liquid compound microecological preparation, a long storage time, a slow decline in the bacterial count during long-term storage, stronger bacterial viability, and greatly promotes production activities. At the same time, the preparation method of the liquid compound microecological preparation of the present invention is simple to operate and has good application prospects. Detailed implementation manners

[0035] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. For those conditions not specified in the embodiments, they are carried out according to conventional conditions or the conditions recommended by the manufacturer. For the reagents or instruments whose manufacturers are not specified, they are all conventional products that can be obtained through commercial purchase.

[0036] The features and properties of the present invention will be further described in detail below in conjunction with the embodiments.

[0037] In the embodiments of the present invention, the Enterococcus faecalis used has an accession number of ATCC29212; the Lactobacillus plantarum has an accession number of ATCC8014; the Pediococcus acidilactici has an accession number of ATCC804; the Bacillus coagulans has an accession number of ATCC7050; the Clostridium butyricum has an accession number of ATCC19398; the Saccharomyces boulardii has a product number of SHBCC D25151.

[0038] Example 1

[0039] This example provides a liquid compound microecological preparation, and its preparation method includes the following steps:

[0040] 1. Activation and subculture of strains

[0041] The refrigerated strains are respectively activated and subcultured. Among them, after the strains of Enterococcus faecalis, Lactobacillus plantarum, and Pediococcus acidilactici are respectively activated, they are jointly subcultured until a lactic acid bacteria suspension of 6×10 9 cfu / ml is obtained.

[0042] After the Bacillus coagulans is activated, it is cultured until a Bacillus coagulans suspension of 2×10 9 cfu / ml is obtained.

[0043] After the Clostridium butyricum is activated, it is cultured until a Clostridium butyricum suspension of 8×10 8 cfu / ml is obtained.

[0044] After the Saccharomyces boulardii is activated, it is cultured until a Saccharomyces boulardii suspension of 3×10 9 cfu / ml is obtained.

[0045] 2. Preparation of Liquid Compound Microecological Preparation

[0046] Mix the lactic acid bacteria suspension, Bacillus coagulans suspension, Clostridium butyricum suspension, and Saccharomyces boulardii suspension prepared in Step 1 in a weight ratio of 0.9:1:2:0.9 to obtain a compound bacteria suspension. Among them, the viable bacteria count ratio of lactic acid bacteria, Bacillus coagulans, Clostridium butyricum, and Saccharomyces boulardii is: 11.3:4.2:3.3:5.6. Then add a protective agent and adjust the pH to 4.5 with 10% sodium hydroxide to obtain the liquid compound microecological preparation.

[0047] Among them, the protective agent composition is: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, 0.2% nucleoside phosphorylase.

[0048] Note: The above percentages are the mass ratios based on the compound bacteria suspension.

[0049] Example 2

[0050] This example has the same other step parameters as Example 1, and the only difference is:

[0051] In Step 2, the viable bacteria count ratio of lactic acid bacteria, Bacillus coagulans, Clostridium butyricum, and Saccharomyces boulardii is: 12.8:3.8:3.1:6.4.

[0052] Example 3

[0053] This example has the same other step parameters as Example 1, and the only difference is:

[0054] In Step 2, adjust the pH to 4.4 with 10% sodium hydroxide.

[0055] Example 4

[0056] This example has the same other step parameters as Example 1, and the only difference is:

[0057] In Step 2, the protective agent composition is: 0.9% glucose, 1.8% soybean meal, 0.4% peptone, 0.6% sodium sulfite, 1.65% disodium hydrogen phosphate, 1.16% citric acid, 0.6% calcium carbonate, 0.5% glycerol, 1.2% sodium butyrate, 1.8% sodium lactate, 0.6% D-sodium propionate, 0.18% nucleoside phosphorylase.

[0058] Comparative Example 1

[0059] This comparative example has the same other step parameters as Example 1, and the only difference is:

[0060] In Step 2, the composition of the protective agent is as follows: 1% glucose, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0061] Comparative Example 2

[0062] This comparative example is the same as Example 1 in other step parameters, and the only difference is that:

[0063] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0064] Comparative Example 3

[0065] This comparative example is the same as Example 1 in other step parameters, and the only difference is that:

[0066] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0067] Comparative Example 4

[0068] This comparative example is the same as Example 1 in other step parameters, and the only difference is that:

[0069] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0070] Comparative Example 5

[0071] This comparative example is the same as Example 1 in other step parameters, and the only difference is that:

[0072] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0073] Comparative Example 6

[0074] This comparative example is the same as Example 1 in other step parameters, with the only difference being that:

[0075] In Step 2, the composition of the protective agent is: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 5% skim milk powder, 0.6% soybean oil, 2% sodium glutamate, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, 0.2% nucleoside phosphorylase.

[0076] Comparative Example 7

[0077] This comparative example is the same as Example 1 in other step parameters, with the only difference being that:

[0078] In Step 2, the composition of the protective agent is: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 5% skim milk powder, 0.6% soybean oil, 2% sodium glutamate, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, 0.2% nucleoside phosphorylase.

[0079] Comparative Example 8

[0080] This comparative example is the same as Example 1 in other step parameters, with the only difference being that:

[0081] In Step 2, the composition of the protective agent is: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% sodium carbonate, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, 0.2% nucleoside phosphorylase.

[0082] Comparative Example 9

[0083] This comparative example is the same as Example 1 in other step parameters, with the only difference being that:

[0084] In Step 2, the composition of the protective agent is: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% sodium carbonate, 1% sodium butyrate, 2% sodium lactate, 0.5% D-sodium propionate, 0.2% nucleoside phosphorylase.

[0085] Comparative Example 10

[0086] This comparative example is the same as Example 1 in other step parameters, with the only difference being that:

[0087] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 2% sodium lactate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0088] Comparative Example 11

[0089] This comparative example has the same parameters for other steps as Example 1, with the only difference being that:

[0090] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 1% sodium butyrate, 0.5% D-sodium propionate, and 0.2% nucleoside phosphorylase.

[0091] Comparative Example 12

[0092] This comparative example has the same parameters for other steps as Example 1, with the only difference being that:

[0093] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, and 0.2% nucleoside phosphorylase.

[0094] Comparative Example 13

[0095] This comparative example has the same parameters for other steps as Example 1, with the only difference being that:

[0096] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, 0.6% glycerol, 1% sodium butyrate, 2% sodium lactate, and 0.5% D-sodium propionate.

[0097] Comparative Example 14

[0098] This comparative example has the same parameters for other steps as Example 1, with the only difference being that:

[0099] In Step 2, the composition of the protective agent is as follows: 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, and 0.6% glycerol.

[0100] Comparative Example 15

[0101] This comparative example has the same parameters for other steps as Example 1, with the only difference being that:

[0102] In Step 2, the composition of the protective agent is as follows: 1% glucose, 2% soybean meal, 0.5% peptone, 0.5% sodium sulfite, 1.6% disodium hydrogen phosphate, 1.15% citric acid, 0.5% calcium carbonate, and 0.6% glycerol.

[0103] Comparative Example 16

[0104] The difference between this comparative example and Example 1 lies in that no protective agent is added in Step 2.

[0105] Experimental Example 1

[0106] The liquid compound microecological agents of Examples 1-4 and Comparative Examples 1-15 were placed in a cool place for 30 days and 60 days, and the bacterial counts of lactic acid bacteria, Bacillus coagulans, Clostridium butyricum, and yeasts in the liquid compound microecological agents were detected by the plate dilution coating counting method. The results are shown in Tables 1-4.

[0107] Table 1 Viable count of lactic acid bacteria

[0108]

[0109]

[0110] Table 2 Viable count of Bacillus coagulans

[0111]

[0112]

[0113] Table 3 Viable count of Clostridium butyricum

[0114]

[0115]

[0116] Table 4 Viable count of yeasts

[0117]

[0118]

[0119] As can be seen from the results of the examples in Table 1, Table 2, Table 3, and Table 4, for the formulation variation ranges in Examples 2-4, the impact on the preservation effect is not significant. From Comparative Example 1, it can be seen that when the protective agent does not contain soybean meal, the decline rate of the amounts of lactic acid bacteria and yeast accelerates during long-term preservation; from Comparative Example 2, it can be seen that when the protective agent does not contain sodium sulfite, the decline rates of the amounts of the four types of bacteria all accelerate during long-term preservation, especially for lactic acid bacteria, Bacillus coagulans, and Clostridium butyricum, which are more obvious; from Comparative Examples 3, 4, and 5, it can be seen that when calcium carbonate and glycerol are respectively removed from the protective agent, the decline rates of the amounts of the four types of bacteria all accelerate significantly during long-term preservation, but when both are removed, compared with removing only one of them, the reduction situation is not obvious; from the comparison between Comparative Examples 4 and 6, it can be seen that when glycerol is replaced with conventional liquid protective components (skim milk powder, soybean oil, sodium glutamate), the preservation effect decreases significantly, only slightly higher than the situation of removing glycerol; from the comparison between Comparative Examples 3 and 8, it can be seen that when calcium carbonate is replaced with sodium carbonate, the preservation effect also decreases significantly, only slightly higher than the situation of removing calcium carbonate; from the comparison between Comparative Examples 3 and 7, it can be seen that when the protective agent does not contain calcium carbonate and glycerol in the protective agent is replaced with conventional liquid protective components (skim milk powder, soybean oil, sodium glutamate), the preservation effect only decreases slightly and is almost unaffected; from the comparison between Comparative Examples 4 and 9, it can be seen that when the protective agent does not contain glycerol and calcium carbonate in the protective agent is replaced with sodium carbonate, the preservation effect is also almost unaffected; from Comparative Examples 10 and 11, it can be seen that when sodium butyrate is removed from the protective agent, the impact on the decline rate of the amount of Clostridium butyricum during long-term preservation is very significant, and when sodium lactate is removed from the protective agent, the impact on the decline rates of the amounts of lactic acid bacteria and Bacillus coagulans during long-term preservation is also very significant; from Comparative Examples 12 and 13, it can be seen that when D-sodium propionate and nucleoside phosphorylase are respectively removed from the protective agent, the decline rates of Bacillus coagulans and Clostridium butyricum during long-term preservation both accelerate significantly; from Comparative Examples 14 and 15, it can be seen that when nutrients and substances inhibiting spore germination are removed from the protective agent, the decline rates of the amounts of lactic acid bacteria and yeast accelerate very severely during long-term preservation, while the amounts of Bacillus coagulans and Clostridium butyricum are only slightly affected, and when only substances inhibiting spore germination are removed from the protective agent, the amounts of Bacillus coagulans and Clostridium butyricum decline very severely during long-term preservation, while the decline rates of the amounts of lactic acid bacteria and yeast are almost unaffected; from Comparative Example 16, it can be seen that without adding a protective agent, the amounts of lactic acid bacteria and yeast both have a huge decline, especially lactic acid bacteria are the most severe, and the amounts of Clostridium butyricum and Bacillus coagulans also both have a certain decline, and the increase rate of the decline rate of the amount of Bacillus coagulans is relatively slight.

[0120] In summary, the preparation method of the liquid compound microecological preparation of the present invention is simple to operate, has a high total bacterial count, and the prepared liquid compound microecological preparation only has a small decrease in the bacterial count within the storage time limits of 30 days and 60 days. The storage effect is better than that of the current similar liquid compound microecological preparations, greatly promoting production activities.

[0121] The foregoing are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A liquid compound microecological preparation, characterized in that, The liquid compound microecological preparation comprises a compound bacterial suspension and a protective agent; the compound bacterial suspension contains lactic acid bacteria, Bacillus coagulans, Clostridium butyricum and yeast; the preservation number of Bacillus coagulans is ATCC7050, and the preservation number of Clostridium butyricum is ATCC19398; The viable count ratio of lactic acid bacteria, Bacillus coagulans, Clostridium butyricum and yeast in the liquid compound microecological preparation is 9.2 - 13:3.7 - 6.5:3.0 - 3.8:3.7 - 6.5; The added mass of the protective agent is 10.83% - 12.11% of the mass of the compound bacterial suspension; The protective agent, by weight, comprises: 0.9 - 1 part of glucose, 1.8 - 2 parts of soybean meal, 0.4 - 0.5 part of peptone, 0.5 - 0.6 part of sodium sulfite, 1.6 - 1.65 parts of disodium hydrogen phosphate, 1.15 - 1.16 parts of citric acid, 0.5 - 0.6 part of calcium carbonate, 0.5 - 0.6 part of glycerol, 1 - 1.2 parts of sodium butyrate, 1.8 - 2 parts of sodium lactate, 0.5 - 0.6 part of D - sodium propionate, 0.18 - 0.2 part of nucleoside phosphorylase.

2. The liquid compound microecological preparation according to claim 1, characterized in that, The protective agent, by weight, comprises: 0.95 - 1 part of glucose, 1.9 - 2 parts of soybean meal, 0.45 - 0.5 part of peptone, 0.5 - 0.55 part of sodium sulfite, 1.6 - 1.62 parts of disodium hydrogen phosphate, 1.15 - 1.155 parts of citric acid, 0.5 - 0.55 part of calcium carbonate, 0.5 - 0.55 part of glycerol, 1.1 - 1.2 parts of sodium butyrate, 1.9 - 2 parts of sodium lactate, 0.5 - 0.55 part of D - sodium propionate, 0.19 - 0.2 part of nucleoside phosphorylase.

3. The liquid compound microecological preparation according to claim 1, characterized in that, The lactic acid bacteria are selected from any one or more of Enterococcus faecalis, Lactobacillus plantarum and Pediococcus acidilactici; the preservation number of Enterococcus faecalis is ATCC29212, the preservation number of Lactobacillus plantarum is ATCC8014, and the preservation number of Pediococcus acidilactici is ATCC8042.

4. The liquid compound microecological preparation according to claim 1, characterized in that, The yeast includes Saccharomyces boulardii.

5. A preparation method of the liquid compound microecological preparation according to any one of claims 1-4, characterized in that, It includes adding the protective agent to the compound bacterial suspension in proportion, and adjusting the pH value to obtain the liquid compound microecological preparation.

6. The preparation method of a liquid compound microecological preparation according to claim 5, characterized in that, The pH value of the liquid compound microecological preparation is 4 - 4.

5.

7. The preparation method of the liquid compound microecological preparation according to claim 5, characterized in that, The preparation steps of the compound bacterial suspension include separately activating and culturing the strains of lactic acid bacteria, Bacillus coagulans, Clostridium butyricum and yeast to obtain a lactic acid bacteria suspension, a yeast suspension, a Bacillus coagulans suspension and a Clostridium butyricum suspension, and then mixing them to obtain the compound bacterial suspension.

8. The preparation method of the liquid compound microecological preparation according to claim 7, characterized in that, The viable count of lactic acid bacteria in the composite bacterial suspension is 9.2×10 8 -1.3×10 9 cfu / ml, the viable count of Bacillus coagulans is 3.7×10 8 -6.5×10 8 cfu / ml, the viable count of Clostridium butyricum is 3.0×10 8 -3.8×10 8 cfu / ml, the viable count of yeast is 3.7×10 8 -6.5×10 8 cfu / ml.

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