CN113729109A - Fermented feed rich in antibacterial substances and preparation method thereof - Google Patents
Fermented feed rich in antibacterial substances and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a fermented feed rich in antibacterial substances and a preparation method thereof. The fermented feed is obtained by fermenting raw materials comprising a carbon source, a nitrogen source, dipotassium hydrogen phosphate, magnesium sulfate, water and paenibacillus polymyxa SP 1; the paenibacillus polymyxa SP1 is preserved in Guangdong province microorganism strain preservation center with the preservation date of 2021, 4 months and 16 days, and the preservation number is GDMCC No: 61605. the preparation method of the feed is simple in process, and the fermented feed obtained by the method is rich in antibacterial substances and can replace part of antibiotics; meanwhile, the content of available phosphorus, protein solubility and acid soluble protein is increased, the content of crude fat is obviously reduced, the absorption and utilization of animals, especially young animals are facilitated, and the method has good popularization and application values.
Description
Technical Field
The invention relates to the technical field of fermented feeds, in particular to a fermented feed rich in antibacterial substances and a preparation method thereof.
Background
In the existing breeding feed technology, antibiotics are used in large quantity, a series of problems are caused, the application of the antibiotics can increase the drug resistance of applied target microorganisms and parasites, and the dosage and the type of the antibiotics are required to be further increased when people use the antibiotics again due to the occurrence of the drug resistance, so that the generation of the drug resistance is aggravated, a vicious circle is formed, and the abuse of the antibiotics is caused; not only does this add significant cost and harm to breeding, but the antibiotics are difficult to eliminate in a short time during the operation of the food chain, and humans become victims of antibiotic drug residues as the top of the food chain. On the premise of comprehensive implementation of the policy of banning antibiotics for livestock and poultry feeds in China, a feed formula for avoiding the problems of drug resistance and drug residue caused by antibiotics is urgently needed.
The microbial fermented feed has the outstanding functions of improving the utilization rate and palatability of animal feed, maintaining the microecological balance of intestinal flora, enhancing the immunity of organisms, improving the disease resistance and production performance of animals and the like, and highlights the potential effects of replacing the feed, reducing the use of antibiotics, improving the immunity of hosts, resisting the occurrence of diseases and the like in the process of animal breeding. If antibacterial additives such as antibacterial peptide (Chinese patent CN102150752A), terramycin, formate and the like are added into the feed, the feed can play an antibacterial role (Chinese patent CN1242689C), but the food-borne pathogenic microorganisms have strong viability and are often distributed in various food raw materials and sewage, the cost is high after the antibacterial additives are used for a long time, and the feed has a certain toxic effect on cultured animals.
In addition, the bred animals, especially young animals, have the defects that the intestinal tract function is not well developed, the high-content protein and the coarse fat in the food are difficult to digest and absorb, the digestion disorder is generated, the symptoms such as vomit, inappetence, gastroenteritis and the like exist, the health of the animals is influenced, the animals can die in serious cases, and the survival rate of the breeding is influenced. The phosphorus is not easy to be absorbed by animals, the available phosphorus in the feed becomes effective phosphorus, and the higher the content of the available phosphorus, the more beneficial the animals to take the phosphorus. For this problem, absorption by animals is often promoted by adding enzyme preparations such as lipase, protease, and phytase (CN103719596A and CN104621413A), but the cost for adding enzyme preparations is high.
Disclosure of Invention
The invention provides a fermented feed rich in antibacterial substances and a preparation method thereof, aiming at solving the technical defects of complex process and high cost caused by drug resistance, drug residue, drug toxicity and addition of enzyme preparations when antibiotics and antibacterial additives are applied in the feed at present.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention provides a fermented feed rich in antibacterial substances and a preparation method thereof, which are used for improving the utilization rate of feed raw materials, have effective action on common food-borne pathogenic microorganisms and can be used for replacing part of antibiotics, have simple process and low cost, are safe and non-toxic, can be widely applied to livestock and poultry breeding, have broad-spectrum antibacterial effect on bacteria used for fermentation, have inhibiting effect on common livestock and poultry pathogenic bacteria such as staphylococcus aureus, escherichia coli K12D31, escherichia coli O78, salmonella typhimurium, salmonella choleraesuis and the like, have good antibacterial effect on common pathogenic bacteria such as bacillus thuringiensis and septicemia melanothorax of insect breeding objects such as silkworms and the like, and can be used as feed additives for insect breeding, particularly silkworms. The bacteria SP1 used for fermentation can produce protease, lipase and phytase, has good fermentation effect on protein, fat and phosphorus, can increase the content of effective phosphorus, protein solubility and acid soluble protein of the feed, and obviously reduce the content of crude fat. The feed fermentation utilizes Paenibacillus polymyxa with efficient fermentation and bacteriostasis functions.
Specifically, the raw material components for preparing the fermented feed comprise: carbon source, nitrogen source, dipotassium hydrogen phosphate, magnesium sulfate, water and paenibacillus polymyxa SP 1. The paenibacillus polymyxa SP1 is preserved in Guangdong province microorganism strain preservation center with the preservation date of 2021, 4 months and 16 days, and the preservation number is GDMCC No: 61605.
further, the components are as follows by weight: 10-13 parts of carbon source, 1-5 parts of nitrogen source, 0.0025-0.25 part of dipotassium hydrogen phosphate, 0.000073-0.0073 parts of magnesium sulfate, 15-25 parts of water and 2-4 parts of paenibacillus polymyxa SP 1.
Preferably, the carbon source is bran or corn flour, and the nitrogen source is soybean meal or soybean flour.
Further, dipotassium hydrogen phosphate adopts hydrate of hydrogen phosphate: k2HPO4·2H2O, magnesium sulfate as magnesium sulfate hydrate: MgSO (MgSO)4·7H2O。
Furthermore, the weight parts of the components are as follows: 12 parts of bran, 3 parts of soybean meal and K2HPO4·2H2O0.03 part, MgSO4·7H20.0015 part of O, 20 parts of water and 3 parts of paenibacillus polymyxa SP 1.
Furthermore, the water adopted by the fermented feed is sterile water.
Further, the fermented feed is prepared by using the paenibacillus polymyxa SP1 in a form of fermentation liquor, wherein the bacterial density is not lower than 1 x 106cfu。
Preferably, the bacterial density of the fermentation liquor of the paenibacillus polymyxa SP1 is (1 × 10)6)-(1×1010)cfu。
The optimal initial adding density is matched with other materials, and the optimal feed finished product can enable the feed to achieve the optimal bacteriostasis and fermentation effects under the corresponding temperature condition.
In order to achieve the purpose, the preparation method of the fermented feed is shown in figure 1, and the preparation method comprises the following specific steps:
s1, pretreatment of raw materials: uniformly mixing a carbon source, a nitrogen source, dipotassium hydrogen phosphate and magnesium sulfate with water; then sterilizing;
s2, culturing the SP1 strain to obtain fermentation liquor;
s3, fermenting feed: inoculating SP1 bacteria fermentation liquor to the mixture sterilized in the step S1 for fermentation under the sterile environment, and turning over water materials intermittently in the fermentation process; and after the fermentation is finished, obtaining the fermented feed rich in the antibacterial substances.
Wherein, the sterilization temperature in the step S1 is preferably 100-150 ℃, and the sterilization time is preferably 15-30 min.
Preferably, the fermentation conditions of step S3 are: controlling the temperature at 29-31 ℃, fermenting for 30-72h, and turning over the materials frequently: every 12 hours 1 time.
The invention has the beneficial effects that:
the invention adds Paenibacillus polymyxa (Paenibacillus polymyxa) SP1 into feed raw materials to be fermented, wherein the Paenibacillus polymyxa is listed as a first-level strain without safety identification in the department of agriculture in China, SP1 is a strain of Paenibacillus polymyxa capable of generating various bioactive substances such as polypeptide antibiotics, antagonistic proteins, enzyme proteins and the like, which is obtained by separation of the subject components of the inventor, and the Paenibacillus polymyxa has good bacteriostatic action on various strains such as staphylococcus aureus, Escherichia coli K12D31, Escherichia coli O78, Salmonella typhimurium, Salmonella choleraesuis, Bacillus thuringiensis, Patrinia nigricans and the like, and does not generate hydrogen sulfide gas during fermentation. Compared with the common fermented feed which mainly improves the disease resistance of livestock and poultry by maintaining the microecological balance of animal intestinal flora through microorganisms, the fermented feed provided by the invention not only can adjust the microecological balance of animal intestinal flora and is rich in antibacterial substances and can directly kill pathogenic bacteria, but also can complete fermentation and bacteriostasis through one bacterium, so that the obtained fermented feed has an inhibiting effect on common intestinal pathogenic bacteria of livestock and poultry, particularly has a good inhibiting effect on pathogenic bacteria of silkworms of insect breeding objects, and can realize the prevention of silkworm cataplexy and septicemia through inhibiting bacillus thuringiensis and septicemia.
Moreover, the strain can secrete a plurality of enzyme proteins such as lipase, phytase and the like, so that the content of effective phosphorus, protein solubility and acid soluble protein in the raw materials is increased, the content of crude fat is obviously reduced, and the absorption and utilization of animals, particularly young animals are facilitated.
The fermented feed is safe and harmless to cultured organisms, short in fermentation time, simple in preparation process and suitable for popularization and application.
Drawings
FIG. 1 is a flow chart of the preparation method of the fermented feed
FIG. 2 is a graph showing the effect of different carbon sources on the fermentation of SP1 bacteria
FIG. 3 comparison of the effect of different nitrogen sources on the colony count of SP1
FIG. 4 is a graph comparing the effect of mass ratio of bran to soybean meal on the colony count of SP1
FIG. 5 addition of different concentrations K2HPO4Comparative plot of the Effect on colony count of SP1
FIG. 6 addition of MgSO (MgSO) at different concentrations4Effect on colony count of SP1
FIG. 7 addition of different concentrations of MnSO4Effect on colony count of SP1
FIG. 8 is a graph showing the effect of different water ratios on the number of viable bacteria fermented
FIG. 9 is a graph comparing the effect of different inoculum sizes on viable counts of fermentations
FIG. 10 is a graph comparing the effect of different fermentation durations on the number of viable bacteria in fermentation
FIG. 11 is a graph showing the bacteriostatic activity of the fermented supernatant and the fermented feed extract
Detailed Description
The invention is further described with reference to the drawings and the following detailed description, which are not intended to limit the invention in any way. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
Unless otherwise indicated, reagents and materials used in the following examples are commercially available.
Examples 1-2 are examples of feeds and examples 3-11 are examples of the present fermented feed in combination with a feed preparation method.
EXAMPLE 1 fermentation Strain screening
The applicant obtains a strain with the highest bacteriostatic activity by comparison and screening from the soil of a mulberry field of southern China agricultural university by adopting an agar hole diffusion method and taking escherichia coli and staphylococcus aureus as indicator bacteria, and the strain is marked as the strain SP 1.
Through the comprehensive results of morphological identification and molecular identification, the identified strain SP1 belongs to Paenibacillus polymyxa (Paenibacillus polymyxa), and is preserved in Guangdong province microorganism strain preservation center in 2021 year 4 and 16 days, the preservation unit address is Guangdong province academy of sciences microorganism research institute (No. 59 building 5 building of Michelia Tourette 100, Guangzhou city), the preservation number is GDMCC No: 61605.
example 2 preparation of fermented feed
1. Weighing the following components in parts by weight:
12 portions of carbon source bran,
3 parts of nitrogen source soybean meal,
Dipotassium hydrogen phosphate hydrate (K)2HPO4·2H2O)0.03 part,
Magnesium sulfate hydrate (MgSO)4·7H2O)0.0015 part,
20 parts of water, namely, water,
3 parts of paenibacillus polymyxa, wherein the paenibacillus polymyxa is SP1 strain, and the density of SP1 strain is 1 multiplied by 106cfu per serving.
2. The preparation method of the fermented feed comprises the following steps:
s1, pretreatment of raw materials: adding water into a carbon source, a nitrogen source, dipotassium phosphate hydrate and magnesium sulfate hydrate and uniformly mixing; sterilizing at 120 deg.C for 20 min;
s2, culturing the SP1 strain to obtain fermentation liquor;
s3, fermenting feed: under aseptic conditions, 3mL of SP1 strain fermentation broth (1X 10) per 30g of the mixture sterilized in step S1 was inoculated6cfu/mL)，Fermenting at constant temperature of (30 +/-1) deg.C for 48h, and intermittently turning over the water material at a frequency of 1 per 12 hr; and after the fermentation is finished, obtaining the fermented feed rich in the antibacterial substances.
Example 3 preparation of fermented feed
1. Weighing the following components in parts by weight:
12 parts of carbon source corn flour,
3 parts of nitrogen source bean powder,
Dipotassium hydrogen phosphate hydrate (K)2HPO4·2H2O)0.03 part,
Magnesium sulfate hydrate (MgSO)4·7H2O)0.0015 part,
20 parts of water, namely, water,
3 parts of paenibacillus polymyxa, wherein the paenibacillus polymyxa is SP1 strain, and the density of SP1 strain is 1 multiplied by 106cfu per serving.
2. The fermented feed was prepared in the same manner as in example 2.
Example 4 preparation of fermented feed
1. Weighing the following components in parts by weight:
10 portions of carbon source bran,
1 part of nitrogen source soybean meal,
Dipotassium hydrogen phosphate hydrate (K)2HPO4·2H2O)0.0025 parts,
Magnesium sulfate hydrate (MgSO)4·7H2O)0.000073 parts,
15 parts of water, namely adding water into the mixture,
2 parts of paenibacillus polymyxa, wherein the paenibacillus polymyxa is SP1 strain, and the density of SP1 strain is 1 multiplied by 106cfu per serving.
2. The fermented feed was prepared in the same manner as in example 2.
Example 5 preparation of fermented feed
1. Weighing the following components in parts by weight:
13 parts of carbon source corn flour,
5 parts of nitrogen source bean powder,
Dipotassium hydrogen phosphate hydrate (K)2HPO4·2H2O)0.25 part,
Magnesium sulfate hydrate (MgSO)4·7H2O)0.0073 part,
25 parts of water, namely, water,
4 parts of paenibacillus polymyxa, wherein the paenibacillus polymyxa is SP1 strain, and the density of SP1 strain is 1 multiplied by 106cfu per serving.
2. The fermented feed was prepared in the same manner as in example 2.
Example 6 preparation of fermented feed
1. Weighing the following components in parts by weight:
10 portions of carbon source bran,
5 parts of nitrogen source soybean meal,
Dipotassium hydrogen phosphate hydrate (K)2HPO4·2H2O)0.0025 parts,
Magnesium sulfate hydrate (MgSO)4·7H2O)0.0073 part,
20 parts of water, namely, water,
3 parts of paenibacillus polymyxa, wherein the paenibacillus polymyxa is SP1 strain, and the density of SP1 strain is 1 multiplied by 106cfu per serving.
2. The fermented feed was prepared in the same manner as in example 2.
Example 7 index test of fermented feed
1. Physical and chemical properties
The results of observing physicochemical properties and measuring related indexes before and after fermentation of the feeds obtained in examples 2 to 6 show that: the fermented feed prepared by the invention is yellow brown, has bean fragrance, and has viable count as high as 6 multiplied by 109cfu/g or more, wherein the number of viable bacteria of the fermented feed prepared in example 2 was 6.33X 109cfu/g, and has good bacteriostatic effect.
2. Effect of fermentation
The fermented feed prepared in example 2 was used as an example to test the changes of relevant indexes before and after fermentation.
The results show that the fermented feed has good fermentation effect, compared with the fermented feed before fermentation, the content of available phosphorus after fermentation is increased by 48.44%, the content of nitrogen in protein solubility is increased by 11.11%, the content of nitrogen in acid-soluble protein is increased by 35.29%, the content of crude fat is reduced by 43.79%, the fermented feed is more beneficial to the absorption and utilization of fat and protein by animals, particularly young children with poor gastrointestinal functions, and the vomiting and dyspepsia caused by excessive fat are reduced (Table 1).
TABLE 1 Change of indexes before and after fermentation
Example 8 selection of carbon sources in fermented feed stocks
Experiments were performed with carbon source shifting based on the method of example 2. The method comprises the following steps: respectively taking 12g of rice hull, 12g of bran and 12g of corn flour as carbon sources, respectively adding 3g of soybean flour as nitrogen source into each group, and then adding a certain mass of K2HPO4·2H2O and MgSO4·7H2O, after stirring and mixing evenly, adding 15mL of sterile water into each component, then sterilizing at 120 ℃ for 20min, and inoculating 3mL (about 10) of sterile water after the temperature is reduced to about 30 DEG C6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture, sampling after stopping fermentation and determining the bacterial content of the solid fermentation product and the antibacterial activity of the product.
As a result, as shown in FIG. 2, in the two groups of culture media containing bran and corn flour, the number of viable bacteria produced was 2.0X 108cfu/g、1.55×108cfu/g, it can be seen that bran is more suitable than corn flour for solid fermentation of the strain SP 1. The effect of solid fermentation by using bran is better than that of corn flour and is better than that of rice hull.
Example 9 selection of Nitrogen sources in fermented feed stocks
Experiments were performed by changing the nitrogen source based on the method of example 2. The method comprises the following steps: taking 12g of bran as a carbon source, respectively taking 3g of yeast powder, 3g of soybean meal and 3g of soybean meal as nitrogen sources, and then adding a certain mass of K2HPO4·2H2O and MgSO4·7H2O, after stirring and mixing evenly, adding 15mL of sterile water into each component, then sterilizing at 120 ℃ for 20min, and inoculating 3mL (about 10) of sterile water after the temperature is reduced to about 30 DEG C6cfu/mL) SP1 seed solution, stirring, placing in incubator, fermentingCulturing, stopping fermentation, sampling, and measuring the bacterial content of the solid fermentation product and the antibacterial activity of the product.
The results are shown in fig. 3, SP1 bacteria did not grow in bran containing yeast powder. The number of viable bacteria detected by bran containing soybean meal and soybean meal is 2.53 × 108cfu/g、3.67×108cfu/g, therefore, the bean pulp is better than the soybean meal, and the paenibacillus polymyxa SP1 can not utilize yeast powder.
Example 10 optimization of the ratio of carbon to nitrogen sources in fermented feed stocks
Preparing 6 groups of bran and bean pulp with mass ratio of 1: 2, 1: 1, 2: 1, 4: 1, 6: 1 and 8: 1 respectively, and adding K with certain mass2HPO4·2H2O and MgSO4·7H2O, mixing uniformly, adding 15mL of sterile water, sterilizing at 120 deg.C for 20min, cooling to about 30 deg.C, adding 3mL (about 10%6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture, and sampling after stopping fermentation to determine the bacterial content of the solid fermentation product. Controlling the ratio of material to water to be 1: 1, the mass ratio of the bran to the bean pulp is 1: 2 respectively, namely 5g of bran and 10g of bean pulp; the mass ratio of the bran to the bean pulp is 1: 1 respectively, namely 7.5g of bran and 7.5g of bean pulp; the mass ratio of the bran to the bean pulp is 2: 1 respectively, namely 10g of bran and 5g of bean pulp; the mass ratio of the bran to the bean pulp is 4: 1 respectively, namely 12g of bran and 3g of bean pulp; the mass ratio of the bran to the bean pulp is 6: 1 respectively, namely 12.9g of bran and 2.1g of bean pulp; the mass ratio of the bran to the soybean meal is respectively 8: 1, namely 13.3g of bran and 1.7g of bean pulp.
The result is shown in figure 4, and the optimal mass ratio is 12g of bran and 3g of soybean meal, namely the bran: the mass ratio of the soybean meal is 4: 1.
Example 11 fermentation of feed Material K2HPO4Selection of content
Weighing 5 parts of bran (12g) and soybean meal (3g) raw materials, and respectively adding 2%, 0.2%, 0.02%, 0.002% and 0% (w/w) K2HPO4·2H2O, mixing uniformly, adding 15mL of sterile water, sterilizing at 120 deg.C for 20min, cooling to about 30 deg.C, adding 3mL (about 10%6cfu/mL) SP1 seed solution, stirring and mixing evenly,placing in an incubator for fermentation culture, sampling after stopping fermentation, and determining the bacteria content of the solid fermentation product.
The results are shown in FIG. 5, optimal addition of K2HPO4The hydrate concentration was 0.2%.
Example 12 MgSO in fermented feed Material4Selection of content
Weighing 5 parts of bran (12g) and soybean meal (3g) raw materials, and adding 30mg K2HPO4·2H2O, and adding 1%, 0.1%, 0.01%, 0.001%, 0% (w/w) MgSO respectively4·7H2O, mixing uniformly, adding 15mL of sterile water, sterilizing at 120 deg.C for 20min, cooling to about 30 deg.C, adding 3mL (about 10%6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture, sampling after stopping fermentation and determining the bacterial content of the solid fermentation product and the antibacterial activity of the product.
The results are shown in FIG. 6, optimal MgSO4The hydrate was added at a concentration of 0.01%, i.e., 1.5 mg.
Example 13 MnSO in fermented feed Material4Screening of (2)
MnSO4Screening. Weighing 5 parts of bran (12g) and soybean meal (3g) raw materials, and adding 30mg K2HPO4·2H2O，1.5mg MgSO4·7H2O, mixing uniformly, and adding 1%, 0.1%, 0.01%, 0.001%, 0% (w/w) MnSO4Adding 15mL of sterile water, sterilizing at 120 deg.C for 20min, cooling to about 30 deg.C, and adding 3mL (about 10%6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture, and sampling after stopping fermentation to determine the bacterial content of the solid fermentation product.
As a result, as shown in FIG. 7, unlike other Paenibacillus polymyxa, Mn ions have an inhibitory effect on the growth of the present Paenibacillus polymyxa SP1, and the growth without addition is the best.
Example 14 feed-to-water ratio selection in fermented feed stocks
Weighing 3 parts of bran (12g) and soybean meal (3g) raw materials, adding 30mg K2HPO4·2H2O，1.5mg MgSO4·7H2O according to the ratio of material to waterAdding distilled water 10mL, 15mL, 20mL, 25mL at a ratio of 3: 2, 1: 1, 3: 4(g/mL), sterilizing at 120 deg.C for 20min, cooling to about 30 deg.C, and adding 3mL (about 10%6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture, and sampling after stopping fermentation to determine the bacterial content of the solid fermentation product.
The results are shown in FIG. 8, with an optimum feed-water ratio of 3 g: 4mL, i.e., 20mL of distilled water.
Example 15 fermentation initial inoculum size screening
Weighing 4 parts of bran (12g) and soybean meal (3g) raw materials, adding 30mg K2HPO4·2H2O、1.5mg MgSO4·7H2O, 20mL of sterile water, sterilizing at 120 deg.C for 20min, cooling to about 30 deg.C, and adding 1mL, 2mL, 3mL, and 4mL (about 10)6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture, and sampling after stopping fermentation to determine the bacterial content of the solid fermentation product.
The results are shown in FIG. 9, with an optimal initial inoculum size of 3 mL.
Example 16 fermentation time selection
Weighing 3 parts of bran (12g) and soybean meal (3g) raw materials, adding 30mg K2HPO4·2H2O、1.5mg MgSO4·7H2O, 20mL of sterile water, sterilizing at 120 ℃ for 20min, and inoculating 3mL (about 10) of sterile water after the temperature is reduced to about 30 DEG C6cfu/mL) SP1 seed liquid, stirring and mixing uniformly, placing in an incubator for fermentation culture for 24h, 48h and 72h, sampling and determining the bacterial content of the solid fermentation product.
The results are shown in FIG. 10, with an optimal fermentation time of 48 h.
Example 17 verification of bacteriostatic Effect
1. Experimental methods and procedures
And (4) detecting the size of the bacteriostatic zone by an agar hole diffusion method to judge the bacteriostatic effect of the fermentation liquor and the fermented feed.
The LB medium includes LB liquid medium and LB solid medium.
LB liquid medium (1000 mL): 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride, diluting to 1L with distilled water, adjusting pH to 6.0-7.0, and autoclaving at 121 ℃ for 20 min.
LB solid medium (1000 mL): 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and 1.5% agar powder, wherein the volume is determined to be 1L by using distilled water, the pH value is 6.0-7.0, and the agar powder is sterilized at 121 ℃ for 20min under high pressure.
The method comprises the following steps: and (3) placing the sterilized LB culture medium at room temperature, cooling to 48-50 ℃, sucking 100 mu L of indicator bacterium staphylococcus aureus diluent, adding the indicator bacterium diluent into 10mL of LB solid culture medium which is dissolved and cooled to 40-50 ℃, immediately pouring the indicator bacterium staphylococcus aureus diluent into a culture dish with the diameter of 90mm, quickly shaking and shaking up to uniformly disperse the thalli, and after the indicator bacterium diluent is cooled and solidified, punching 3 holes by using a puncher with the diameter of 2.7mm for later use. Adding 5uL of sterile water, 5uL of SP1 fermentation liquor and 5uL of fermented feed extracting solution into each hole respectively, standing for 30min, culturing at 37 ℃ for 24h, observing and measuring the size of a bacteriostatic circle, and recording data.
2. Test results
The bacteriostatic effect against staphylococcus aureus is shown in fig. 11, where CK1 is a sterile water control (2.7mm, i.e. the diameter of the spot well); CK2 is the inhibition zone (12.78mm) of SP1 fermentation liquor; 1 is the bacteriostatic zone (18.86mm) of the fermented feed extract.
The result shows that the fermentation broth of the paenibacillus polymyxa SP1 has good bacteriostatic effect, and the bacteriostatic effect of the fermented feed prepared by the method is obviously improved.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (10)
1. The fermented feed rich in antibacterial substances is characterized by being obtained by fermenting raw materials comprising a carbon source, a nitrogen source, dipotassium hydrogen phosphate, magnesium sulfate, water and paenibacillus polymyxa SP 1; the paenibacillus polymyxa SP1 is preserved in Guangdong province microorganism strain preservation center with the preservation date of 2021, 4 months and 16 days, and the preservation number is GDMCC No: 61605.
2. the fermented feed according to claim 1, characterized in that the components are, in parts by weight: 10-13 parts of carbon source, 1-5 parts of nitrogen source, 0.0025-0.25 part of dipotassium hydrogen phosphate, 0.000073-0.0073 parts of magnesium sulfate, 15-25 parts of water and 2-4 parts of paenibacillus polymyxa SP 1.
3. The fermented feed according to claim 2, wherein the carbon source is bran or corn flour and the nitrogen source is soybean meal or soybean flour.
4. The fermented feed according to claim 2, wherein the dipotassium hydrogen phosphate is selected from the group consisting of hydrated hydrogen phosphate: k2HPO4·2H2O, magnesium sulfate as magnesium sulfate hydrate: MgSO (MgSO)4·7H2O。
5. The fermented feed according to claim 2, wherein the components are selected from the group consisting of 12 parts by weight of bran, 3 parts by weight of soybean meal, and K2HPO4·2H2O0.03 part, MgSO4·7H20.0015 part of O, 20 parts of water and 3 parts of paenibacillus polymyxa SP 1.
6. The fermented feed according to claim 4, wherein the Paenibacillus polymyxa SP1 is used in the form of a fermentation broth having a density of not less than 1X 106cfu/mL。
7. The fermented feed according to claim 4, wherein the Paenibacillus polymyxa SP1 is used in the form of a fermentation broth having a cell density of (1X 10)6)-(1×1010)cfu/mL。
8. The fermented feed according to any one of claims 1 to 7, which is prepared by a method comprising the steps of:
s1, pretreatment of raw materials: uniformly mixing a carbon source, a nitrogen source, dipotassium hydrogen phosphate and magnesium sulfate with water; then sterilizing;
s2, culturing the SP1 strain to obtain fermentation liquor;
s3, fermenting feed: inoculating SP1 bacteria fermentation liquor to the mixture sterilized in the step S1 for fermentation under the sterile environment, and turning over water materials intermittently in the fermentation process; and after the fermentation is finished, obtaining the fermented feed rich in the antibacterial substances.
9. The fermented feed according to claim 8, wherein the sterilization temperature in step S1 is 100-150 ℃ and the sterilization time is 15-30 min.
10. The fermented feed according to claim 8, wherein the fermentation conditions of step S3 are: controlling the temperature at 29-31 ℃, fermenting for 30-72h, and turning over the materials frequently: every 12 hours 1 time.
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