CN114651913A

CN114651913A - Feed additive containing synbiotic for aquatic animals

Info

Publication number: CN114651913A
Application number: CN202011528857.2A
Authority: CN
Inventors: 黄美莹; 朱惠真; 曾亮玮; 叶信明; 曾福生
Original assignee: Agricultural Technology Research Institute
Current assignee: Agricultural Technology Research Institute
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-06-24

Abstract

The invention relates to a feed additive containing synbiotic for aquatic animals. The invention provides a feed additive for improving growth and health of aquatic animals, a feed containing the feed additive and a preparation method of the feed additive. Wherein the feed additive is characterized in that a feed additive rich in synbiotic and good in palatability is obtained by using a mixture of a staphylococcus aureus strain or a bacillus strain capable of producing glucan and oligosaccharide and a raw material containing disaccharide source.

Description

Feed additive containing synbiotic for aquatic animals

Technical Field

The invention relates to a feed additive which can improve the growth and health of aquatic animals and is rich in synbiotic and good in palatability, a feed containing the feed additive and a preparation method of the feed additive.

Background

More than half of the yield of aquatic animals as food comes from artificial farming, and an important factor affecting the yield of aquaculture is disease. When faced with the problem of bacterial infection of aquatic animals, farmers often use antibiotics to avoid infection, and in asian countries, shrimp farming densities are too high and abuse of antibiotics causes shrimp production to decrease. In addition, the abuse of antibiotics can also cause the appearance of drug-resistant strains, and the strains with drug resistance can also harm human beings. Studies have shown that genes of specific drug resistance can enter the bacteria that the human body may come into contact with in a specific manner, which means that drug resistant strains from farms are likely to enter the human body through the bacteria that may come into contact with, causing health problems for humans, which is currently a considerable issue.

The invasion of the infection source by the aquatic animals such as fish can start a nonspecific and specific immune mechanism, and especially, nonspecific immunity is important, so that the autoimmunity of the aquatic animals such as fish can be improved, and the health condition of the aquatic animals can be improved. Many research reports show that the appropriate use of probiotics can increase nonspecific and specific immune response and enhance disease resistance of fish, thereby improving the breeding effect of aquatic animals and the prevention and resistance of diseases (He et al., 2003; Gupta et al., 2008; Geng et al., 2011).

Probiotics (probiotics) are generally defined as microorganisms used to promote host health, while aquatic probiotics include microorganisms that improve water quality (Nayak, 2010). The range of probiotics used in aquatic products is wider than that of terrestrial animals, and the main species are Bacillus sp, lactic acid bacteria (lactic acid bacteria) and yeast (yeast). The use of different probiotics in aquaculture provides different functions including (1) improving the aquaculture environment, (2) promoting the growth of the aquaculture organisms, (3) reducing the occurrence of diseases in the aquaculture organisms, (4) increasing the disease resistance, (5) reducing the mortality rate, and (6) reducing the abuse of antibiotics.

Probiotics (prebiotics) are defined as non-digestible food materials that selectively stimulate the growth of probiotics in vivo and thus promote host health; prebiotics are primarily non-digestible carbohydrates, including oligosaccharides and polysaccharides, among others. Research reports indicate that the growth and the immunity of fishes and shrimps can be effectively enhanced by taking the probiotics as the feed additive, such as: fructo-oligosaccharides (fructo-oligosaccharides), mannooligosaccharides (mannan-oligosaccharides), gluco-oligosaccharides (gluco-oligosaccharides), polyglucose (beta-glucan), levan (levan), and the like, which can enhance the resistance of aquatic animals to growth and bacterial diseases.

Probiotics for aquatic animal feed additives are widely available in the market today, and the production method thereof is different according to the raw materials, except that soybean oligosaccharide is extracted from soybean, lactulose (lactulose) is chemically synthesized, and other oligosaccharides are mostly obtained by using enzymes. In the aspect of glycan, polydextrose is mainly extracted and purified from cell walls of microorganisms such as fungi and yeasts or part of algae; however, the preparation method needs complicated steps such as extraction and purification, and is difficult to obtain, so the price is high, and the preparation method is difficult to popularize because the preparation method has good efficacy as an immune activator of cultured organisms and has a high price.

In addition, on-site farmers are used to expand and culture probiotics before using the probiotics in feed, so as to save the cost for purchasing the probiotics.

Disclosure of Invention

In view of the problems of the prior art, the present invention provides a low cost feed supplement containing synbiotic that has excellent flavor to improve palatability to aquatic animals, thereby improving growth and health of aquatic animals and preventing infection by pathogens.

Accordingly, the present invention is directed to a feed supplement that can improve the growth, immunocompetence, health, reduce drug use, shorten the cultivation time, and reduce the cost of aquatic animals by feeding the aquatic animals.

In one aspect, the present invention provides a feed supplement for preventing infection of aquatic animals by pathogens, which is rich in synbiotic and has good palatability, characterized by comprising a strain of Leuconostoc or Bacillus strain producing glycans and oligosaccharides, and a mixture of disaccharide-containing source materials, fermented before use; wherein the disaccharide source is sucrose, maltose, lactose, or a combination thereof.

According to the present invention, the feedstock may further comprise a nitrogen-containing source, which may be milk powder, soybean meal, or a combination thereof.

According to an embodiment of the present invention, the Leuconostoc mesenteroides strain B4(Leuconostoc mesenteroides B4), hereinafter referred to as strain B4.

According to another embodiment of the present invention, the Bacillus strain is a Bacillus licheniformis strain FRI MY-55(Bacillus licheniformis FRI MY-55), hereinafter referred to as FRI MY-55 strain.

In another aspect, the invention provides an aquatic animal feed product comprising the feed supplement of the invention.

In one embodiment, the aquatic animal is selected from the group consisting of fish, shrimp, shellfish, crab, and mollusk. The aquatic animal is preferably fish or shrimp.

According to an embodiment of the invention, the feed supplement of the invention has the effect of enhancing the efficacy of aquatic animals in the prevention of pathogen infections. Such pathogens include, but are not limited to, viruses, bacteria, fungi, and parasites.

In a further aspect, the invention provides the use of a feed supplement of the invention or a feed containing a feed supplement of the invention for enhancing the health and growth of aquatic animals.

In yet another aspect, the present invention provides a method for promoting the health and growth of an aquatic animal comprising administering to the aquatic animal a feed supplement of the present invention or a feed product comprising a feed supplement of the present invention.

The invention further provides a feed additive which is used for aquatic animals, is rich in synbiotic and has good palatability, and is characterized by comprising a mixture of the enterococcus faecalis strain B4 and a raw material containing a nitrogen source and a disaccharide source, and the mixture is fermented before use; wherein the disaccharide source is sucrose, or a combination of sucrose and maltose; wherein the nitrogen-containing source is milk powder, soybean meal, or a combination thereof.

The invention also provides a feed additive which is used for aquatic animals, is rich in synbiotic and has good palatability, and is characterized in that the feed additive comprises a mixture of a bacillus licheniformis strain FRI MY-55 and raw materials containing disaccharide source, and the mixture is fermented before use; wherein the disaccharide source is a combination of sucrose and lactose.

Drawings

FIG. 1 shows that the survival rates of the groupers (spotted halibut) after being attacked by Vibrio harveyi after being fed with the control group and the feeds containing the FRI MY-55 strain, lactulose, the FRI MY-55 strain and levan for 10 weeks are respectively 38.6%, 90.5% and 67.3%. Columns with different superscript letters are distinctly different (P < 0.05).

Detailed Description

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used herein, the singular forms "a", "an" and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to "a sample" includes a plurality of such samples and equivalents thereof known to those skilled in the art.

As used herein, "probiotic" is a microorganism used to promote host health, while aquatic probiotics also include microorganisms that can improve water quality. The probiotic bacteria of aquatic products have functions including, but not limited to, the following: increase nutrition, improve feed digestibility, improve biological immunity, compete with pathogenic bacteria to reject bacteria, improve water quality, resist viruses and the like. Therefore, the probiotics used in aquaculture can promote the growth of aquaculture organisms, reduce the abuse of antibiotics, reduce the occurrence of diseases of aquaculture organisms, increase the disease resistance, reduce the death rate, improve the aquaculture environment and the like.

As used herein, "prebiotics" refers to food materials defined as non-digestible that selectively stimulate the growth of probiotics in vivo and thereby promote host health; prebiotics are mainly non-digestible carbohydrates including oligosaccharides and polysaccharides. Can effectively improve the growth and the immunity of fishes and shrimps.

As used herein, "synbiotic" refers to a combination of probiotics and probiotics, which can improve the growth and bacterial diseases of aquatic animals, and increase the growth and survival rate of aquatic animals, and improve the feed efficiency and disease resistance of aquatic animals.

The invention provides a feed additive for preventing aquatic animals from being infected by pathogens, which is rich in synbiotic and has good palatability, and is characterized by comprising a Leuconostoc strain or a bacillus strain capable of producing glycan and oligosaccharide and a mixture containing disaccharide source raw materials, wherein the mixture is fermented before use; wherein the disaccharide source is sucrose, maltose, lactose, or a combination thereof. The feed additive rich in synbiotic and good in palatability can be obtained according to the invention, and has good development potential in aquaculture industry.

According to the invention, Leuconostoc spp (Leuconostoc spp) capable of producing glucan and oligosaccharide exists in intestinal tracts of a plurality of aquatic animals, has the function of inhibiting a plurality of pathogenic bacteria, and has the characteristic of being used as a probiotic. According to a preferred embodiment of the present invention, the Leuconostoc mesenteroides strain B4(L. mesenteroides strain B4) is selected from the intestinal tract of Lateolabrax japonicus (Leuconostoc mesenteroides strain B4) and the strains selected from the intestinal tract of Lateolabrax japonicus (Aquifex luteus et al), 25(2) (23-33, 2017), and the strains are called B4 strain, which can produce glucan and oligosaccharide under special saccharide conditions. This document is incorporated herein by reference in its entirety.

According to another embodiment of the present invention, the Bacillus strain is Bacillus licheniformis strain FRI MY-55(Bacillus licheniformis FRI MY-55), selected from seawater wugu fish culture ponds for screening fructan-producing bacteria, aquaculture research, 19(2):77-91, 2011), selected from seawater wugu fish culture ponds for strain FRI MY-55, which produces fructan and oligosaccharide under special carbohydrate conditions. This document is incorporated herein by reference in its entirety.

According to the present invention, the raw material may further comprise a nitrogen-containing source, which may be milk powder, soybean meal or a combination thereof, suitably formulated according to the general knowledge in the art to achieve the same or similar feed additives and effects thereof of the present invention. According to the preferred embodiment of the present invention, the nitrogen-containing source is milk powder, soybean meal, or a combination thereof, so as to reduce the cost.

According to embodiments of the present invention, the disaccharide source can include, but is not limited to, sucrose, maltose, lactose, or a combination thereof. Preferred examples of disaccharide sources for strains of Leuconostoc are sucrose or a combination of sucrose and maltose; for bacillus strains, a preferred example of a disaccharide source is a combination of sucrose and lactose.

Therefore, the invention further provides a feed additive for aquatic animals, which is rich in synbiotic and has good palatability, and is characterized in that the feed additive comprises a mixture of the enterococcus faecalis strain B4 and raw materials containing a nitrogen source and a disaccharide source, and the mixture is fermented before use; wherein the disaccharide source is sucrose, or a combination of sucrose and maltose; wherein the nitrogen-containing source is milk powder, soybean meal, or a combination thereof. After fermentation, glucan is generated to be the prebiotics, and B4 strain is used as the probiotic bacteria to combine into the synbiotic.

In addition, the invention further provides a feed additive which is used for aquatic animals, is rich in synbiotic and has good palatability, and is characterized by comprising a mixture of the bacillus licheniformis strain FRI MY-55 and raw materials containing a nitrogen source and a disaccharide source, and the mixture is fermented before use; wherein the disaccharide source is a combination of sucrose and lactose; wherein the nitrogen-containing source is milk powder, soybean meal, or a combination thereof. Fructan will be produced as a prebiotic and the FRI MY-55 strain as a probiotic binding synbiotic.

The feed supplement of the present invention may further comprise a carrier that is widely used in aquatic animals. For example, but not limited to, solvents, buffers, emulsifiers, suspending agents, disintegrating agents, dispersing agents, binders, excipients, stabilizers, chelating agents, diluents, gelling agents, wetting agents, absorption delaying agents, liposomes, and the like.

The feed supplement of the present invention may be mixed with or dissolved in other feed supplements as necessary, and processed into various forms such as powder, granule, pellet, pastille, gel, or various liquid forms.

In another aspect, the invention provides an aquatic animal feed product comprising a feed supplement according to the invention.

The feed supplement provided by the invention or the feed containing the feed supplement of the invention has the activity of promoting the health and growth of aquatic (or marine) animals including, but not limited to, fishes, shrimps, shellfishes, crabs and mollusks according to the invention. The fish is preferably selected from carp, grass carp, silver carp, herring, bighead carp, crucian carp, rainbow trout, medusa, larkspur, jupiter, gilles bass, Japanese eel, sea eel, yellow-skinned carangid, grouper, louse, silver-skinned fish

Fish of the family Lutjanus, Pisces nigricans, and Lutjidae. The so-called shrimps are preferably selected from the group consisting of white shrimps, spotted shrimps and prawns.

The feed supplement of the invention or a feed comprising the feed supplement of the invention is not restricted to the developmental stage of the aquatic animal fed by it, but the timing of feeding is determined in accordance with the size of the individual to be administered or the effect to be achieved. The feeding time point can be given along with the time of the individual feed supply, such as once a day, twice a day, or three times a day, etc., while the feeding times can be increased for aquatic animals in need, and the invention is not limited thereto.

The feed additive provided by the invention can be directly used, and is also suitable for being mixed with other components to prepare feed for later application, so that the prepared feed can further comprise a carrier for aquatic animals, such as buffer solution, bran, fish meal, protein, amino acid, vitamin or fish oil and other lipids, or comprises other additives, leavening agents, fillers, disintegrating agents, bonding agents or coating materials capable of maintaining the specific shape of the feed, which are helpful for protecting the activity of antioxidants to reach the acidic environment such as the gastrointestinal tract of the aquatic animals, besides the feed additive provided by the invention. May be made by techniques or methods known to those skilled in the art to which the invention pertains.

In yet another aspect, the present invention provides a method for improving the health and growth of an aquatic animal comprising administering to the aquatic animal a feed supplement according to the present invention or a feed comprising a feed supplement according to the present invention.

The feed additive of the present invention has an effect of enhancing the prevention of infection of an aquatic animal with a pathogen, including pathogens of prokaryotes (prokaryotes) or eukaryotes (eukaryotes). In a specific embodiment, the pathogen is a virus, bacterium, fungus or parasite. The virus is selected from the group consisting of midgut gland necrosis baculovirus, prawns baculovirus, hepatopancreatic parvovirus, reovirus-like, taura virus, yellowhead virus, leukoderma virus, infectious pancreatic necrosis virus, infectious hematopoietic necrosis virus, nervous necrosis virus and iridovirus. More preferably, the bacterium is selected from the group consisting of Vibrio, Streptococcus, Edwardsiella, Aeromonas, Pseudomonas fluorescens, Staphylococcus, and Mycobacterium. More preferably, the fungus is selected from the group consisting of saprolegnia, pispora and gill mold. More preferably, the parasite is selected from the group consisting of cryptotrichia, nematodes, Trichostoma, Trichoderma, Trypanosoma cruzi, Trichuris ovalis, dactylogyrus and amoeba.

The invention also provides the use of the feed supplement of the invention or of a feed comprising the feed supplement of the invention for improving the health and growth of aquatic animals.

The invention is further illustrated by the following examples which are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent.

Examples

1. Preparation of feed supplement from B4 strain

(1) Preparation of the feed supplement of the invention with the B4 Strain

The composition containing B4 strain, milk powder and granulated sugar (sucrose) was cultured for 48 hours, the bacterial count of B4 strain reached 109CFU/g, and about 50mg/mL of glucan was produced, and this contained B4 strain and the glucan product produced by it as a feed additive in an amount of 1% of the feed.

(2) Field experiments in the Tilapia aquaculture farm

The field test is carried out on the culture farm of the Tilapia rupoensis in the lake of Gao-Xiong city, the stocking weight of a control pond and a test pond is respectively 0.5 hectare and 0.3 hectare, the fry are stocked in the pond in 2019, 2 months and 26 days, the stocking weight of the control pond and the test pond is respectively 1,949 kilogram and 1,161 kilogram, the stocking number of the control pond and the test pond is respectively 25,900 and 15,480 tails, the average stocking amount is 51,800 and 51,600 tails per hectare, and the average weight of the fry is about 75 grams. The experiment was carried out with feed supplemented with feed supplement containing B4 strain and glucan produced by it (hereinafter referred to as "B4 feed supplement"), starting at 2.4.2019, B4 feed supplement was prepared as described in example 1, at the beginning of the experiment (4.4.2019), the daily feed intake of the control pond and the test pond was 150 kg and 90 kg, respectively, the control pond fish were fed with only dao-pai nile feed, the test pond was fed with 1 kg of B4 feed supplement in 30 kg of dao-pai nile feed, after mixing well, the fish were fed manually, and the remaining 60 kg of feed was fed with the automatic feed-spraying barrel. Gradually adjusting the feeding amount along with the growth and feeding conditions of the fish, taking pond water at 5, 6, 8, 10 and 12 weeks for water quality analysis, performing experiments for 3-4 months (6 months end in 2019), or stopping when the fish only reaches the shape of the market, harvesting the fish, collecting harvest amount data, and calculating the feeding amount of the total Feed to calculate the Feed benefit (FE). The disease condition of the fish was observed and recorded during the test. And collecting door air temperature data of a meteorological department in the Taiwan area as reference information during the test.

The feed efficiency is that the weight of fish is increased (kilogram)/the amount of feed ingested (kilogram)

Water quality analysis

The analytical items are pH, ammonia nitrogen and nitrous acid.

Collecting a water sample about 5 centimeters below the water surface, carrying the water sample back to a laboratory after being frozen, and analyzing items such as a pH value, ammonia-nitrogen, nitrous acid-nitrogen and the like. The analytical method and the instrument used are as follows:

the pH was measured with a pH meter (model METTLER DELTA 350). Water samples of ammonia-nitrogen and nitrous acid-nitrogen analysis items are filtered by Whatman GF/C (Whatman GF/C) glass fiber filter paper, and filter liquor is collected for analysis.

Ammonia-nitrogen was measured by phenol hypochlorite (phenol hypochlorite) method; the principle is that a water sample containing ammonia nitrogen and ammonium ions is added with hypochlorite (hypochlorite) and a phenol solution to react to generate dark blue indophenol (indophenol), and the color of the solution is stronger after the catalysis of sodium nitrosyl ferricyanide solution (sodium nitroprusside). And (4) carrying out colorimetric analysis at the wavelength of 640nm by using a spectral brightness meter to obtain the concentration of ammonia-nitrogen in the water sample.

The nitrite-nitrogen method is characterized in that nitrite and sulfanilamide (sulfanilamide) generate diazo compound under acidic condition, and the diazo compound and naphthalene diethylamine (N, N-diethyl-1-naphthylamine) generate azo action to form red purple azo compound. The absorbance was measured at 543nm with a spectrophotometer to determine the quantity.

The average temperature during the test period (4-6 months in 2019) is 25.5-29.7 ℃, and the maximum temperature is 34.3 ℃.

In terms of water quality, the pH values (pH values) of the control pool and the test pool water at week 6 were 8.64 and 8.41, respectively, and the pH values of the control pool and the test pool water at week 12 were 8.12 and 8.14, respectively. In the case of ammonia-nitrogen and nitrite-nitrogen, the control cell and the test cell water in week 5 were each 0.1ppm in ammonia-nitrogen and nitrite-nitrogen, the control cell and the test cell water in week 6 were each 0.5ppm in ammonia-nitrogen and nitrite-nitrogen, and the test cell water was each 1.0ppm in ammonia-nitrogen and nitrite-nitrogen. The control and test cell waters were 0.5ppm ammonia-nitrogen and nitrous acid-nitrogen at weeks 10 and 12 (tables 1 and 2).

TABLE 1 pond water pH and Ammonia-Nitrogen (ppm) content during the test

TABLE 2 nitrite-nitrogen (ppm) content of pond water during the test

The test cell was similar to the control cell in ammonia-nitrogen and nitrous acid-nitrogen contents at the rest of the time points, except that at week 6, the ammonia-nitrogen and nitrous acid-nitrogen contents were 1.0ppm higher than the control cell.

The fry are released into the pond in 2019, 2 months and 26 days, the releasing amount of the control pond and the test pond is 25,900 and 15,4800 tails respectively, the average releasing amount is 51,800 and 51,600 tails per hectare, and the average weight is about 75 grams. The test is carried out by using the feed added with the B4 feed additive, starting at 4 and 2 days in 2019, the size of the Wugou fish is 5 kg/kg, the body weight is about 120 g, the daily feed feeding amount of the control pond and the test pond is 150 kg and 90 kg respectively, and the feed of each fish is about 6.0 g.

During the test, the feeding condition of the fish in the test pond is better than that of the control pond, the fish grows fast, the health condition of the fish is good, so the feeding amount of the test pond is increased faster than that of the control pond, the daily feeding amount of the feed in the control pond and the feed in the test pond are 210 kg and 150 kg respectively at week 9, and the feed in each fish in the control pond and the feed in each fish in the test pond are about 8.1 g and 9.7 g respectively.

In the control group, at week 14, the fish were found to be infected with streptococci, treated with antibiotics, and harvested at week 16 (30 days at 6 months) for 124 days of co-cultivation, with a total feed intake of 17,670 kg, a harvest of approximately 12,060 kg, a unit throughput of 24,120 kg/ha, an average weight of 467 g for the fish, and a feed efficiency of 0.57 (Table 3).

During the test period, the feeding condition of the fish in the test pond is better than that of the control pond and the fish grows faster, the fish reaches the marketable body type at week 12 and is cultured for 93 days, so the fish is fished in advance, and the fish in the test pond is shown by fishermen and workers to be particularly active. The total feed feeding amount is 11,520 kg, the harvest yield is about 8,820 kg, the unit production amount is 29,400 jin/ha, the average body weight is 570 g, and the feed benefit is 0.66. Compared with the control group, the test group is harvested 1 month earlier, the culture effect is good, and the body of the fish is strong during the test period, and all the fish do not suffer from diseases (Table 3).

TABLE 3 Fish stocking amount, bait feeding amount, growth and morbid state

The experiment shows that in the test pond with the B4 feed additive added into the feed of the tilapia, the fish grows faster than the control group, and reaches the shape of the marketed fish at week 12, so that the fish is caught in advance. The unit production is 29,400 kg/hectare, which is improved by 5,280 kg/hectare compared with a control group (24,120 kg/hectare); the average body weight of the test group is 570 grams, the body weight is increased by 103 grams compared with the control group (467 grams), the feed efficiency is 0.66, and the body weight is improved by 0.09 compared with the control group (0.57); during the test period, the bodies of the fishes in the test group are strong, the fishes are not sick, the streptococci are infected by the fishes in the control group at the final stage of culture, the fishes in the test group are harvested 1 month earlier than the fishes in the control group, and the culture effect is good.

Regarding the cost analysis, the fishes in the test group were harvested 1 month earlier than the control group, and estimated that about 4,500 kg of feed is required for 1 month, about 16 yuan per kg of feed, about 7.2 ten thousand yuan (16 yuan × 4,500 kg-7.2 ten thousand yuan) of feed cost is saved, and about 6,000 yuan of electricity cost per month is saved while the cultivation risk for 1 month is shortened. The cost of B4 feed supplement added to these 2 month feeds was estimated to be about 100 yuan per kg of B4 feed supplement, 1% feed supplement, 1 yuan per kg of feed, 9,000 kg per month of 2 month feed, and about 9,000 yuan per month (1 yuan × 4,500 × 2 months — 9,000 yuan) (table 4).

TABLE 4 comparison of the cultivation costs and growth of fish between the test and control groups

Comparison of test group with control group	Reduction of	Increase of
			Total days of rearing (day)	31
Feed expense (Yuan)	72,000
			Electric charge (Yuan)	6,000
B4 feed supplement cost (Yuan)		9,000
			Specific production capacity (kilogram/hectare)		5,280
Efficiency of feed		0.09
			Average fish weight (gram)		103
The fish is suffering from a disease	Morbid free

(3) Effect of feed supplemented with B4 feed supplement on growth of fish and shrimp

A field test is carried out on a farm in the Jiayi region between 8 and 1 month and 15 days in 2019, the test has 3 pools, and a B4 feed additive is added into the feed of the fish and the shrimp in the 1 st pool, which is called a B4 test pool. 1% of B4 feed additive by weight of the feed is added into the 1 st pond, B4 feed additive is prepared by the method of the embodiment 1, and 1% of Bacillus pumilus for resisting aquatic pathogenic bacteria is directly added into the 2 nd pond fish and shrimp feed. Wherein the carangid and the silver of the black star are all raised in each pond

Bean fish and white shrimp. After the test is finished, 2,267.4 kg of B4 test pool is harvested. However, the control pond resulted in massive mortality due to Trichuris ovata infection in 2019, 10, 28 months, and only 663.4 kg was harvested (Table 5). The results of this experiment show that the B4 feed supplement has the efficacy of enhancing the growth of aquatic animals and preventing or combating parasitic infections.

TABLE 5 comparison of harvest and morbidity of aquatic animals with B4 feed supplement

(4) Influence of milk powder-added feed additive on growth of fish

B4 feed additive prepared from B4 strain, milk powder and granulated sugar composition, and MRS culture medium and granulated sugar culture product are added into feed as feed additive.

The results show that the percentage (%) of the weight gain of the fish with the feed supplement of B4 and the product cultured in MRS medium was 22.06 and 2.81%, respectively. The feed additive of the invention B4 and the feed additive cultured in the culture medium had feed-increasing benefits of 15.79 and 6.74% respectively for fish (Table 6), indicating that the addition of milk powder could produce unexpected efficacy.

TABLE 6 comparison of growth of fish with milk powder

A: MRS culture medium for culturing B4 strain and glucan product produced by same

B: fermenting the composition containing milk powder, granulated sugar and B4 strain to obtain B4 feed additive containing B4 strain and glucan produced by the strain

(5) Feed added with B4 feed additive can improve palatability of fish

The carangid takes 15 minutes for feeding 1 kg of feed containing the B4 feed additive, while the carangid is generally fed with other probiotics for 25 minutes, which shows that the B4 feed additive is beneficial to the palatability of the feed, namely, the fish only prefers to feed the feed added with the B4 feed additive.

2. Preparation of feed additive from FRI MY-55 strain

(1) Production of feed

The experimental feeds total 3 groups, comprising:

(a) a control group,

(b) A culture solution containing FRI MY-55 strain (107CFU/g) and lactulose (0.15%), and

(c) culture broth of FRI MY-55 strain (107CFU/g) and levan (0.15%) was added.

Inoculating the FRI MY-55 strain into a culture solution containing 10% lactose and 10% sucrose, and performing shake culture at 28 deg.C for 48hr to obtain a culture solution containing FRI MY-55 strain and lactulose with bacteria number of above 109CFU/mL, wherein the culture solution containing FRI MY-55 strain and lactulose is used as additive source of probiotic and lactulose in feed.

Inoculating the FRI MY-55 strain into a culture solution containing 20% sucrose, and shake-culturing at 28 deg.C for 48hr to obtain a culture solution containing FRI MY-55 strain and levan, wherein the number of bacteria is more than 109CFU/mL, and the culture solution containing FRI MY-55 strain and levan is used as an additive source of FRI MY-55 strain and levan in the feed.

(2) Influence of feed additives containing FRI MY-55 strain, lactulose and levan on short-chain fatty acid in feces of fish

(I) laboratory animals

300-point rocky spots (Epinephalus coioides) used in the experiment are purchased from a folk farm, stored in 4 circulating FRP (500 liters) barrels in an aquatic product test station for 2 weeks, and the water temperature is maintained at 28 +/-1 ℃.

(II) the FRI MY-55 strain, lactulose and levan are added into the feed to raise the spot with the rocky spot

The experiment is carried out for 3 groups, 180 fishes with the size of about 34g are taken for the experiment, 20 fishes in each jar are repeated for 3 fishes, and the fishes are randomly selected and placed in 9 glass jars with the size of 82 liters. Feeding the fish with feed at 9 am and 5 pm every day, wherein the feeding amount is 2.0% of the weight of the fish, measuring the weight of the fish once every 2 weeks, adjusting the feeding amount, fishing out the residual bait which is not ingested after feeding for 30 minutes every time, counting the quantity of the residual bait, and deducting the residual bait from the feeding amount. During the experiment, the water temperature is controlled at 28 +/-1 ℃, and one third of water is changed every day. The test was carried out for 10 weeks.

(III) influence of feed additives containing FRI MY-55 strain, lactulose and levan on short-chain fatty acid in feces of fish

At 6 and 10 weeks of animal experiment, collecting fish feces of each control group and test group, centrifuging (5,000 Xg, 15min), collecting supernatant, filtering with 0.22 μm membrane, analyzing the composition of each short chain fatty acid (lactic acid, acetic acid, propionic acid, butyric acid and isobutyric acid) by HPLC (Waters,1515isocratic HPLC pump), wherein the detector is refractometer (Waters,2414), the separation column is ICSep ICE-ION-300(Transgenomic Inc., USA), the operation conditions are that the flow rate is 0.4 mL/min, the mobile phase is 0.01N sulfuric acid, the column temperature is 65 ℃, comparing with standard substance (Merck) of short chain fatty acids (lactic acid, acetic acid, propionic acid, butyric acid and isobutyric acid), analyzing, calculating retention time with Breeze software (Waters), the content of each component was converted to mM in accordance with a standard curve for each short-chain fatty acid.

(3) The influence of probiotics, lactulose and levan added into the feed on the nonspecific immune response of the fish

The influence of probiotics, lactulose and fructan on short-chain fatty acid in feces of fish is added into the feed of experimental animals and breeding animals (3), the fish are respectively fed with 3 groups of feed for 10 weeks and 9 days after pathogenic bacteria attack, and the serum of 9 fish in each group is collected for immune index analysis.

(3.1) serum Collection

Collecting blood from tail handle with 25G needle in 1mL plastic syringe, standing at 4 deg.C overnight, centrifuging (3000 Xg, 10min,4 deg.C), sucking upper layer serum, storing in-80 deg.C freezer, and analyzing immune index such as total serum protein, albumin, globulin, protease resistance (antiprotease) and lysozyme activity.

(3.2) collecting blood and separating leukocytes

Blood was collected from Epinephelus, white blood cells were separated, 0.7mL of blood was collected from the tail stem using a 1mL plastic syringe equipped with a 25G needle, mixed with 1mL L-15 medium (containing 50. mu.L of 125mM EDTA), and after layering by adding 4mL of 50% Parcoll (Percoll) chromatography (Sigma), centrifugation (400 Xg, 15min,4 ℃) was performed, 2mL of the layered interface liquid was extracted, 3mL of Hank's Balance Salt Solution (HBSS) (Sigma) was added, centrifugation (600 Xg, 10min,4 ℃) and supernatant was removed, and after repeating the washing step three times, 1mL of L-15 medium was dissolved and white blood cell concentration was calculated using a blood cell counting plate. The separated white blood cells were used for measurement of Respiratory Burst Activity (RBA).

(3.3) analysis of the immune index

(a) Determination of serum Total protein, albumin and globulin

The determination of total protein and albumin was performed using the serum total protein and albumin test kit (RANDOX Laboratories, Antrim, UK), and the globulin content was estimated as the difference between total protein and albumin.

(b) Determination of protease resistance in serum

mu.L of serum was mixed with 35. mu.L of 1mg/mL Trypsin solution (Trypsin bone cultures in 0.01M Tris-HCl (pH 8.2) buffer), 500. mu.L of 2mM BAPNA (Sodium-benzyl-DL-arginine-p-nitroanilide HCl) was added to the mixture in a volume of 1mL with 0.1M Tris-HCl (pH 8.2) buffer, and the mixture was reacted at 28 ℃ for 25 minutes; after the reaction was terminated with 150. mu.L of 30% acetic acid, the absorbance value was measured at a wavelength of 415nm with a disk spectrophotometer (microphotometer, Benchmark plus, Bio-Rad Laboratories, Inc., Calif., USA). The antiprotease activity in serum is expressed as the Percentage of trypsin inhibition (percent trypsin inhibition) and is calculated by the following formula:

percent inhibition trypsin ═ (absorbance of blank trypsin-absorbance of sample)/absorbance of blank trypsin × 100%

(c) Lysozyme activity assay

Lysozyme standard solutions were prepared by dissolving hen egg white lysozyme in 0.05M sodium phosphate buffer (pH 6.2) at concentrations of 0. mu.g/mL, 2. mu.g/mL, 4. mu.g/mL, 6. mu.g/mL, 8. mu.g/mL, 16. mu.g/mL, and 32. mu.g/mL, respectively. A0.2 mg/mL culture of Micrococcus luteus (Micrococcus lysodeiikus) was prepared using 0.05M sodium phosphate buffer (pH 6.2). Taking 10 mu L of lysozyme standard solution, adding 200 mu L of micrococcus luteus, reacting at 28 ℃, recording the light absorption values at the wavelength of 530nm by a disc type spectrophotometer at 1 st minute and 6 th minute respectively, and plotting the lysozyme concentration according to the change of the light absorption values per minute of the standard solutions of lysozyme with different concentrations to obtain a detection line. The steps are repeated by using the fish serum instead of the standard solution of lysozyme, the light absorption values at 1 st and 6 th minutes are recorded, the concentration of lysozyme in the serum is estimated by a detection line according to the change of the light absorption values, the concentration of chicken egg white lysozyme is expressed as lysozyme activity, and 1 unit of lysozyme activity is defined as chicken egg white lysozyme with the concentration of 1 mu g/mL.

(d) Determination of respiratory burst

The method for measuring the breath burst is to measure by a tetrazolium blue (NBT) staining method. In a 96-well flat-bottom microtiter plate, 100. mu.L of 0.2% poly-L-lysine was added per well and covered for 30 minutes, and then poly-L-lysine was taken out to increase the adsorption surface of the blood spheres. 100. mu.L of the macrophage suspension was added to a flat-bottomed microtiter plate of a 96-well treated cell, incubated at room temperature for 2 hours to allow blood cells to adhere to the bottom of the cell, 100. mu.L of zymosan (0.2%) and HBSS were added after removing the supernatant, reaction was induced at room temperature for 30 minutes, the supernatant was removed, 100. mu.L of NBT (0.3%) was added and allowed to act at room temperature for 30 minutes, and 100% ethanol was added to terminate the reaction. After washing with 70% ethanol three times, 120. mu.L of 2M KOH and 140. mu.L of Dimethyl sulfoxide (DMSO) were added to the mixture by air drying to dissolve cytoplasmic trityl ester (cytoplasma formazone), and the absorbance value was measured at a wavelength of 630nm with a disk spectrophotometer. Basal Activity (BA) of the uninduced immunostimulatory group by HBSS treatment and Stimulated Activity (SA) of the induced immunostimulatory group by zymosan treatment, and the difference between them is Respiratory Burst Activity (RBA) (Pick and Mizel,1981) indicating increase or decrease of superoxide anion production by macrophages.

(4) Test of fish pathogenic bacteria resistance by adding FRI MY-55 strain, lactulose and levan into feed

(4.1) preparation of bacterial suspension

Inoculating pathogenic bacteria-Vibrio harveyi to Tryptic Soy Broth (TSB), culturing at 28 deg.C for 24 hr, centrifuging the culture solution (8000 Xg, 10min,4 deg.C) to remove supernatant, washing the precipitated bacteria with physiological saline for 2 times, measuring with spectrophotometer at 600nm, and adjusting the concentration of the bacteria solution to obtain 1X 107CFU/mL suspension.

(4.2) challenge test

After 3 groups of different feeds are fed for 10 weeks, the fish is injected into abdominal muscles of the fish by pathogenic bacteria suspension, the injection amount is 0.5 percent of the weight of the fish, and the attack infection dose is 5.5 multiplied by 104 CFU/g; and the other fish is injected with the same amount of normal saline as a control, and the death number of the fish is observed and recorded for 2 weeks after injection.

(5) Statistical analysis method

The results of each experiment were analyzed statistically by one way analysis of variance (ANOVA) using SAS software package (Version 14.0), and tested by Duncan's test for significant differences among treatment groups, with a significance level p <0.05 for all experiments.

(6) Results

(6.1) Effect of the feed supplement of the invention on the composition of short-chain fatty acids in feces of Fish

The feces of the fish in the 6 th week of the spotted grouper test were collected and analyzed for the content of short chain fatty acids, and the results showed that the total content of four short chain fatty acids (lactic acid, acetic acid, propionic acid, butyric acid, etc.) in the spotted grouper feces was the highest (28.84mM) in the feeding group with the FRI MY-55 strain and the lactulose group, followed by the feeding group with the FRI MY-55 strain and the levan group (22.81mM), while the total content of short chain fatty acids was the lowest (15.11mM) in the control group. At week 10, the fish feces of the FRI MY-55 strain and fructan-containing group were fed with highest total short-chain fatty acids (62.46mM), which were the FRI MY-55 strain and lactulose group (50.33mM) and lowest control group (41.28 mM).

Lactulose and levan produced by the FRI MY-55 strain added into the feed of the research test group can increase the content of short-chain fatty acid in the spotted grouper manure, can increase the action matrix of probiotics, and can proliferate the probiotics to produce more short-chain fatty acid, so that the intestinal tract can cause a more acidic environment, inhibit the reproduction of harmful microorganisms, and is beneficial to the health of the fed organisms.

(6.2) influence of probiotics, lactulose and fructan added into feed on immune index of fish

After 3 different feeds are fed for 10 weeks respectively for stippling the groupers, the total protein amount and globulin amount in the blood serum of the fish in the control group and the fish in the groups added with the FRI MY-55 strain and lactulose, the FRI MY-55 strain and fructan feed are 53.55, 63.32, 62.82 and 45.91, 55.07 and 52.22 mg/mL respectively, statistically, the total protein amount and globulin amount in the blood serum of the groupers in the test group are higher than those in the control group (p is less than 0.05) (Table 7), after pathogenic bacteria challenge, the total protein amount and globulin amount in the blood serum of the fish fed with the control group, the fish fed with the FRI MY-55 strain and the fructosan, the FRI MY-55 strain and the fructosan feed group are respectively 51.79, 50.25, 61.59, 42.13, 40.69 and 53.26mg/mL, and the total protein amount in the blood serum of the fish fed with the FRI MY-55 strain and the fructosan feed group is higher than that of the control group (p is less than 0.05) (Table 7).

TABLE 7 Total protein, albumin and globulin levels in serum before and after challenge with Vibrio harveyi by feeding groupers (Epinephelus punctatus) with control and feed containing FRI MY-55 strain and lactulose, FRI MY-55 strain and levan for 10 weeks, respectively.

In the control group, the protease resistance activities in the sera of the fish fed with the FRI MY-55 strain and lactulose, the FRI MY-55 strain and levan feed group were 42.8, 47.8 and 46.3%, respectively, and statistically, the protease resistance activities in the sera of the fish fed with the control group, the fish fed with the FRI MY-55 strain and lactulose, the fish fed with the FRI MY-55 strain and levan feed group were 62.1, 69.9 and 61.1%, respectively (table 8).

The lysozyme activity in the serum of the fish in the control group, the fish in the added FRI MY-55 strain and lactulose, the fish in the added FRI MY-55 strain and levan feed group is respectively 2.63, 15.03 and 14.87U/mL, on the basis of statistics, the lysozyme activity in the serum of the fish in the test group is obviously higher than that in the control group (p <0.05) (Table 8), while the lysozyme activity in the serum of the fish in the control group, the fish in the added FRI MY-55 strain and lactulose, the fish in the added FRI MY-55 strain and levan feed group is respectively 3.61, 12.50 and 11.33U/mL after the fish is attacked by pathogenic bacteria, on the basis of statistics, the lysozyme activity in the serum of the fish in the test group is also obviously higher than that in the control group (p <0.05) (Table 8).

The breath bursts in the blood of the fish in the control group and the group added with the FRI MY-55 strain and the lactulose, the FRI MY-55 strain and the levan feed are respectively 0.47, 0.50 and 0.52, statistically, the test group and the control group have no obvious difference (p >0.05) (table 8), and after the fish is attacked by the pathogenic bacteria, the breath bursts in the blood of the fish in the control group and the group added with the FRI MY-55 strain, the lactulose, the FRI MY-55 strain and the levan feed are respectively 0.47, 1.07 and 1.03, statistically, the test group is obviously higher than the control group (p <0.05) (table 8).

TABLE 8 comparison of immune index of fish with feed supplement fermented with FRI MY-55 Strain

The research test group feed is added with lactulose and levan produced by the FRI MY-55 strain, so that the immune indexes such as total protein, globulin, lysozyme, respiratory burst and the like in the spotted grouper serum are obviously improved, and multiple immune indexes of fish and shrimp can be effectively improved.

(6.3) the feed is added with probiotics, lactulose and levan to resist the influence of pathogenic bacteria of fish

The grouper is fed with the control group, the feed containing the FRI MY-55 strain and lactulose, the FRI MY-55 strain and levan for 10 weeks respectively, after the grouper is attacked by Vibrio harveyi, the survival rates of the control group, the FRI MY-55 strain, the lactulose, the FRI MY-55 strain and the levan are respectively 38.6%, 90.5% and 67.3% (figure 1), wherein the survival rate of the fish fed with the FRI MY-55 strain and the lactulose group is the highest, and the survival rate of the fish fed with the FRI MY-55 strain and the levan group is the second, and the statistical difference with the control group is obvious (p is less than 0.05).

Lactulose and levan produced by the FRI MY-55 strain are added into the feed of the research test group, so that the survival rate of the epinephelus malabaricus after being attacked by vibrio harveyi is obviously improved, and the survival rate of fishes and shrimps after being attacked by pathogenic bacteria is effectively improved.

In conclusion, the FRI MY-55 strain has the characteristics of inhibiting vibrio harveyi and various human pathogenic bacteria, can ferment fructose and generate short-chain fatty acid, can be combined with probiotics such as lactulose and levan to serve as synbiotic, can increase the immunity of the rockspot, can enhance the capability of resisting pathogenic bacteria, can improve the palatability, and is favorable for maintaining the health of the feeding organisms.

It will be appreciated by those skilled in the art that changes could be made to the specific embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications within the spirit and scope of the present invention as defined by the appended claims.

Claims

1. A feed additive for preventing aquatic animals from being infected by pathogens, which is rich in synbiotic and has good palatability, is characterized by comprising a Leuconostoc strain or Bacillus strain capable of producing glycan and oligosaccharide, and a mixture containing disaccharide source raw materials, which are fermented before use; wherein the disaccharide source is sucrose, maltose, lactose, or a combination thereof.

2. The feed supplement of claim 1, wherein the feedstock further comprises a nitrogen-containing source that is milk powder, soybean meal, or a combination thereof.

3. The feed supplement of claim 1 wherein the Leuconostoc strain is Leuconostoc mesenteroides strain B4(Leuconostoc mesenteroides B4).

4. The feed supplement of claim 1 wherein the Bacillus strain is Bacillus licheniformis strain FRI MY-55(Bacillus licheniformis FRI MY-55).

5. The feed supplement of claim 1 wherein the source of disaccharide used is sucrose, or a combination of sucrose and maltose when the strain of Leuconostoc mesenteroides is Leuconostoc mesenteroides strain B4.

6. The feed supplement of claim 1 wherein when the bacillus strain is bacillus licheniformis strain FRI MY-55 the disaccharide source used is a combination of sucrose and lactose.

7. A feed supplement according to any one of claims 1 to 5 wherein the aquatic animal is a fish or shrimp.

8. A feed additive for preventing aquatic animals from being infected by pathogens, which is rich in synbiotic and has good palatability, is characterized by comprising a mixture of a Leuconostoc mesenteroides strain B4 and a nitrogen-containing source and a disaccharide-containing source, and is fermented before use; wherein the disaccharide source is sucrose, or a combination of sucrose and maltose; wherein the nitrogen-containing source is milk powder, soybean meal, or a combination thereof.

9. A feed additive for preventing aquatic animals from being infected by pathogens, which is rich in synbiotic and has good palatability, is characterized in that the feed additive comprises a mixture of a Bacillus licheniformis strain FRI MY-55 and a raw material containing disaccharide source, and the mixture is fermented before use; wherein the disaccharide source is a combination of sucrose and lactose.

10. An aquatic animal feed product comprising a feed additive according to any one of claims 1-6, 8 and 9.