Broad-spectrum additive for inhibiting bottle expansion of liquid microecological preparation and application of broad-spectrum additive

Abstract

The invention discloses a broad-spectrum additive for inhibiting bottle expansion of a liquid microecological preparation and application of the broad-spectrum additive, and belongs to the technical field of agricultural biology. The broad-spectrum additive comprises the following components: 10%-30% of calcium carbonate, 10%-30% of calcium chloride, 10%-30% of potassium chloride, 10%-50% of sodium chloride, 1%-10% of manganese sulfate and 1%-3% of polylysine; the broad-spectrum additive with the concentration of 5%-15% is added into the liquid microecological preparation, and then the liquid microecological preparation is sealed and preserved at the temperature of 10-45 DEG C. The raw materials are cheap and easy to obtain and free of toxic and side effects, the product using method is simple, the bottle expansion phenomenon inhibiting effect is remarkable, and the growth capacity of microorganisms can be completely recovered when the product is diluted for use. Therefore, the broad-spectrum additive can improve the stability of the liquid microecological preparation and prolong the shelf life of the liquid microecological preparation. The method can be widely applied to preservation of liquid micro-ecological / environment-friendly preparations in multiple fields, and has huge economic and social benefits.

Description

Technical Field

[0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a broad-spectrum additive for inhibiting bottle swelling of a liquid microecological preparation and application thereof. Background Art

[0002] As a means of preventing and treating diseases and promoting growth, antibiotics were once widely used in animal husbandry, which led to the increasingly prominent problem of antibiotic abuse, seriously threatening the health of livestock and poultry, humans, and the safety of the ecological environment. Seeking alternatives to antibiotics has become a hot topic in the animal nutrition and health industry.

[0003] "Alternative antibiotic products" include fermented feed and enzyme preparations that improve feed digestibility, antimicrobial peptides and plant extracts that inhibit the growth of harmful microorganisms, and microecological preparations that improve intestinal function. Microecological preparations, also known as microecological regulators, are preparations made from microorganisms that are beneficial to the host and their metabolites and growth-promoting substances. They can balance intestinal microorganisms through antibacterial, adhesion and colonization, and biological barrier effects, inhibit pathogenic growth and the production of harmful metabolites, and improve the health of the host; they can also produce beneficial substances such as digestive enzymes, making them ideal "alternative antibiotic products." In addition to the feed industry, microecological preparations are also widely used in clinical treatment and health care, biological pesticides, wastewater treatment and other fields.

[0004] Probiotic preparations include two forms: solid and liquid. The advantages of solid preparations are that they are easy to transport, store and use, but the disadvantages are that they require special drying equipment during the production process, the production process is complex, and the production cost is high. Therefore, liquid probiotic preparations have a broad market, especially in direct drinking by livestock and poultry and making silage. Although the production process of liquid preparations is simple, the problem of "bottle swelling" may occur during transportation and storage at room temperature. On the one hand, "bottle swelling" affects the appearance of the product, and on the other hand, it causes changes in the pH of the probiotic preparation, reduces the concentration of live bacteria in the product, and shortens the shelf life. The resulting quality disputes and even returns seriously affect the economic benefits of the company. Therefore, the "bottle swelling" problem is an industry problem faced by the microecological field, and it is of great significance to develop a broad-spectrum additive that can effectively inhibit "bottle swelling".

[0005] The "bottle swelling" of liquid microecological preparations is mainly caused by the production of gas by the growth and metabolism of microorganisms. Common microorganisms that cause "bottle swelling" include yeast, Bacillus (Bacillus subtilis, Bacillus licheniformis and Clostridium, etc.), and lactic acid bacteria (such as Lactobacillus pobuzihii, Lactobacillus acidophilus, and Lactobacillus halophilus). Methods for inhibiting microbial growth include physical methods (heating, filtration, radiation, low temperature, high pressure, drying, and high osmotic pressure) and chemical methods (adding antibacterial agents or preservatives). Commonly used antibacterial agents include phenols, halogens, alcohols, aldehydes, peroxides, heavy metals, surfactants, etc. Commonly used preservatives include chemical preservatives such as potassium sorbate, sodium nitrite, sodium lactate, citric acid, sodium diacetate, polylysine, etc.; natural preservatives such as nisin, lysozyme, chitosan, rosemary extract, tea polyphenols, and auxiliary materials such as garlic, cloves, and propolis. These methods are suitable for different objects. Heating, filtration and radiation in physical methods irreversibly inhibit or even kill microorganisms; low-temperature preservation requires cold chain transportation and low-temperature equipment, which is costly; high pressure and drying are suitable for solid products; most chemical antibacterial agents are toxic to humans and livestock.

[0006] Salting and sugaring based on the principle of hypertonicity and adding chemical preservatives are commonly used food preservation methods, and the added substances are harmless or have little toxic side effects. Literature reports that polylysine and nisin have good antibacterial effects on Clostridium perfringens, which causes food corruption, followed by rosmarinic acid; a patent has announced that adding a fresh-keeping solution composed of salt, cooking wine, sodium dehydroacetate, potassium sorbate, citric acid, acetic acid, lactic acid and sodium ethylenediaminetetraacetic acid can effectively inhibit the bulging of pickled pepper products (patent number 201611238808.9). These substances can only inhibit the growth of microorganisms within a certain concentration range, and they will lose their effect after dilution. It can be understood that their inhibitory effect is reversible and is an additive that is more suitable for inhibiting the "bottle swelling" of liquid microecological preparations. A mixture of potassium sorbate, glycine, borax and trehalose is used to improve the "bottle swelling" phenomenon of liquid microecological preparations (patent number 202011222349.1). However, the products reported in the literature have limited application scope, and it is of great significance to develop broad-spectrum anti-swelling bottle additives suitable for commonly used liquid probiotic preparations. Summary of the invention

[0007] In view of this, the present invention provides a broad-spectrum additive for inhibiting the swelling of liquid microecological preparations and its application. The raw materials of the broad-spectrum additive are cheap and readily available and have no toxic side effects. The product is simple to use, has a significant effect on inhibiting the swelling of the bottle, and the microorganisms can fully restore their growth ability when diluted for use. Therefore, the broad-spectrum additive can improve the stability of liquid microecological preparations, extend their shelf life, and can be widely used in the preservation of liquid microecological / environmentally friendly preparations in multiple fields, with huge potential for economic and social benefits.

[0008] In order to achieve the above object, the present invention adopts the following technical solutions:

[0009] One of the purposes of the present invention is to provide a broad-spectrum additive for inhibiting bottle swelling of a liquid probiotic preparation. The broad-spectrum additive comprises the following components by weight percentage: 10% to 30% of calcium carbonate, 10% to 30% of calcium chloride, 10% to 30% of potassium chloride, 10% to 30% of sodium chloride, 10% to 50% of manganese sulfate, and 1% to 3% of polylysine.

[0010] Preferably, the broad-spectrum additive comprises the following components by weight percentage: 10% to 15% calcium carbonate, 10% to 20% calcium chloride, 20% to 30% potassium chloride, 30% to 50% sodium chloride, 3% to 5% manganese sulfate and 1% to 3% polylysine.

[0011] More preferably, the broad-spectrum additive comprises the following components by weight: 15% calcium carbonate, 15% calcium chloride, 30% potassium chloride, 34% sodium chloride, 5% manganese sulfate and 1% polylysine.

[0012] The second object of the present invention is to provide a method for inhibiting bottle swelling of a liquid probiotic preparation, the method comprising using the broad-spectrum additive.

[0013] Preferably, the broad-spectrum additive is added to the liquid microecological preparation at a concentration of 5% to 15%.

[0014] More preferably, the broad-spectrum additive is added to the liquid probiotic preparation at a concentration of 7.5%.

[0015] Preferably, the broad-spectrum additive is added to the liquid probiotic preparation and then sealed for storage.

[0016] More preferably, the storage temperature is 10-45°C.

[0017] More preferably, the storage temperature is 22-40°C.

[0018] The third object of the present invention is to provide an application of the broad-spectrum additive or the method in inhibiting bottle swelling of liquid probiotic preparations.

[0019] The liquid microecological preparation may be of any type or types of common gas-producing microorganisms, such as Clostridium butyricum liquid bacterial agent, Clostridium butyricum-lactic acid bacteria composite liquid bacterial agent, EM liquid bacterial agent, Bacillus subtilis liquid bacterial agent, Bacillus licheniformis liquid bacterial agent, nitrifying bacteria liquid bacterial agent, etc.

[0020] The liquid microecological preparation can be applied in multiple fields, such as health care products, veterinary drugs, feed, pesticides and the like.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention aims to create an unfavorable living environment for liquid microecological preparations, so that they stop growing or maintain a spore state, thereby inhibiting the "bottle swelling" phenomenon. Calcium carbonate, calcium chloride and potassium chloride can keep the liquid microecological preparation at a high pH value, which is not conducive to the growth of microorganisms; manganese sulfate is conducive to the production of spores or the maintenance of spores by Bacillus; sodium chloride can inhibit the growth of most salt-intolerant microorganisms; polylysine has a good antibacterial effect on Clostridium. The raw materials of the broad-spectrum "anti-swelling bottle" additive obtained by compounding these substances are cheap, easy to obtain and have no toxic side effects. The product is simple to use and has a good inhibitory effect on the "bottle swelling" phenomenon of liquid microecological preparations. In addition, the microorganisms can fully restore their growth ability when the product is diluted. Therefore, the broad-spectrum additive in the present invention can improve the stability of liquid microecological preparations and extend their shelf life. The product in the present invention can be widely used in the preservation of liquid microecological preparations or liquid environmentally friendly bacterial agents in multiple fields such as health products, veterinary drugs, feed, pesticides, etc., and has huge potential economic and social benefits. DETAILED DESCRIPTION

[0023] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention are within the scope of the present invention. The products, reagents, equipment, etc. used in the following examples are commercially available unless otherwise specified, and the methods used are consistent with conventional methods unless otherwise specified.

[0024] The technical solution of the present invention is further described in detail below in conjunction with embodiments.

[0025] Example 1 Screening of stabilizers

[0026] At present, the preservatives commonly added in food and feed include potassium sorbate, sodium benzoate, glycine, borax, trehalose, sodium propionate and chitosan. It is reported in the literature that citric acid, acetic acid and lactic acid can inhibit the growth of bacteria in food and prevent food from "swelling bags". Food is often sterilized before preservation, and the initial concentration of microorganisms is low. However, the concentration of microorganisms in liquid microbial preparations is very high, so the substances that prevent food from "swelling bags" are not necessarily suitable for inhibiting the "swelling bottles" of liquid microecological preparations. On the other hand, some microorganisms in liquid microecological preparations can form spores, and the addition of metabolizable substances such as citric acid, glycine, trehalose and chitosan may stimulate the germination of spores, which is not conducive to the long-term preservation of products. In addition, some microorganisms contained in microecological preparations can produce substances such as butyric acid and lactic acid, and no additional addition is required. Therefore, these substances are not ideal choices either. The present invention aims to create an unfavorable living environment for liquid microecological preparations, so that they stop growing or maintain a spore state, thereby inhibiting the "swelling bottle" phenomenon. Calcium carbonate, calcium chloride and potassium chloride can keep the pH value of liquid microecological preparations at a higher level, which is not conducive to the growth of microorganisms; manganese sulfate is conducive to the production of spores or the maintenance of spores in the bacillus state; sodium chloride can inhibit the growth of most salt-intolerant microorganisms; polylysine has a good antibacterial effect on Clostridium; borax has an inhibitory effect on a variety of microorganisms.

[0027] The present invention takes liquid Clostridium butyricum agent (strain preservation number is CCTCC No: M2022151) as the research object, and studies the inhibitory effect of these alternative stabilizers on "bottle swelling" and the recovery of microbial growth after product dilution. The liquid Clostridium butyricum preparation is obtained by conventional anaerobic fermentation process for 40 hours. According to literature and experience, three concentration groups of high, medium and low are set for each stabilizer, and the control group without stabilizer, each group of 50 bottles, is subjected to accelerated storage experiment at 37°C. After 10 days and 20 days, the odor and bottle swelling rate of the samples are statistically analyzed. Three bottles of samples are randomly selected to detect the live bacteria concentration and spore concentration of Clostridium butyricum, and the average value is calculated.

[0028] The test results are as follows: after 10 days of storage at 37°C, the control group produced an obvious odor, while the test group had no obvious odor after 20 days of storage. The results of the bottle swelling rate, live bacteria concentration and spore concentration of the liquid Clostridium butyricum preparation after storage with different stabilizers are shown in Table 1.

[0029] Table 1 Preliminary study of stabilizer concentration

[0030]

[0031]

[0032] Note: The concentration of live Clostridium butyricum in the initial sample was 1.35×10 9 CFU / mL, spore concentration 1.28×10 9CFU / mL

[0033] From the results in Table 1, it can be concluded that the seven stabilizers have a certain inhibitory effect on the "bottle swelling" of liquid Clostridium butyricum preparations, and the bottle swelling rate is less than 55% after being stored at 37°C for 20 days. The comparison of the inhibitory effect on bottle swelling is as follows: calcium chloride>potassium chloride>sodium chloride>calcium carbonate>polylysine>manganese sulfate>borax. The live bacteria concentration in the control group decreased seriously, and the spore concentration decreased slowly; after adding stabilizers for preservation, the decreasing trend of live bacteria concentration and spore concentration slowed down significantly, and the final live bacteria concentration and spore concentration of most test groups were greater than that of the control group. This shows that the inhibitory effects of these stabilizers on microorganisms are reversible. In subsequent studies, the six stabilizers with better effects were used for composite tests.

[0034] Example 2 Stabilizer compounding test

[0035] In order to improve the stabilization effect, the six stabilizers selected in Example 1 were compounded and tested. 10% of the composite stabilizer of the formula shown in Table 2 was added to the liquid Clostridium butyricum preparation, and the control group was a control group without stabilizer. Each formula group had 50 bottles. After being stored at 37°C for 20 days, the odor and bottle swelling rate of the liquid Clostridium butyricum preparation were counted, and 3 bottles were randomly selected for Clostridium butyricum live bacteria concentration and spore concentration detection, and the average value was calculated. The results are shown in Table 3.

[0036] Table 2 Ratio of different stabilizers (%)

[0037]

[0038]

[0039] Table 3 Preservation results of different stabilizer formulations

[0040]

[0041] Note: The concentration of live Clostridium butyricum in the initial sample was 1.39×10 9 CFU / mL, spore concentration 1.27×10 9 CFU / mL

[0042] As can be seen from Table 3, the above six formulas all have good control effects on the "bottle swelling" of liquid Clostridium butyricum preparations, and the bottle swelling rate is less than 10% after being stored at 37°C for 20 days. Formulas 1 to 3 have good effects, and the bottle swelling rate is 0 after being stored at 37°C for 20 days. Among them, formula 3 has the best effect, and its viable bacteria and spore survival rates are also higher than those of the control group, so formula 3 is further studied.

[0043] Example 3 Study on the amount of stabilizer

[0044] Different concentrations of Formula 3 stabilizer were added to the liquid Clostridium butyricum preparation, and the control group was the one without stabilizer. Each concentration group had 50 bottles. After being stored at 37°C for 20 days, the odor of the liquid preparation and the "bottle swelling" rate of the packaging bottle were counted, and 3 bottles were randomly selected to test the live bacteria concentration and spore concentration of Clostridium butyricum, and the average values ​​were calculated. The results are shown in Table 4.

[0045] Table 4 Effect of stabilizer dosage on preservation effect

[0046]

[0047]

[0048] Note: The concentration of live Clostridium butyricum in the initial sample was 1.41×10 9 CFU / mL, spore concentration 1.32×10 9 CFU / mL

[0049] As can be seen from Table 4, the above five concentrations all have a good control effect on bottle swelling of the liquid Clostridium butyricum preparation, and the bottle swelling rate is less than 2% after being stored at 37°C for 20 days. Among them, 7.5% has the best effect, and the bottle swelling rate is 0 after being stored at 37°C for 20 days.

[0050] After dilution, the concentration of live Clostridium butyricum and the concentration of spores were higher than those in the control group. The loss rate of live Clostridium butyricum in the control group was 44.04%, and the loss rate of spores was 49.17%. The loss rate of live Clostridium butyricum in the 7.5% experimental group was 41.63%, and the loss rate of spores was 40.38%. Considering the cost and effect comprehensively, the dosage of 7.5% was selected for subsequent research.

[0051] Example 4 Study on the storage of liquid Clostridium butyricum-lactic acid bacteria composite microecological preparation at room temperature

[0052] The test group added 7.5% of the No. 3 formula stabilizer to the liquid Clostridium butyricum preparation, the control group did not add the stabilizer, and the control group added the stabilizer prepared according to the formula in Patent No. 202011222349.1 (1L liquid microecological preparation added 0.08g potassium sorbate, 0.08g glycine, 0.15g borax, and 0.02g trehalose). Each group of 50 bottles was subjected to a long-term storage test at 25°C. After 60 days, the odor and the "bottle swelling" rate of the packaging bottles were counted, and 3 bottles were randomly selected for the Clostridium butyricum live bacteria concentration and spore concentration detection, and the average value was calculated.

[0053] The results showed that after 60 days of storage at room temperature (25°C), the experimental group had no odor and the bottle swelling rate was 0%, the control group had a bad smell, the bottle swelling rate was 100%, and the comparison group had a slight odor and a bottle swelling rate of 30%. The loss rate of live bacteria count of Clostridium butyricum in the experimental group was 42.37%, and the spore loss rate was 45.12%. The loss rate of live bacteria count of Clostridium butyricum in the control group was 46.08%, and the spore loss rate was 51.19%. The loss rate of live bacteria count of Clostridium butyricum in the comparison group was 45.38%, and the spore loss rate was 70.36%. It shows that the composite stabilizer of the present invention has an ideal inhibitory effect on the "bottle swelling" of the composite Clostridium butyricum preparation under room temperature storage conditions, and the product stability is also significantly improved. Although the formula 202011222349.1 in the patent is suitable for Bacillus subtilis and Bacillus licheniformis, it is not suitable for acid-producing and gas-producing Clostridium butyricum liquid preparations.

[0054] In addition, in addition to the liquid Clostridium butyricum preparation, the above stabilizer in the present invention also achieved ideal results in the preservation of liquid bacterial agents such as lactic acid bacteria, Clostridium butyricum-lactic acid bacteria composite microecological preparations, EM bacteria, Bacillus subtilis, Bacillus licheniformis, Mudingbao, and nitrifying bacteria, and there was no bottle swelling phenomenon after being stored at room temperature for 60 days. It shows that the product of the present invention can inhibit the growth of most microorganisms, thereby inhibiting the swelling of the liquid microecological preparation.

[0055] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A broad-spectrum additive for inhibiting bottle swelling of liquid microecological preparations, characterized in that: The broad-spectrum additive comprises the following components by weight percentage: 10% to 30% calcium carbonate, 10% to 30% calcium chloride, 10% to 30% potassium chloride, 10% to 50% sodium chloride, 1% to 10% manganese sulfate and 1% to 3% polylysine.

2. The broad-spectrum additive according to claim 1, characterized in that The broad-spectrum additive comprises the following components by weight percentage: 10% to 15% calcium carbonate, 10% to 20% calcium chloride, 20% to 30% potassium chloride, 30% to 50% sodium chloride, 3% to 5% manganese sulfate and 1% to 3% polylysine.

3. The broad-spectrum additive according to claim 2, characterized in that: The broad-spectrum additive comprises the following components by weight percentage: 15% calcium carbonate, 15% calcium chloride, 30% potassium chloride, 34% sodium chloride, 5% manganese sulfate and 1% polylysine.

4. A method for inhibiting bottle swelling of a liquid probiotic preparation, characterized in that: The method comprises using the broad-spectrum additive according to any one of claims 1 to 3.

5. The method according to claim 4, characterized in that The broad-spectrum additive is added in a concentration of 5% to 15% in the liquid microecological preparation.

6. The method according to claim 5, characterized in that The broad-spectrum additive is added to the liquid microecological preparation at a concentration of 7.5%.

7. The method according to claim 4, characterized in that The broad-spectrum additive is added into the liquid probiotic preparation and then sealed for storage.

8. The method according to claim 7, characterized in that The storage temperature is 10-45°C.

9. The method according to claim 8, characterized in that The storage temperature is 22-40°C.

10. Use of the broad-spectrum additive according to any one of claims 1 to 3 or the method according to any one of claims 4 to 9 in inhibiting bottle swelling of liquid probiotic preparations.

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