Application of feed additives in preparing animal feed

Abstract

The present application provides an application of a feed additive. Among them, the feed additive is Surfactin with a content of ≥60 mg / g. The feed additive has strong stability and resistance to enzymolysis, and can play a better role in the intestines of animals. It can also greatly reduce the feed-to-weight ratio, enhance the anti-allergic, anti-inflammatory and antibacterial capabilities of the animal body, enhance the immunity of the animal body, thereby promoting the growth of the animal body and reducing breeding costs. The aforementioned feed additive can protect antimicrobial peptides and improve the retention rate of antimicrobial peptides in the presence of trypsin.

Description

Technical Field

[0001] The present application relates to the field of microbial technology, and in particular to the application of a feed additive in the preparation of animal feed. Background Art

[0002] In the relevant technologies, antibiotics are widely used and used in high doses. A large amount of them remain in livestock and poultry, which not only threatens animal food safety and affects the ecological environment, but also makes many bacteria resistant. In recent years, studies have also found that antibiotics have an inhibitory effect on beneficial bacteria, causing flora disorders in animals, reducing the absorption and utilization rate of various nutrients by the animal body, and thus causing diarrhea and even death. At present, feed production companies have stopped producing commercial feed containing growth-promoting drug feed additives (except traditional Chinese medicine).

[0003] Therefore, there is an urgent need for a new type of feed additive that can achieve green, healthy and ecological farming. Summary of the invention

[0004] In view of this, the purpose of the present application is to provide a Bacillus subtilis, a feed additive and a preparation method and application thereof.

[0005] Based on the above purpose, the embodiments of the present application provide the use of a feed additive in the preparation of animal feed, wherein the feed additive is Surfactin with a content of ≥60 mg / g.

[0006] In some of these embodiments, the animal feed has an antimicrobial peptide that is not resistant to enzymatic degradation.

[0007] In some embodiments, the antimicrobial peptide that is not resistant to enzymatic hydrolysis is yeast peptide or plectasin.

[0008] In some embodiments, the animal feed is freshwater fish feed.

[0009] In some embodiments, the freshwater fish feed is carp feed.

[0010] In some of the embodiments, the feed additive is added in an amount of 200 to 300 grams per ton of carp feed.

[0011] As can be seen from the above, the feed additive with a Surfactin content of ≥60 mg / g provided by the present application has strong stability, resistance to enzymolysis, and can play a better role in the animal intestine. And it can greatly reduce the feed-to-weight ratio, enhance the anti-allergic, anti-inflammatory and antibacterial capabilities of the animal body, enhance the immunity of the animal body, thereby promoting the growth of the animal body and reducing breeding costs. The antimicrobial peptides can be protected by the aforementioned feed additives, and the retention rate of the antimicrobial peptides can be improved in the presence of trypsin. DETAILED DESCRIPTION

[0012] In order to make the objectives, technical solutions and advantages of the present application more clearly understood, the present application is further described in detail below in conjunction with specific embodiments.

[0013] It should be noted that, unless otherwise defined, the technical terms or scientific terms used in the embodiments of the present application should be understood by people with ordinary skills in the field to which the present application belongs. The words "including" or "comprising" and the like used in the embodiments of the present application mean that the elements or objects appearing before the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. The words "connect" or "connected" and the like are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect.

[0014] In view of the current residues of antibiotics in livestock and poultry and the harm they cause, the "comprehensive ban on antibiotics in feed and reduction and limitation of antibiotics in breeding" is gradually being implemented. Feed production companies should stop producing commercial feed containing growth-promoting drug feed additives (except traditional Chinese medicine). After antibiotics are withdrawn from feed, protecting the health of animals and promoting their growth are urgent issues. In order to ensure the healthy development of the feed industry, it is necessary to take safety precautions and develop suitable feed raw materials to protect animal health, especially feed additives that can replace antibiotics to achieve green, healthy and ecological breeding.

[0015] Surfactin is mainly produced by Bacillus subtilis through non-ribosomal metabolism. Its molecular structure includes a β-hydroxy fatty acid chain with a carbon chain length of 12-16 at the hydrophobic end and a cyclic anionic heptapeptide at the hydrophilic end.

[0016] The present application embodiment provides a use of a feed additive in preparing animal feed. The feed additive is Surfactin with a content of ≥60 mg / g.

[0017] In some embodiments, the animal feed contains antimicrobial peptides that are not resistant to enzymatic hydrolysis, such as yeast peptides or plectasin, and the aforementioned feed additives can protect the antimicrobial peptides and improve the retention rate of the antimicrobial peptides in the presence of trypsin, thereby extending the action time of the antimicrobial peptides in the animal intestine and fully exerting the probiotic effect.

[0018] In some embodiments, the animal feed is freshwater fish feed. The feed additive rich in Surfactin is used in freshwater fish feed to improve the growth performance of freshwater fish, reduce mortality, enhance body immunity, and reduce freshwater fish breeding costs.

[0019] In some embodiments, the freshwater fish feed is carp feed. In some embodiments, the feed additive is added in an amount of 200 to 300 grams per ton of carp feed. This can better improve the growth performance of freshwater fish, reduce mortality, enhance body immunity, and reduce freshwater fish farming costs.

[0020] The technical solution of the present invention is further described below in conjunction with specific implementation methods.

[0021] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0022] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0023] Example 1 Preparation of feed additives

[0024] (1) Preparation of culture medium

[0025] Fermentation medium: 1050 g brown sugar, 15 g yeast extract, 75 g urea, 4.995 g KH2PO4, 15 g Na2HPO4·12H2O, 2.25 g MgSO4·7H2O, 7 mg / L CaCl2, 6 mg / L MnSO4·H2O, 6 mg / L FeSO4·7H2O, 5% soybean oil, 15 L liquid volume in a 30 L fermentation tank, adjust the pH to 7.0, sterilize at 115 °C with steam for 20 min, and cool to 37 °C for use.

[0026] Sugar-supplemented culture medium: 525 g brown sugar and 37.5 g urea, first dissolved in 1 L deionized water, fixed to 1.5 L, sterilized at 115 ° C for 20 min for use.

[0027] (2) Inoculation and fermentation

[0028] Activate Bacillus subtilis, pick a larger single colony of Bacillus subtilis spores from the plate and inoculate it into a seed medium made of LB, and culture it at 37°C and 200 rpm for 16-18 hours to obtain a seed solution. Inoculate the seed solution into a 30L fermenter containing the fermentation medium in step (1), with the inoculation amount being 5% of the mass of the medium, stir evenly, introduce sterile air, rotate at 300 rpm, control the fermentation temperature at 37°C, and ferment for 48 hours.

[0029] (3) Purification and concentration

[0030] After the fermentation is completed, the fermentation liquid is concentrated and purified to obtain pure Surfactin.

[0031] (4) Testing the Surfactin content and spore count of the product obtained in step (3)

[0032] Spore count determination method: weigh 1g of the above sample, add sterile water to make up to 100mL, treat in 80℃ water bath for 10min, dilute the bacterial solution 10 times and spread on LB plate for culture, then count by plate colony counting method, the counting result is the spore count.

[0033] Surfactin determination method: HPLC quantitative determination method was used.

[0034] HPLC quantitative determination:

[0035] (1) Chromatographic conditions: chromatographic column Shimadzu C18 ODS-3 (4.6 mm×250 mm, 5 μm), flow rate 1.0 mL / min, detection wavelength 205 nm, column temperature at room temperature; mobile phase: acetonitrile containing 0.1% TFA-0.1% TFA; instrument: Shimadzu L-033 chromatograph; gradient setting: 0-7 min, A (10%-90%), B (90%-10%); 7-24 min, A (90%), B (10%); 24-29 min, A (90%-10%), B (10%-90%); 29-36 min, A (10%), B (90%).

[0036] (2) Determination method: Under the above chromatographic conditions, the concentration of the surfactin standard solution gradient was detected to prepare a standard curve of the relationship between the surfactin standard concentration and the peak area. The relationship function between the surfactin standard peak area and the concentration (mg / mL) was obtained, Y = 115.96X + 5.2147, R 2 =0.99774 (Y is the peak area, X is the concentration). Weigh 0.5 g of the sample prepared from the product obtained in step (3), add 25 mL of methanol, and perform closed ultrasonication for 10 min. After the system is mixed, the clear liquid is passed through the membrane. The clear liquid is measured for the Surfactin content according to the above-mentioned HPLC quantitative determination method, and the Surfactin content per gram of sample (product obtained in step (3)) is calculated, and the average value is taken after 3 measurements.

[0037] The final test results were: Surfactin content was 60 mg / g, spore count was 7.9×10 9 cfu / g.

[0038] Test Example 1 In vitro test of antibacterial activity of feed additives

[0039] Test principle: Surfactin (i.e., feed additive) obtained in Example 1 was used to conduct an in vitro antibacterial test.

[0040] Surfactin was used as indicator bacteria for antibacterial tests, using Clostridium perfringens ATCC13124 (purchased from Guangdong Huankai Microbiological Technology Co., Ltd.), Aeromonas hydrophila, and Aeromonas vernix (isolated from the intestines of sick grass carp by Yinghuier Microecological Laboratory). Clostridium perfringens ATCC13124 was cultured anaerobically at 37°C using fresh RCM medium; Aeromonas hydrophila and Aeromonas vernix were cultured in LB medium and shake flasks at 37°C. Fresh bacterial liquid was spread on the corresponding agar medium plate, 5mm holes were punched with a hole puncher, 100μL Surfactin was added, and cultured in a 37°C incubator for 24h, the results were observed, and the size of the inhibition zone was determined (Table 1).

[0041] Test results: See Table 1

[0042] Table 1 Inhibitory effect of Surfactin on several pathogenic bacteria

[0043]

[0044] Result analysis: According to the data in Table 1, feed additives with a Surfactin content of ≥60 mg / g have a good inhibitory effect after direct contact with Clostridium perfringens and Aeromonas hydrophila.

[0045] Test Example 2 Anti-allergic properties of feed additives

[0046] Test principle: Hyaluronidase is a participant in type I allergic reactions. Hyaluronidase is strongly correlated with inflammation and allergies. Various drugs that release histamine from mast cells can regulate the activity of hyaluronidase. Some anti-allergic drugs have strong inhibitory effects on hyaluronidase activity. Therefore, the inhibition of hyaluronidase activity is used as an indicator of the anti-allergic effect of feed additives.

[0047] Test design: The treatment group and the control group were repeated 5 times, the treatment group used the feed additive prepared in Example 1, and the control group used an equal amount of Surfactin-sodium standard (purchased from McLean). The hyaluronidase content was tested by measuring the absorbance value.

[0048] Test method: as shown in Table 2. The treatment group and the control group were tested using the method shown in Table 2. The difference between the treatment group and the control group is that in the treatment group, the sample is the feed additive prepared in Example 1. In the control group, the sample is Surfactin-sodium standard.

[0049] Table 2 Test methods for anti-allergic properties of feed additives

[0050]

[0051]

[0052] Test results: See Table 3.

[0053] Table 3 Results of hyaluronidase in vitro inhibition test

[0054]

[0055] Note: Hyaluronidase inhibition rate (%) = [(CD)-(AB)] / (CD)×100%.

[0056] Result analysis: As shown in Table 3, the data in Table 3 show that the inhibition rate of hyaluronidase by the feed additive rich in Surfactin in the test group is ≥70%, showing a strong anti-allergic effect. Substances that inhibit the activity of hyaluronidase also have anti-allergic and anti-inflammatory effects. The data results show that the feed additive rich in Surfactin prepared in Example 1 has great application potential in the treatment of animal inflammation and allergies.

[0057] Test Example 3 Anti-enzymatic stability of feed additives

[0058] Test method: Take an appropriate amount of Surfactin prepared in Example 1, adjust pH to 8.0, and detect the Surfactin content of 20 g / L in liquid phase. Add 0.1% trypsin, shake at 37°C, take samples at 4, 6, and 8 hours, and detect the change of Surfactin content.

[0059] Test results: as shown in Table 4.

[0060] Table 4 Liquid phase detection of Surfactin content changes

[0061]

[0062] Result analysis: From the data in Table 4, it can be seen that the Surfactin obtained from the fermentation broth of Bacillus subtilis in the present invention can resist the hydrolysis of trypsin, which is beneficial for it to play a better role in the animal intestine and improve the feed utilization rate.

[0063] Test Example 4: Improvement of the anti-enzymatic performance of antimicrobial peptides by feed additives

[0064] Test method: Surfactin (i.e., feed additive) obtained in Example 1 was compounded with yeast peptide (Yinghuier yeast peptide product Huitaikang), and the mixture was treated with trypsin. Samples with different treatment times were taken to detect the yeast peptide content in the compound system, and the yeast peptide alone was used as a control group to detect the yeast peptide content and retention rate.

[0065] Test results: as shown in Table 5.

[0066] Table 5 Changes in the content of antimicrobial peptides (same as above) detected by liquid phase

[0067]

[0068] Results analysis: As shown in Table 5, under trypsin treatment, compared with the control group, the feed additive rich in Surfactin can significantly increase the content and retention rate of yeast peptides under enzyme treatment, and play a protective role. Therefore, for antimicrobial peptides that are not resistant to enzymatic hydrolysis, their combined use with Surfactin can increase the effect, thereby better playing a role in the animal intestine, and is also conducive to the development of new compatible products.

[0069] Test Example 5: Improvement of the anti-enzymatic performance of mycelium by feed additives

[0070] Test principle: Staphylococcus aureus is an indicator of plectomycin, not surfactin. The content of plectomycin can be characterized by detecting the diameter of the inhibition zone of Staphylococcus aureus.

[0071] Test method: The Surfactin feed additive prepared in Example 1 was compounded with plectasin, and the mixture was treated with trypsin as the test group; the plectasin plus trypsin treatment was used as the control group. Samples with different treatment times were taken, and Staphylococcus aureus was used as the indicator bacteria. The antibacterial activity was determined by the punching method, and the diameter of the antibacterial zone was recorded.

[0072] Test results: as shown in Table 6.

[0073] Table 6 Antibacterial diameter of different samples against Staphylococcus aureus (mm)

[0074]

[0075] Results analysis: From the data in Table 6, it can be seen that under trypsin treatment, compared with the control group, Surfactin can significantly improve the antibacterial effect of plectasin on Staphylococcus aureus under enzyme treatment. Therefore, for plectasin that is not resistant to enzymatic hydrolysis, its combined use with Surfactin can increase the effect and thus play a better role in the animal intestine.

[0076] Test Example 6 Effect of feed additives added to feed on the growth of freshwater fish

[0077] Test materials: Imported fish meal, domestic fish meal, soybean meal, rapeseed meal, cottonseed meal, chicken meal as protein source, flour as carbon source, soybean oil as fat source, and appropriate minerals and cellulose are added as carp compound feed. For specific ingredients, please refer to Table 7. Carps with strong physique, normal appearance, and average tail weight of 57±0.25g were selected as test fish.

[0078] Table 7 Carp test feed formula and test design

[0079]

[0080]

[0081] Note: The above feed formula table shows that the control group and the experimental group were prepared with a total feed volume of 90 and 70 kg respectively, unit: g.

[0082] Test method: three treatment groups were set up, in which 0.20g, 0.25g and 0.30g of the Surfactin-rich feed additive prepared in Example 1 were added per kilogram of compound feed respectively. A blank control group was used without the feed sample. A circulating aquaculture system was used to divide the animals into four groups, in which three replicates were set up for each treatment group and control group, 40 fish in each replicate, and a total of 12 breeding barrels. The temperature was controlled at 26-28°C. The breeding barrels of each group were fed four times a day, at 8:00, 11:00, 14:00 and 17:00, respectively, and the fish were fed to their full stomach each time for 6 weeks. A pre-test group for attacking poison without the feed sample was set up, and the pre-test group had 90 fish and a total of 1 temporary breeding barrel.

[0083] Two weeks before the end of the breeding, two groups of carp with uniform appearance and weight were selected from the temporary holding barrels, with 40 fish in each group, for a pre-test of the challenge to determine the challenge strain and its median lethal dose. The carp were infected with different concentrations of Aeromonas hydrophila and Aeromonas vernix by intraperitoneal injection. After that, no more feeding was done, and the cumulative mortality rate after the challenge was calculated for 7 days. The results are shown in Table 8. According to the mortality rate, the challenge dose was determined to be 0.5 mL of bacterial solution (4.7×10 8 cfu / mL).

[0084] After the feeding period of carp was over, feeding was stopped 24 h before the end of the breeding experiment. After weighing the carp in each group, carp with uniform appearance and weight were selected from each treatment group and control group, and 3 replicates were set up for the challenge test with Aeromonas hydrophila.

[0085] Test results: as shown in Table 8 and Table 9.

[0086] Table 8: Results of the pre-test of the virus attack

[0087]

[0088]

[0089] Table 9 Statistics of carp breeding experiment data

[0090]

[0091] Result analysis: As shown in Table 9, compared with the control group, the average weight, weight gain rate and specific growth rate of each Surfactin-added group were significantly improved; moreover, the mortality rates of Srf group 1, Srf group 2 and Srf group 3 were reduced by 33.3%, 37.0% and 29.6% respectively compared with the control group. It can be seen that the Surfactin feed additive prepared by the present invention is added to the basic diet, which can significantly improve the growth performance of carp, improve the body's immunity, reduce the mortality rate and reduce the cost of carp breeding.

[0092] The Surfactin feed additive provided in the examples of the present application is highly stable and resistant to enzymolysis, and can play a better role in the animal intestine; it can also protect the mycelial antimicrobial peptide and yeast peptide, and improve the retention rate of these two antimicrobial peptides in the presence of trypsin. It is of great significance for the antimicrobial peptides to extend the action time in the animal intestine and give full play to their probiotic effects. The preparation of the feed additive has a short production cycle by fermenting the metabolite Surfactin, and is not affected by external environments such as seasons and climate. The production cost is greatly reduced and can be mass-produced industrially. The Surfactin feed additive in the examples of the present application is used in carp feed, which can improve the growth performance of carp, reduce mortality, enhance the body's immunity, and reduce the cost of carp farming.

[0093] Those skilled in the art should understand that the discussion of any of the above embodiments is merely illustrative and is not intended to imply that the scope of the present disclosure (including the claims) is limited to these examples. Based on the concept of the present disclosure, the technical features in the above embodiments or different embodiments may be combined, the steps may be implemented in any order, and there are many other variations of the different aspects of the embodiments of the present disclosure as described above, which are not provided in detail for the sake of simplicity.

[0094] While the disclosure has been described in conjunction with specific embodiments thereof, many alternatives, modifications and variations of these embodiments will be apparent to those skilled in the art in light of the foregoing description.

[0095] The embodiments of the present disclosure are intended to cover all such substitutions, modifications and variations that fall within the broad scope of the appended claims. Therefore, any omissions, modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the embodiments of the present disclosure should be included in the scope of protection of the present disclosure.

Claims

1. Use of a feed additive in preparing animal feed, characterized in that: The feed additive is Surfactin with a content of ≥60 mg / g; the animal feed contains antimicrobial peptides that are not resistant to enzymolysis; the antimicrobial peptides that are not resistant to enzymolysis are yeast peptides or plectasin; and the application includes improving the retention rate of the antimicrobial peptides that are not resistant to enzymolysis in the presence of trypsin.

2. The use according to claim 1, characterized in that: The animal feed is freshwater fish feed.

3. The use according to claim 2, characterized in that: The freshwater fish feed is carp feed.

4. The use according to claim 3, characterized in that: The added amount of the feed additive is 200-300 grams per ton of carp feed.

Images