CN112790281A

CN112790281A - Preparation method of feed additive rich in surfactin and functional microorganisms

Info

Publication number: CN112790281A
Application number: CN202110013347.XA
Authority: CN
Inventors: 胡美忠; 陈敏; 郁建生; 张华琦; 邬小兰
Original assignee: Tongren Polytechnic College
Current assignee: Tongren Polytechnic College
Priority date: 2021-01-06
Filing date: 2021-01-06
Publication date: 2021-05-14

Abstract

The invention relates to the technical field of bioengineering, in particular to a preparation method of a feed additive rich in surfactin functional microorganisms. The soybean meal is used as a main formula of a culture medium, and Bacillus subtilis S21 is used asThe leaven is fermented in a solid state fermentation mode, so that the purpose of producing the surfactin-rich functional microbial feed additive by utilizing the solid state fermentation of the soybean meal is realized, the economic benefit of the soybean meal is greatly improved, the surfactin content of a fermentation product is more than or equal to 0.6mg/g, and the number of spores is more than 1 multiplied by 10¹¹cfu/g。

Description

Preparation method of feed additive rich in surfactin and functional microorganisms

Technical Field

The invention relates to the technical field of bioengineering, in particular to a preparation method of a feed additive rich in surfactin and functional microorganisms.

Background

Animal food is a very important and excellent source of protein supplementation. The animal food is obtained by converting plant foods such as corn, wheat bran, rice bran, silage and the like in the livestock breeding process, so that how to reduce the consumption of the plant foods and accelerate the production of the animal food, namely, the feed-meat ratio is reduced becomes the focus of important attention in the current breeding industry.

At present, in order to improve the utilization rate of feed and promote the growth of livestock and poultry, growth-promoting antibiotics are usually added into the feed; however, the use of growth-promoting antibiotics causes antibiotic residues, environmental pollution and drug resistance enhancement, so that animals are in a sub-health state for a long time, the immunity is reduced, the breeding cost is higher, and even growth-promoting antibiotics are remained in produced animal food, thereby greatly influencing the body health of eaters. Therefore, the search for a novel feed additive capable of replacing growth-promoting antibiotics becomes a major research direction of current aquaculture industry.

Surfactin is a cyclic lipopeptide with surface activity produced by bacillus by a non-ribosome pathway metabolism, and is formed by esterifying a cyclic peptide consisting of 7 amino acid residues and beta-hydroxy fatty acid with the chain length of 13-15 carbon atoms. The bacillus subtilis is separated from bacillus subtilis by Arima and the like for the first time in 1968, has excellent surface activity, has good effects of promoting growth, resisting oxidation, enhancing immunity of animal organisms and the like on livestock and poultry, and is widely applied to the field of livestock and poultry feed addition, for example: the patent No. CN201510617789.X discloses a compound feed additive, which is prepared by compounding bacillus antibacterial peptide and bacillus probiotics to obtain the feed additive, wherein the content of the bacillus antibacterial peptide is 2-20mg/g, and the content of the bacillus probiotics is 1.0-5.0 x 10⁹cfu/g, the bacillus antibacterial peptide is selected from surfactant active (Surfactin) and mustineAt least one of iturin, plipastatin and bacillomycin; the bacillus probiotics is at least one of bacillus subtilis and bacillus amyloliquefaciens. The bacillus antibacterial peptide and the bacillus probiotics are prepared into a mixture, the effect of the feed additive is exerted to the maximum extent, the good synergistic effect in vivo is ensured, the dosage of the antibacterial peptide in the feed is greatly reduced, the production performance, the immunity, the disease resistance and the feed conversion rate of the fed animals are improved, the antibiotic is replaced, the antibiotic residue is eliminated, and the product quality of the fed animals is ensured. The patent document sufficiently demonstrates the effect of the feed additive containing surfactin, but it is not clear how to efficiently and easily prepare the feed additive containing surfactin.

For this reason, the present researchers conducted 2018 fermentation of Bacillus subtilis S21 as seed to prepare microecologics, such as: a process for preparing the microecological preparation of negundo chastetree fruit from Bacillus subtilis whose number is 201810608662.5 includes such steps as mixing negundo chastetree fruit, soybean powder, glucose, urea and water, stirring to obtain culture medium, inoculating, fermenting, spraying aseptic calcium chloride to supplement calcium, drying, pulverizing and bagging to obtain the product with spore number up to 1X 10¹¹～1×10¹²cfu/g, the purposes of increasing the daily gain of the poultry and reducing the death rate are achieved, and the material-to-weight ratio is improved; however: in the prepared negundo chastetree fruit microecological preparation, the content of surfactin is extremely low, and negundo chastetree fruit is required to be used as a raw material, so that the preparation cost is high.

Disclosure of Invention

In order to solve the technical problems in the prior art, the invention provides a preparation method of a feed additive rich in surfactin and functional microorganisms.

The method is realized by the following technical scheme:

one of the purposes of the invention is to provide a preparation method of a feed additive rich in surfactin and functional microorganisms, which is prepared by fermenting soybean meal with Bacillus subtilis S21 and comprises the following steps:

(1) preparing a culture medium: taking 1000 parts of soybean meal, 20-30 parts of sugar, 1 part of monopotassium phosphate, 0.5 part of potassium chloride, 0.5 part of magnesium sulfate and 1200 parts of water by mass, mixing and stirring uniformly, standing and storing for 8-12h, steaming for 1h by using water vapor under the normal temperature and pressure environment, and transferring to a tray for cooling for later use;

(2) inoculating and fermenting: activating Bacillus subtilis S21, preparing a seed solution by using an LB (lysogeny broth) culture medium, inoculating the seed solution into the tray filled with the culture medium in the step (1), wherein the inoculation amount is 8-12% of the mass of the culture medium, uniformly stirring, introducing sterile air, controlling the fermentation temperature to be 25-40 ℃, and fermenting for 60-70 h;

(3) drying and crushing: drying the product obtained by fermentation at 50-70 deg.C until the water content is less than 10%, pulverizing, and packaging.

The sugar is selected from one or more of sucrose, glucose, fructose and brown sugar. The main purpose is to ensure the rationalization of nutrient components in the culture medium and accelerate the fermentation process.

The above water had dissolved therein 5ppm of copper sulfate, 5ppm of manganese sulfate, 5ppm of ferrous sulfate and 5ppm of zinc sulfate. The method is beneficial to improving the fermentation effect after inoculation of the seed liquid, increasing the quantity of surfactin generated in the fermentation process of Bacillus subtilis S21 and improving the effect of the feed additive.

Preferably, the culture medium is transferred to a tray for cooling, and the thickness of the culture medium after being steamed for 1 hour by water vapor is 3-5 cm. In the step (2), the fermentation time is 60 h.

The second purpose of the invention is to provide the feed additive rich in surfactin and functional microorganisms prepared by the method, wherein the content of surfactin in the feed additive is more than or equal to 0.6mg/g, and the number of spores is more than 1 multiplied by 10¹¹cfu/g. The content of surfactin is increased, the content of spore number in the feed additive is increased, the mixing uniformity of surfactin and microorganisms is guaranteed, and the formed feed additive has excellent efficacy, and the specific embodied advantages are described later.

Preferably, in the feed additive, the content of surfactin is 0.6579mg/g, and the number of spores is 8.9 multiplied by 10¹¹cfu/g. Can greatly reduce the material-weight ratio, enhance the immunity and reduce the breeding cost.

The invention also aims to provide the feed additive rich in surfactin and functional microorganisms, which is applied to preparation of feeds for sheep, chickens and pigs.

Preferably, the feed additive is added into the feed in an amount of 0.10-0.50% of the mass of the basic ration.

Preferably, the feed additive is added into the feed in an amount of 0.30 percent of the mass of the basic ration.

Compared with the prior art, the invention has the technical effects that:

firstly, bean pulp is used as a main raw material of a culture medium, a solid state fermentation mode is adopted, firstly, the economic benefit of the bean pulp is greatly improved, the aim of applying the bean pulp to the production of a surfactin-containing functional feed additive is achieved, secondly, the fermentation condition and the nutrient content of the culture medium are improved, the quantity of surfactin produced by fermenting Bacillus subtilis S21 is promoted, the economic value is improved, the surfactin content is not less than 0.6mg/g, and the number of spores is more than 1 multiplied by 10¹¹cfu/g。

Secondly, the feed additive belongs to a surfactin and functional microorganism composite additive, and fully ensures that Bacillus subtilis S21 can produce surfactin in large quantity in the fermentation process by utilizing the adjustment of the preparation process and the control of the formula, so that the production quantity of the surfactin is greatly improved.

The invention utilizes the solid fermentation technology, can be used after fermentation and drying, shortens the process flow, simplifies the process, ensures the reliability of the process and ensures that the equipment investment in industrial large-scale production is less.

The feed additive has the effects of fattening, promoting growth, regulating intestinal tracts and improving immunity, and is beneficial to reducing the feed conversion ratio and the death rate, so that the livestock breeding cost is reduced, and the breeding economic benefit is improved.

Detailed Description

The technical solution of the present invention is further defined below with reference to the specific embodiments, but the scope of the claims is not limited to the description.

The Bacillus subtilis adopted in the invention is Bacillus subtilis S21, and is preserved in China general microbiological culture Collection center at 04.02.2018, wherein the strain preservation numbers are as follows: CGMCC No: 15544. the strain was filed in 2018 on 13.06.8 and is disclosed in the patent application publication No. 201810608662.5. The current situation that the strain produces antibacterial peptide, surfactin and the like is disclosed in the prior literature, and redundant description is omitted.

The LB medium was: 5.0g/L yeast powder, 10.0g/L tryptone, 10.0g/L NaCl10.0 g/L, 1L water, and pH 7.

The invention creates a research foundation based on the previous stage, considers the conditions of different culture medium formulas, fermentation processes and the like, can influence the growth performance of the strains, causes the difference of effective components generated by the strains, synthesizes the existing research foundation, researches the production of the compound additive rich in surfactin, and realizes the unification of surfactin high content and fermentation process simplification.

In view of this, the present investigators have conducted the following detailed study:

example 1

The preparation method of the feed additive is prepared by fermenting soybean meal with Bacillus subtilis S21 and comprises the following steps:

(1) preparing a culture medium: taking 10kg of soybean meal, 200g of brown sugar, 10g of monopotassium phosphate, 5g of potassium chloride, 5g of magnesium sulfate and 10L of pure water by mass, mixing and stirring uniformly, standing and storing for 8-12h, steaming for 1h by using water vapor under the normal temperature and pressure environment, transferring to a tray for cooling, and stacking in the tray to a thickness of 3-5cm for later use;

(2) inoculating and fermenting: activating Bacillus subtilis S21, preparing a seed solution by using an LB (lysogeny broth) culture medium, inoculating the seed solution into the tray filled with the culture medium in the step (1), wherein the inoculation amount is 8 percent of the mass of the culture medium, uniformly stirring, introducing sterile air, controlling the fermentation temperature to be 25-40 ℃, and fermenting for 70 hours;

Example 2

(1) preparing a culture medium: taking 10kg of bean pulp, 300g of brown sugar and fructose mixture sugar, 10g of monopotassium phosphate, 5g of potassium chloride, 5g of magnesium sulfate and 10L of pure water according to the equal proportion by mass, mixing and stirring uniformly, standing and storing for 8-12h, steaming for 1h by using water vapor under the normal temperature and pressure environment, transferring to a tray for cooling, and stacking in the tray to a thickness of 3-5cm for later use;

(2) inoculating and fermenting: activating Bacillus subtilis S21, preparing a seed solution by using an LB (lysogeny broth) culture medium, inoculating the seed solution into the tray filled with the culture medium in the step (1), uniformly stirring the seed solution, introducing sterile air, controlling the fermentation temperature to be 25-40 ℃, and fermenting for 60 hours, wherein the inoculation amount is 12% of the mass of the culture medium;

Example 3

(1) preparing a culture medium: taking 10kg of soybean meal, 250g of glucose, 10g of monopotassium phosphate, 5g of potassium chloride, 5g of magnesium sulfate and 10L of water, dissolving 50mg of copper sulfate, 50mg of manganese sulfate, 50mg of ferrous sulfate and 50mg of zinc sulfate in the water, mixing and stirring uniformly, standing and storing for 8-12h, steaming for 1h by using water vapor under normal temperature and pressure environment, transferring to a tray for cooling, and stacking on the tray to be 3-5cm in thickness for later use;

(2) inoculating and fermenting: activating Bacillus subtilis S21, preparing a seed solution by using an LB (lysogeny broth) culture medium, inoculating the seed solution into the tray filled with the culture medium in the step (1), uniformly stirring, introducing sterile air, controlling the fermentation temperature to be 25-40 ℃, and fermenting for 60 hours, wherein the inoculation amount is 10% of the mass of the culture medium;

1. Determination test

The feed additives prepared in examples 1 to 3 were used as samples to conduct surfactin and spore number determination tests.

The spore number measuring method comprises the following steps: weighing 1.00g of fermented surfactin functional microbial feed additive, adding sterile water to a constant volume of 100mL, carrying out water bath at 80 ℃ for 10min, and counting by adopting a plate colony counting method to obtain a counting result, namely the number of spores.

Surfactin assay methods: determined by HPLC quantitative assay.

Quantitative determination by HPLC:

chromatographic conditions are as follows: chromatographic column 5TC-C18, wavelength 210nm, flow rate 1mL/min, time 0-20min, mobile phase: 90% acetonitrile (both organic and aqueous phases containing 0.1% trifluoroacetic acid); the instrument comprises the following steps: agilent model 1100 chromatograph.

The determination method comprises the following steps: and detecting the concentration of the precisely prepared surfactin (sigma) standard substance gradient solution under the chromatographic condition, and preparing a standard curve of the relation between the concentration of the surfactin standard substance and the peak area. Obtaining a relation function of the surfactin standard peak area and the concentration (mg/mL), wherein Y is 6920.7x +404.36, R²0.9976(Y is peak area and x is concentration). Adding 10mL of methanol into each 5g of samples prepared in examples 1-3, sealing and performing ultrasonic treatment for 10min, centrifuging at 8000r/min for 10min, taking methanol supernatant, measuring surfactin content of the supernatant according to the HPLC quantitative determination method, calculating surfactin content in each kg of sample, and averaging by 3 times of measurement.

The results of the above measurement tests are reported in table 1 below.

TABLE 1

	Example 1	Example 2	Example 3
				surfactin content (mg/kg)	613.52	647.81	657.90
Number of spores (cfu/g)	5.2×10¹¹	4.3×10¹¹	8.9×10¹¹

2. Cultivation application test

In order to verify the effect of the feed additive produced by the invention on the growth performance of animals by immunity influence, the researchers directly apply the samples prepared in the above examples 1-3 to corresponding feed preparation in two years of 2019 and 2020, and feed chicks, pigs and sheep, and determine the growth performance of the chicks, pigs and sheep, and the specific experimental research is as follows:

2.1 basic diet

TABLE 2

Item	Content (%)	Item	Content (%)
				Corn flour	60	Bone meal	0.5
Soybean meal powder	29	Fish meal	0.5
				Bran	10

2.2 feeding test

Preparing a feed: the feed for feeding was prepared in a ratio of 3kg of the feed additive prepared in examples 1 to 3 per t of basal diet as an experimental group, and the basal diet alone was used as a control group.

2.21 chick feeding test

2.211 test method

Local variety chicks are selected, and three repeated groups are arranged in each group of experiments, wherein each repeated group comprises 20 chicks. Feeding the chicken in the morning and at night for 1 time, freely taking the chicken, and regularly cleaning excrement to ensure the sanitation in the chicken house; the test groups were weighed on an empty stomach at 20, 35 and 50 days of age of the chicks fed, respectively, and the feed amount and the natural death of each group were recorded.

2.212 test results

The growth performance during the feeding of the chicks was recorded as shown in table 3 below:

TABLE 3

	20d average weight (g)	35d average weight (g)	50d average weight (g)	Daily gain of 20-35 days	Weight gain within 35-50 days
						Example 1	213.4±3.1	309.8±12.3	428.4±11.3	6.43±0.61g/d	7.91±0.07g/d
Example 2	211.1±2.8	310.4±10.1	435.7±22.1	6.62±0.49g/d	8.35±0.80g/d
						Example 3	225.7±3.0	352.7±3.8	501.6±8.3	8.47±0.05g/d	9.93±0.30g/d
Control group	197.2±2.4	271.9±8.5	358.1±10.2	4.98±0.41g/d	5.75±0.11g/d

The calculated data for mortality and feed ratio of the chicks during the feeding of the chicks are recorded as shown in table 4 below:

TABLE 4

	Number of trials	Number of deaths	Mortality (%)	Material to weight ratio
					Example 1	60	3	5.0	1.66±0.21
Example 2	60	4	6.7	1.68±0.06
					Example 3	60	1	1.7	1.59±0.13
Control group	60	9	15.0	1.71±0.21

The data in tables 3 and 4 show that the feed additive prepared by the invention can obviously improve the growth rate of chicks, reduce the morbidity and mortality, enhance the immunity and reduce the breeding cost when being added into basic daily ration.

2.22 piglet feeding test

Selecting 48 Jiangku radish pigs, Duroc pigs and long white pigs which are 30 +/-3 days old and have similar body characteristics such as weight, randomly dividing the three-element hybrid weaned pigs into 4 treatment groups, wherein each treatment group has 12 heads, and each treatment group is repeated for three times, and each repeated group has 4 heads. The feed and the pure ration obtained by adding the feed additives prepared in examples 1 to 3 were fed to 4 treatment groups (control group) as feeds, and the feed was fed 1 time each day in the morning and at the evening, and the feed was taken freely, and after feeding and feeding for 28 days, the feed was weighed on an empty stomach, the average weight gain was calculated, the feed intake was recorded, and the feed-to-weight ratio was calculated, and the results are shown in table 5 below:

TABLE 5

As shown in Table 5, the feed additive provided by the invention is added into basic ration, so that the growth performance of piglets can be improved, the effect of promoting the growth of fertilizer is achieved, the feed-weight ratio is reduced, and the breeding cost is reduced.

2.23 lamb feeding test

Selecting 120 white goats aged 15 +/-4 days and having similar body characteristics such as weight and the like, and randomly dividing the goats into 4 treatment groups, wherein each treatment group comprises 30 goats; three replicates per treatment group, 10 per replicate treatment group. The feed and the pure ration obtained by adding the feed additives prepared in examples 1 to 3 were fed to 4 treatment groups (control group) as feeds, and the feed was fed 1 time in the morning and at the evening of each day, and the feed was freely taken, and after feeding and feeding for 30 days, the feed was weighed on an empty stomach, the average weight gain was calculated, the feed intake was recorded, and the feed-to-weight ratio was calculated, and the results are shown in table 6 below:

TABLE 6

	Average weight gain (kg) after 30 days	Daily average weight gain (kg/d)	Material to weight ratio
				Example 1	8.9±0.43	0.297±0.014	1.69±0.18
Example 2	9.4±0.32	0.313±0.011	1.67±0.09
				Example 3	11.2±0.68	0.373±0.023	1.61±0.22
Control group	7.5±0.75	0.250±0.025	1.73±0.17

As shown in Table 6, the feed additive provided by the invention is added into basic ration, which is helpful for improving the growth performance of lambs, achieving the effect of promoting the growth of fat, reducing the material-weight ratio and reducing the breeding cost.

In conclusion, the feed additive provided by the invention is used in the livestock and poultry breeding process, has the effects of fattening, promoting growth, regulating intestinal tracts, improving immunity, reducing the livestock and poultry breeding cost and improving the livestock and poultry breeding economic benefits.

3. Optimization test of additive amount of feed additive

3.1 test methods

The feed additive prepared in example 3 was used as a feed additive for this experiment, and the feed additive was formulated with a basal diet in the following proportions to give a chick feeding feed.

Basic ration and 0.10 percent of feed additive;

② basic ration plus 0.20 percent of feed additive;

③ basic ration and 0.40 percent of feed additive;

fourthly, basic ration and 0.50 percent of feed additive;

the feed prepared by the method is used for a chick feeding test, and the test method is described in the section of '2.21 chick feeding test', and is not described in detail herein.

3.2 test results

The growth performance during the feeding of the chicks was recorded as shown in table 7 below:

TABLE 7

	20d average weight (g)	35d average weight (g)	50d average weight (g)	Daily gain of 20-35 days	Weight gain within 35-50 days
						0.10％	220.3±2.8	345.9±8.6	490.7±6.2	8.37±0.39g/d	9.65±0.16g/d
0.20％	219.9±3.4	346.7±4.6	487.4±7.3	8.45±0.08g/d	9.38±0.18g/d
						0.30％	225.7±3.0	352.7±3.8	501.6±8.3	8.47±0.05g/d	9.93±0.30g/d
0.40％	221.6±1.8	363.4±2.7	511.3±6.9	9.45±0.06g/d	9.86±0.28g/d
						0.50％	220.9±2.3	365.1±3.3	510.8±7.4	9.61±0.07g/d	9.71±0.27g/d

The calculated data for mortality and feed ratio of the chicks during the feeding of the chicks are recorded as shown in table 8 below:

TABLE 8

	Number of trials	Number of deaths	Mortality (%)	Material to weight ratio
					0.10％	60	0	0.00	1.63±0.11
0.20％	60	1	1.70	1.64±0.27
					0.30％	60	1	1.70	1.59±0.13
0.40％	60	0	0.00	1.65±0.08
					0.50％	60	0	0.00	1.62±0.10

The data in tables 7 and 8 show that the proper addition amount is beneficial to improving the growth performance of the chicks, accelerating the growth of the fattening of the chicks, enhancing the immunity, reducing the death rate, reducing the material-weight ratio and reducing the breeding cost. The surfactin content is 0.6579mg/g and the spore number is 8.9 multiplied by 10¹¹The cfu/g feed additive can greatly improve the growth performance of chicks, improve the fattening and growth promoting effects and reduce the feed-weight ratio.

4. Test conditions for sick animals

The feed additive prepared in the example 3 is added and mixed according to the mass ratio of 0.1 percent of the basic ration, the feed is supplied to the diluted sheep flock as feed for feeding, the feed is fed for 1 time respectively in the morning and evening of each day, the feed is taken freely, after the feed is fed for 1-2 days, the diluted sheep flock is not diluted any more, and compared with the feed additive which is not added, the daily gain is obviously increased when the basic ration is directly used for feeding as a control group, the feed weight ratio is obviously reduced, and no diluted sheep flock is dead.

The feed additive prepared in example 3 was added and mixed in a mass ratio of 0.1% of the basic ration, and fed to the chickens as a feed, wherein the feed was fed 1 time each day in the morning and at the evening, and the chickens were fed with the feed freely. After the chicken is fed for 1-2 days, the diarrhea effect of the chicken is obviously improved, compared with the chicken fed by basic ration directly as a control group without adding feed additives, the mortality of the chicken fed by diarrhea is reduced by more than 80%, the disease resistance of the chicken fed by diarrhea is greatly improved, and the immunity is enhanced.

The invention can be realized by making reference to the prior art or common general knowledge and conventional technical means known to those skilled in the art.

The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.

Claims

1. A preparation method of a feed additive rich in surfactin and functional microorganisms is prepared by fermenting soybean meal with Bacillus subtilis S21, and is characterized by comprising the following steps:

(1) preparing a culture medium: taking 1000 parts of soybean meal, 20-30 parts of sugar, 1 part of monopotassium phosphate, 0.5 part of potassium chloride, 0.5 part of magnesium sulfate and 1200 parts of water by mass, mixing and stirring uniformly, standing and storing for 6-8h, steaming for 1h by using water vapor under the normal temperature and pressure environment, and transferring to a tray for cooling for later use;

2. The method for preparing the feed additive rich in surfactin and functional microorganisms as claimed in claim 1, wherein the sugar is one or a mixture of sucrose, glucose, fructose and brown sugar.

3. The method of preparing a feed additive enriched in surfactin and functional microorganisms as claimed in claim 1, wherein the water has dissolved therein 5ppm copper sulfate, 5ppm manganese sulfate, 5ppm ferrous sulfate and 5ppm zinc sulfate.

4. The method for preparing a feed additive rich in surfactin and functional microorganisms as claimed in claim 1, wherein the transfer into the tray for cooling is carried out by stacking the culture medium steamed with water vapor for 1 hour in the tray to a thickness of 3-5 cm.

5. The method for preparing a feed additive rich in surfactin and functional microorganisms as claimed in claim 1, wherein the fermentation time in the step (2) is 60 hours.

6. A feed additive rich in surfactin and functional microorganisms prepared by the method as claimed in any one of claims 1 to 5, wherein the feed additive has a surfactin content of 0.6mg/g or more and a spore count of 1 x 10 or more¹¹cfu/g。

7. The feed additive rich in surfactin and functional microorganisms as claimed in claim 6, wherein the feed additive has a surfactin content of 0.6mg/g or more and a spore count of 8.9 x 10¹¹cfu/g。

8. The feed additive rich in surfactin and functional microorganisms as claimed in any one of claims 6 to 7 is applied to the preparation of feeds for sheep, chickens and pigs.

9. The use according to claim 8, wherein the feed additive is added to the feed in an amount of 0.10-0.50% by mass of the basal ration.

10. The use according to claim 8, wherein the feed additive is added to the feed in an amount of 0.30% by mass of the basal ration.