CN114891680A

CN114891680A - Predigested feed for improving health and growth of herbivorous and omnivorous aquatic animals

Info

Publication number: CN114891680A
Application number: CN202210552443.6A
Authority: CN
Inventors: 周志刚; 高辰辰; 药园园; 李道君; 李毛毛; 杨雅麟; 冉超; 张震; 栾银银; 张宇婷
Original assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Current assignee: Feed Research Institute of Chinese Academy of Agricultural Sciences
Priority date: 2022-05-20
Filing date: 2022-05-20
Publication date: 2022-08-12
Anticipated expiration: 2042-05-20
Also published as: CN114891680B

Abstract

The invention discloses a predigested feed for improving the health and growth of herbivorous and omnivorous aquatic animals, belonging to the field of aquatic feeds. The invention adopts a leaven consisting of indigenous marine lactobacillus rhamnosus, bacillus subtilis, saccharomyces cerevisiae and whale bacillus sorbii to ferment basic feed to prepare the predigestion feed. The feed can obviously improve the weight gain rate of fish, reduce the feed coefficient, and has higher feed conversion rate, thereby reducing the feed cost.

Description

Predigested feed for improving health and growth of herbivorous and omnivorous aquatic animals

Technical Field

The invention relates to the field of aquatic feeds, in particular to a predigested feed for improving the health and growth of herbivorous and omnivorous aquatic animals.

Background

The aquatic products provide 1/3 high-quality animal protein in China, wherein 2/3 is provided by cultivation. The aquaculture scale of China is the first world, and the yield accounts for 2/3 all over the world. Wherein the aquaculture amount of herbivorous and omnivorous aquatic animals accounts for 3/5 of the total aquaculture amount. At present, the conventional aquaculture feed is generally considered to be rich in bioactive substances such as probiotics after being subjected to microbial predigestion, so that the intestinal health of aquatic animals can be improved, and the immunity can be improved. Therefore, the development of predigested feed is an industrial hotspot.

The feed formula of herbivorous and omnivorous aquatic animals mainly comprises a vegetable protein raw material and an energy raw material, wherein non-thermosensitive anti-nutritional factors in the vegetable protein raw material cannot eliminate the influence of the non-thermosensitive anti-nutritional factors through the processing technologies of crushing, conditioning, puffing and the like, and the high adding proportion of the vegetable protein raw material causes the activity of digestive enzymes such as trypsin secreted by the digestive tracts of herbivorous and omnivorous fishes to be reduced, so that the digestive enzymes cannot be completely digested, and the water environment pollution is caused after the digestive enzymes are discharged.

Disclosure of Invention

The invention provides a predigested feed for improving the health and growth of herbivorous and omnivorous aquatic animals, which expands the raw materials into unconventional feed raw materials such as dregs and the like, adopts the compound compatibility of aquatic indigenous lactobacilli, bacillus, saccharomycetes and whale bacillus as a leaven for fermentation and pretreatment, and can reduce the adverse effect of anti-nutritional factors on the aquatic animals.

The invention firstly provides a microbial composition which comprises lactobacillus rhamnosus, bacillus subtilis, saccharomyces cerevisiae and whale bacillus sovieiensis.

Specifically, the ratio of the number of bacteria of lactobacillus rhamnosus, bacillus subtilis, saccharomyces cerevisiae and whale bacillus sovieiensis is 1:17.5:1: 1.25.

Secondly, the invention provides a leaven, the active ingredients of which comprise lactobacillus rhamnosus, bacillus subtilis, saccharomyces cerevisiae and whale bacillus sovieiensis.

Specifically, the number of viable bacteria of the lactobacillus rhamnosus, the bacillus subtilis and the saccharomyces cerevisiae in each kilogram of the leavening agent is 8000 +/-800 hundred million cfu, 140000 +/-14000 hundred million cfu and 8000 +/-800 hundred million cfu respectively; the number of the bacteria of the whale bacillus sorbii is 10000 plus or minus 1000 hundred million cells.

More specifically, the viable count of the lactobacillus rhamnosus, the bacillus subtilis and the saccharomyces cerevisiae in each kilogram of the leavening agent is 8000 hundred million cfu, 140000 million cfu and 8000 hundred million cfu respectively; the number of the bacteria of the whale bacillus sorbieritis is 10000 hundred million cells.

In the above mentioned leavening agent, said leavening agent further comprises a carrier;

the carrier can be specifically rice hull powder and zeolite powder in a mass ratio of 1: 1;

the lactobacillus rhamnosus is lactobacillus rhamnosus GCC-3;

the bacillus subtilis is bacillus subtilis HGCC-1;

the saccharomyces cerevisiae is saccharomyces cerevisiae GCC-1;

the whale funeralis is whale funeralis XMX-1.

In the above leavening agent, the mass percentage of the carrier is 26-35%, and specifically 28%.

Thirdly, the invention provides a predigested feed, comprising a basal feed and the leavening agent; the basic feed comprises the following raw materials in percentage by mass: 15 +/-1% of wheat flour; 32 +/-1% of soybean meal; cotton dregs 10 plus or minus 1%; 10 plus or minus 1 percent of corn protein powder; 11 +/-1% of rice bran; 15 +/-1% of chicken powder; 3% of soybean oil; 2.5 percent of calcium dihydrogen phosphate; 1.5 percent of premix;

the mass of the leavening agent is 0.3 to 0.7 percent of that of the basic feed; specifically, it may be 0.5%.

Fourthly, the invention provides a preparation method of the predigested feed, which is the following method (1) or (2):

the method (1) comprises the following steps: mixing the basic feed with a leavening agent, adjusting the water content to 30-35 wt%, and fermenting to obtain the predigested feed;

the method (2) comprises the following steps: preparing the basic feed and the leavening agent into granules, then adjusting the water content to 30-35 wt%, and fermenting to obtain the predigested feed.

The method (1) further comprises the steps of granulating the fermented materials and drying; specifically, the drying temperature is 90 +/-5 ℃.

In the method (2), the step of drying is further included after the fermentation; specifically, the drying temperature is 90 +/-5 ℃.

In the preparation method, the fermentation temperature is 20-37 ℃; specifically, the temperature can be 37 ℃; the fermentation time is 48-72 h; specifically, the time can be 72 h.

The fermentation is aerobic fermentation and then anaerobic fermentation, and can be carried out in a closed container.

The application of the predigested feed in breeding herbivorous and omnivorous aquatic animals also belongs to the protection scope of the invention.

The invention has the following advantages:

(1) the leaven used by the invention is from the indigenous bacteria of the aquatic products, and is more suitable for the growth health of the aquatic animals; the predigestion feed formula more suitable for the intestinal tracts of aquatic animals is obtained by screening raw materials and designing and screening fermentation processes;

(2) the predigested feed has good food calling performance, can obviously improve the weight gain rate of fish, reduce the feed coefficient, and has higher feed conversion rate, thereby reducing the feed cost;

(3) the indigenous probiotics and metabolites or inherent components thereof used by the invention are more suitable for the intestinal environment of herbivorous and omnivorous fishes and shrimps, can regulate intestinal flora, enhance the functions of liver and kidney and maintain the healthy and stable state of the fishes and shrimps.

Biological material preservation instructions

Classification nomenclature of biological materials: saccharomyces cerevisiae (Saccharomyces cerevisiae)

Strain number of biological material: GCC-1

Deposit name of biological material: china general microbiological culture Collection center

The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)

Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101

Preservation date of biological material: 2021 year, 3 months and 9 days

Accession number to the collection of biological materials: CGMCC No.21819

Biological material preservation instructions

Classification nomenclature of biological materials: lactobacillus rhamnosus (Lactobacillus rhamnosus)

Strain number of biological material: GCC-3

Preservation date of biological material: 23/2/2021

Accession number to the collection of biological materials: CGMCC No.21821

Biological material preservation instructions

Classification nomenclature of biological materials: bacillus subtilis (Bacillus subtilis)

Strain number of biological material: HGCC-1

Deposit unit address of biological material: beijing, west way No.1 hospital on chaoyang district, No. 3, institute for microbiology, chinese academy of sciences, zip code: 100101

Preservation date of biological material: 2021, 2 months and 23 days

Accession number to the collection of biological materials: CGMCC No.21820

Drawings

FIG. 1 shows the degree of fermentation of cottonseed meal, soybean meal, rapeseed meal, corn gluten meal, guniting corn germ meal and glutamic acid residue by a leavening agent hypochondrium.

Fig. 2 shows the degree of fermentation of wheat flour by the leaven worries-free partner B.

FIG. 3 shows the degree of fermentation of rice bran and wheat bran by the fermentation agent carefree partner B.

FIG. 4 shows the degree of fermentation of tapioca starch and dextrin by the fermentation agent carefree partner B.

FIG. 5 shows the degree of fermentation of the combination of brown sugar and glutamic acid residue, brown sugar and corn gluten meal by the fermentation agent carefree partner B.

FIG. 6 shows the degree of fermentation of the combination of glutamic acid sludge and bran, glutamic acid sludge and corn gluten meal by the fermentation agent carefree partner B.

Fig. 7 is the degree of fermentation of the combination of soybean meal and pork meal by the starter carefree partner B.

Fig. 8 is the degree of fermentation of the combination of soybean meal and chicken meal by the leaven worriess partner B.

FIG. 9 shows the fermentation of different levels of moisture by fermentation partner B.

FIG. 10 shows the degree of fermentation of the fermentation agent carefree partner B at 20 ℃.

FIG. 11 shows the degree of fermentation of the fermentation agent carefree partner B under low temperature conditions.

FIG. 12 is the particle size appearance after 12h in predigested feed water in example 7.

FIG. 13 is a graph of changes in fish Survival Rate (SR), weight gain rate (WG), and feed Factor (FCR) for 2 weeks with predigested feed; a is the survival rate, B is the weight gain rate, and C is the feed coefficient.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Bacillus solitarius XMX-1 in the following examples is CGMCC No.18908, which is a collection number of China general microbiological culture Collection center (CGMCC), and is described in China patent application CN 111321093A.

In the following examples, unless otherwise specified, all percentages refer to mass percentages.

In the following examples, the adjusted moisture content during fermentation refers to the percentage of the total mass of the sum of the raw material moisture and the added moisture.

The fermentation raw materials used in the following examples, i.e., cottonseed meal, soybean meal, rapeseed meal, corn gluten meal, gunite corn germ meal, glutamic acid residue, wheat flour, rice bran, wheat bran, tapioca starch, dextrin, pork powder, chicken powder, etc., were purchased from Beijing Xinghu farming and animal science, Inc.

The rice bran in the following examples is a by-product of rice husking and brown rice whitening; bran is the wheat epidermis.

EXAMPLE 1 acquisition and characterization of Saccharomyces cerevisiae GCC-1

1. Separation and purification of GCC-1

Materials:

enrichment culture medium: 2% glucose, 2% peptone, 1% yeast powder, 0.01% penicillin, and the balance water. Separating a culture medium: cleaning potato, peeling, weighing 200g, cutting into small pieces, adding water, decocting, filtering with eight layers of gauze, heating, adding 20g of agar, adding 20g of glucose after the agar is dissolved, stirring, slightly cooling, diluting to 1000mL, packaging into conical bottles, and sterilizing at 115 deg.C under high temperature and high pressure for 30 min.

Taking sludge at the bottom of a pond with the length of 5-15 cm in a shrimp culture pond, taking 0.5g of sludge in an enrichment medium, and culturing for 2 days at 28 ℃; diluting the enrichment solution to 10 degrees ^-5 Coating 100 mu L of diluent in a separation culture medium, and standing and culturing at 28 ℃; selecting single clone according to colony morphology, and culturing for 48h at 28 deg.C and 200r/min in YPD medium to obtain single colony, which is named as GCC-1.

2. Identification of GCC-1

(1) Morphological identification

Selecting strains, streaking on an YPD culture medium, standing and culturing at 30 ℃ for 48h, and observing colony morphology;

and (3) morphological identification result: the colony size is about 1 ~ 2mm, and the colony texture is even for milk white colour homogeneous, and the edge is neat, and the colony surface is moist smooth, easily picks up.

(2) Molecular identification

The strain sequencing work is completed by Beijing Rui Boxing Corp, and the gene to be tested is 16S rDNA; the sequencing result is shown as sequence 1, and the strain is derived from Saccharomyces cerevisiae by NCBI blast (https:// blast. NCBI. nlm. nih. gov/blast. cgi) alignment. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 3, 9 and 2021 at 3, 9 days, and the preservation number is as follows: CGMCC No.21819, a classification name of the strain Saccharomyces cerevisiae.

Example 2 acquisition and characterization of Lactobacillus rhamnosus GCC-3

1. Separation and purification of GCC-3

Materials: MRS medium was purchased from haibo.

Separating a culture medium: to MRS medium was added 2% agar, 2% calcium carbonate.

The strain is separated from intestinal tracts of the paralichthys olivaceus, and the paralichthys olivaceus is purchased in the market. Dissecting living body of Paralichthys olivaceus, shearing off intestinal tract, squeezing out intestinal tract content, washing with sterile seawater, homogenizing in sterile seawater, and taking homogenate as sample. 50 mu L of homogenate sample is diluted by 100 times, 100 mu L of diluted sample is coated in a separation culture medium, and the mixture is statically cultured at 36 ℃ until a colony grows out. Selecting a single clone with a calcium-dissolving ring according to the colony morphology, and culturing for 48h at the temperature of 200r/min by using an MRS culture medium at 36 ℃ to obtain a single colony, wherein the single colony is named as GCC-3.

2. Identification of GCC-3

(1) Morphological identification

Streaking a laboratory preserved strain in an MRS culture medium, standing and culturing at 36 ℃ for 48h, and observing the colony morphology;

morphological identification shows that: the colony size is about 2mm, and the colony texture is even, and is slightly white, and is moist, and the colour is even, and the edge is neat, and the colony surface is moist smooth, is circularly.

(2) Molecular identification

The strain sequencing work is completed by Beijing Rui Boxing Corp, and the gene to be tested is 16S rDNA; the sequencing result is shown as sequence 2, and the strain is in Lactobacillus rhamnosus through NCBI blast (https:// blast.ncbi.nlm.nih.gov/blast.cgi) alignment. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 23.2.2021, with the preservation number as follows: CGMCC No.21821, and the classification name of the strain is Lactobacillus rhamnosus.

Example 3 obtaining and identification of Bacillus subtilis HGCC-1

1. Separation and purification of HGCC-1

Materials:

enrichment culture medium: 2% of glucose, 2% of peptone, 1% of yeast powder and the balance of water;

separating a culture medium: peptone 1%, sodium chloride 0.5%; 1% of soluble starch; agar 2%, and water in balance. During preparation, the soluble starch is mixed with a small amount of water into paste and added into a melted culture medium.

Taking sludge at the bottom of a pond with the length of 5-15 cm in a shrimp culture pond, taking 0.5g of sludge in an enrichment medium, and culturing for 2 days at 37 ℃; diluting the enrichment solution to 10 degrees ^-5 Coating 100 mu L of diluent in a separation culture medium, and standing and culturing at 37 ℃; selecting single clone according to colony morphology, and culturing overnight at 200r/min with LB culture medium at 37 deg.C to obtain isolate named HGCC-1.

2. Identification of HGCC-1

(1) Morphological identification

The single clone was selected, streaked on LB plate, and cultured statically at 37 ℃ for 24 hours to observe colony morphology.

And (3) morphological identification result: the colony diameter is about 1cm, and the surface with irregular edges is dry, rough and wrinkled.

(2) Molecular identification

The strain sequencing work is completed by Beijing Rui Boxing Corp, and the gene to be tested is 16S rDNA; the sequencing result is shown as sequence 3, and the strain is in Bacillus subtilis after NCBI blast (https:// blast.ncbi.nlm.nih.gov/blast.cgi) alignment. The strain is preserved in China general microbiological culture Collection center (CGMCC) at 23.2.2021, with the preservation number as follows: CGMCC No.21820, and the classification name of the strain is Bacillus subtilis.

Example 4 preparation of leaven carefree partner B

1. The fermentation agent hypochondriac companion B is a 5kg strain package, and the formula is as follows (calculated on a dry basis): 0.80kg of Lactobacillus rhamnosus GCC-3 microbial inoculum (with the viable bacteria content of 50 hundred million cfu/g), 1.40kg of Bacillus subtilis HGCC-1 microbial inoculum (with the viable bacteria content of 500 hundred million cfu/g), 0.40kg of Saccharomyces cerevisiae GCC-1 microbial inoculum (with the viable bacteria content of 100 hundred million cfu/g), 1.00kg of whale Bacillus someriae XMX-1 microbial inoculum (with the bacterial content of 50 hundred million cells/g), and 1.40kg of carriers (rice hull powder and zeolite powder mixed according to the mass ratio of 1: 1).

2. The preparation method of each microbial inoculum comprises the following steps: liquid fermentation is adopted; (1) the GCC-3 selects a single colony to be cultured in an MRS culture medium for 24h at 36 ℃ at 180r/min, the single colony is inoculated in the MRS culture medium according to the inoculation amount of 1 percent (volume percentage), the single colony is cultured for 24h at 36 ℃ at 180r/min, the single colony is inoculated in the MRS culture medium according to the inoculation amount of 5 percent (volume percentage), the single colony is subjected to shake flask fermentation for 48h at 36 ℃ at 180r/min, culture solution is collected, and the Lactobacillus rhamnosus GCC-3 microbial inoculum is obtained through low-temperature drying; the viable bacteria content of the Lactobacillus rhamnosus in the Lactobacillus rhamnosus GCC-3 microbial inoculum is 50 hundred million cfu/g; (2) the method comprises the following steps of (1) selecting a single colony of HGCC-1, culturing the single colony in an LB culture medium at 37 ℃ for 24 hours at 180r/min, inoculating the single colony in the LB culture medium according to the inoculum size of 1 percent (volume percentage), culturing the single colony for 12 hours at 37 ℃ at 180r/min, collecting a culture solution, and drying the culture solution at low temperature to obtain a Bacillus subtilis HGCC-1 microbial inoculum; the viable bacteria content of Bacillus in the Bacillus subtilis HGCC-1 microbial inoculum is 500 hundred million cfu/g; (3) GCC-1 selects a single colony to be cultured in a YPD culture medium for 48h at 30 ℃ and 180r/min, the single colony is inoculated in the YPD culture medium according to the inoculum size of 1 percent (volume percentage), the single colony is cultured for 24h at 30 ℃ and 180r/min, culture solution is collected, and the single colony is dried at low temperature to obtain a Saccharomyces cerevisiae GCC-1 microbial inoculum; the live bacteria content of the Saccharomyces cerevisiae in the Saccharomyces cerevisiae GCC-1 microbial inoculum is 100 hundred million cfu/g; (4) XMX-1 selects a single colony to be placed in GAM culture medium, stands for anaerobic culture for 24h at 28 ℃, inoculates the single colony in the GAM culture medium according to the inoculation amount of 5 percent (volume percentage), stands for anaerobic culture for 12h at 28 ℃, inoculates the secondary seed liquid in the GAM culture medium according to the inoculation amount of 5 percent (volume percentage), stands for fermentation for 48h in an anaerobic culture box at 28 ℃, collects the culture liquid, and dries at low temperature to obtain the Cetobacterium someria XMX-1 microbial inoculum; the bacteria content of the whale bacillus sojae in the whale bacillus sojae XMX-1 bacterial agent is 50 hundred million cells/g.

3. The preparation method of the fermentation agent carefree mate B comprises the steps of uniformly mixing all the raw materials.

Example 5 predigested feed formulation combination screening

1. Fermentation screening of starter carefree partner B on single raw material

(1) Fermentation experiments of the leaven careless partner B on single vegetable protein raw materials of cottonseed meal, soybean meal, rapeseed meal, corn protein powder, guniting corn germ meal and glutamic acid residue.

Respectively weighing 200g of cottonseed meal, 100g of 43% soybean meal (the mass percentage of crude protein in the soybean meal is 43%), 100g of rapeseed meal, 100g of corn protein powder, 200g of guniting corn germ meal and 100g of glutamic acid residue, and filling into 6 fermentation bags; weighing couple of fermentation agent of example 4, such as couple B1g, 0.5g, 1g and 0.5g, respectively, adding water 100mL, 50mL, 100mL and 50mL, and mixing well; respectively pouring water soluble leaven carefree mate B into the fermentation bags, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. The result is shown in figure 1, after 24 hours of fermentation, a moderate amount of gas is generated in the cottonseed meal, a small amount of gas is generated in the soybean meal, the rapeseed meal and the corn protein powder, and the sprayed corn germ meal and the glutamic acid residue are free of gas; after 48 hours of fermentation, the gas production of the cottonseed meal is finished, the cottonseed meal has the fermentation acid fragrance, the soybean meal, the rapeseed meal and the corn protein powder continue to generate gas, the cottonseed meal has the fermentation acid fragrance, and the guniting corn germ meal and the ninthlic acid residue do not generate gas. Therefore, the cottonseed meal, the soybean meal, the rapeseed meal and the corn protein powder can be fermented by using the leavening carefree companion B, the cottonseed meal is superior to the soybean meal, the rapeseed meal and the corn protein powder, but the single fermentation effect is not good enough, and the sprayed corn germ meal and the glutamic acid residue cannot be fermented independently.

In figure 1, cotton dregs, bean dregs, rapeseed dregs, corn protein powder, sprayed corn germ meal and glutamic acid residue indicate no fermentation and no gas production; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

Weighing 100g of wheat flour, and filling into a fermentation bag; weighing 0.5g of the fermentation agent carefree companion B in the embodiment 4, adding 50mL of water, and uniformly mixing; pouring water soluble leaven hypochondrium B into a fermentation bag, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. As shown in FIG. 2, after 18h of fermentation, the flour showed increased gas production, and the air bag was inflated 3/4, which is effective and can be fermented alone.

In FIG. 2, -indicates no fermentation and no gas evolution; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; the product is fermented, has sour and fragrant smell, produces a large amount of gas, and is full of fermentation bags.

(2) Fermentation experiment of fermentation agent hypochondrium B on rice bran and wheat bran which are single-energy raw materials

Respectively weighing 200g of rice bran and 200g of wheat bran, and filling into a fermentation bag; weighing 1g of each fermentation agent carefree mate B in the embodiment 4, adding 100mL of water correspondingly, and mixing uniformly; pouring water soluble leaven hypochondrium B into a fermentation bag, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. The results are shown in figure 3, and after 24 hours of fermentation, the rice bran and the wheat bran have gas production of different degrees; after fermentation for 48h, the gas generation is finished and acid fragrance is generated. Thus, both the rice bran and the wheat bran can be fermented separately.

In FIG. 3, the second rice bran and the third wheat bran indicate that the fermentation cannot be performed and no gas generation phenomenon occurs; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

Respectively weighing 100g of cassava starch and 100g of dextrin, and filling into a fermentation bag; respectively weighing 0.5g of each fermentation agent hypochondrium B in the embodiment 4, adding 50mL of water correspondingly, and uniformly mixing; pouring water soluble leaven hypochondrium B into a fermentation bag, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. As shown in figure 4, after 24 hours of fermentation, the cassava starch and the dextrin generate gas, but the gas generation amount is very small, and the smell is slightly sour; after fermentation for 72h, gas production is increased, sour taste is obvious, but the speed is still slow. Therefore, the cassava starch and the dextrin are not suitable for separate fermentation.

In FIG. 4, -indicates no fermentation and no gas evolution; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

2. Fermentation screening of combined raw materials by fermentation agent carefree partner B

(1) Fermentation experiment of leaven careless mate B on combination of brown sugar, glutamic acid residue, brown sugar and corn protein powder

Weighing 100g of brown sugar and 100g of glutamic acid residue respectively, uniformly mixing and filling into a fermentation bag; weighing 150g of brown sugar and 50g of corn protein powder, uniformly mixing, and filling into another fermentation bag; weighing couple B1g of the leaven in the embodiment 4, adding 100mL of water, and mixing uniformly; pouring water soluble leaven carefree mate B into the two fermentation bags respectively, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. The result is shown in fig. 5, after 24h of fermentation, the (brown sugar) + glutamic acid residue and the (brown sugar) + corn protein powder have no obvious fermentation phenomenon; after 48h of fermentation, there is no fermentation phenomenon and the gas produced in the R (R) is obvious. Therefore, the combination of the brown sugar and the glutamic acid residue can not be fermented, and the combined fermentation effect of the brown sugar and the corn protein powder is good.

In FIG. 5, the red sugar + glutamic acid residue, brown sugar in the red (R) and corn gluten meal indicate no fermentation and no gas production; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

(2) Fermentation experiment of fermentation agent carefree partner B on combination of glutamic acid residue and bran, glutamic acid residue and corn protein powder

Weighing 400g of glutamic acid residue and 100g of bran, uniformly mixing, and filling into a fermentation bag; weighing 400g of glutamic acid residue and 100g of corn protein powder, uniformly mixing, and filling into another fermentation bag; respectively weighing 2.5g of the fermentation agent hypochondrium B in the embodiment 4, adding 250mL of water, and uniformly mixing; pouring water soluble leaven carefree mate B into the two fermentation bags respectively, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. As shown in FIG. 6, after 12 hours of fermentation, the glutamic acid residue and the bran and the glutamic acid residue and the corn protein powder have no obvious fermentation phenomenon; after fermenting for 24 hours, firstly, only a small amount of gas is generated, and secondly, no obvious change is caused; after fermentation for 48h, a small amount of gas is generated, no fermentation smell is generated, and no change is caused. Therefore, the combination of the glutamic acid residue and the bran and the combination of the glutamic acid residue and the corn protein powder can not be fermented.

In FIG. 6, firstly, glutamic acid residue + bran, secondly, glutamic acid residue + corn protein powder, indicates that fermentation cannot be performed and no gas generation phenomenon occurs; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

(3) Fermentation experiment of combination of soybean meal, pork powder, soybean meal and chicken powder by using leaven carefree partner B

Weighing 100g of pork powder and bean pulp respectively, uniformly mixing, and filling into a fermentation bag; weighing the ferment carefree mate B1g in the embodiment 4, adding 100mL of water, and mixing uniformly; pouring water soluble leaven hypochondrium B into a fermentation bag, stirring and mixing uniformly, and putting into a 37 ℃ constant temperature box for fermentation. As shown in FIG. 7, gas was produced after 24 hours of fermentation; and (5) finishing gas production after fermentation for 48 hours. Therefore, the combination of the soybean meal and the pork powder can be fermented.

In FIG. 7, -shows no fermentation and no gassing; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

According to the proportion of 20% of zeolite powder, 80% (chicken powder, soybean meal) and leavening agent/water, the following proportion of chicken powder and soybean meal combined fermentation is designed: 50g of 20 percent of zeolite powder, 125g of 50 percent of chicken powder, 75g of 30 percent of soybean meal and 127.5g of the fermentation agent carefree partner B1.25g/water in the embodiment 4; ② 20 percent of zeolite powder 50g, 40 percent of chicken powder 100g, 40 percent of soybean meal 100g and leaven worries-free companion B1.25g/water 127.5 g; ③ 50g of 20 percent zeolite powder, 75g of 30 percent chicken powder, 125g of 50 percent bean pulp and 127.5g of ferment hypochondriac companion B1.25g/water; 50g of 20% zeolite powder, 50g of 20% chicken powder, 150g of 60% soybean meal and 127.5g of ferment hypochondriac companion B1.25g/water; 20% zeolite powder 50g, 10% chicken powder 25g, 70% bean pulp 175g, leaven carefree partner B1.25g/water 127.5 g. Respectively putting into 5 fermentation bags, and fermenting at 37 deg.C. The results are shown in fig. 8, after fermentation for 19 hours, the fermentation gas production effect of the two combined samples respectively containing 50% and 60% of plant base is better, and the combined fermentation gas production effect of the combined sample containing 70% of plant base is best; the inflation speed is sequentially from fifth to fourth to third to fourth. Therefore, the combined raw materials of 20% of zeolite powder, 10% of chicken powder and 70% of soybean meal can be fermented, and the effect is optimal.

In FIG. 8, -, no fermentation and no gas evolution; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

From the above data, for the formula of the predigested pellet feed for herbivorous and omnivorous aquatic animals, the leavening agent of example 4 is used as the carefree mate B, only rice bran, flour, wheat bran and other raw materials can be fermented singly, most raw materials can be fermented in combination, and the gas production effect of the combined raw materials is greater than that of the single raw material. Therefore, the ferment careless companion B is used as a ferment, rice bran, wheat bran and flour are selected as main raw materials of the predigested feed, and corn protein powder, soybean meal and the like are combined according to a proper proportion.

Example 6 fermentation Process Condition study of predigested feed

The raw materials used for preparing the predigested feed are as follows: the basic feed is prepared from soybean meal, cotton meal, corn protein powder and chicken meal as main protein sources and soybean oil as a main fat source. The formula is as follows (the following are mass percent): 15% of wheat flour; 32% of soybean meal; 10% of cottonseed meal; 10% of corn protein powder; 11% of rice bran; 15% of chicken powder; 3% of soybean oil; 2.5 percent of calcium dihydrogen phosphate; 1.5 percent of premix (Beijing New road combined aquatic product science and technology limited, 1 percent of compound premix feed for aquatic animals); 5g of hypochondrium B is added into each 1kg of basic group feed.

The following fermentation method comprises the following steps: mixing the above materials, placing into a fermentation bag, and fermenting at a certain temperature.

1. Study on fermentation moisture content and time

The temperature was set at 36 ℃, moisture 27%, 30%, 32%, 35%,

time

24h, 48h and 72 h. The results are shown in fig. 9, the fermentation can be successfully carried out at the temperature of 36 ℃ and the water content of more than 30%, the fermentation is basically finished within 72h, and the strain count has no significant difference. 27% moisture was not fermentable.

In FIG. 9, -shows no fermentation and no gas evolution; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

2. Study of fermentation temperature

The temperature is set to be 20 ℃, the water content is 30 percent, and the time is 24h and 48 h. The results are shown in FIG. 10, and the fermentation was successful at a temperature of 20 ℃ and a moisture of 30%.

In FIG. 10, — indicates no fermentation and no gas evolution; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

Setting the temperature at 15 ℃ or 18 ℃, the water content at 32 percent, and the time at 48h, 96h, 108h and 120 h. The results are shown in FIG. 11, the temperature of the laboratory is constant at 18 ℃, and the fermentation speed of 30% moisture is slow. In practice, heat dissipation is slow and possibly better due to accumulation and heat generation, and in order to ensure the effect, heat preservation measures are recommended to be taken when the temperature is lower than 20 ℃.

In FIG. 11, -shows no fermentation and no gas evolution; + can be fermented, has acid fragrance and produces a small amount of gas; + indicates that the fermented product has acid fragrance and moderate gas production; + + + + indicates fermentation, acid smell and large amount of gas production; + + + + + + indicates fermentation, acid smell, large amount of gas generation, and full fermentation bag.

Example 7 predigested feed stability study

(1) The preparation method of the fermentation material comprises the following steps: the raw materials are mixed according to the raw material proportion in the formula described in the embodiment 6, the mixture is made into granules by a small noodle machine, the moisture content of the granules is adjusted to 30 percent and the granules are put into a fermentation bag and fermented for 72 hours in a thermostat at 37 ℃. After fermentation, the mixture is made into particles with the diameter of 0.42mm by a small noodle making machine and dried in a constant temperature oven at 90 ℃.

(2) The preparation method of the normal material comprises the following steps: uniformly mixing a small amount of raw materials according to the raw material formula of the basic feed in the embodiment 6, and then uniformly mixing a large amount of raw materials in a step-by-step enlarged uniform mixing manner, adding no leavening agent worry-free mate B in the embodiment 6, adding a proper amount of water after the raw materials are prepared for the feed, uniformly mixing, making the mixture into particles with the diameter of 0.42mm by using a small noodle machine, and drying the particles in a constant-temperature oven at the temperature of 90 ℃;

(3) the experimental method comprises the following steps: a water body static group and a water body movement group are arranged, 500mL of water is filled in two 500mL beakers of each group, 100g of normal materials and 100g of fermentation materials are respectively scattered, small oxygen pumps are placed in two beakers of the movement group, and the air flow of the movement group is 1.5L/min, so that bubbles are generated in the water body and flow. And observing the feed state within 12 h.

The result is shown in fig. 12, the granularity of the feed is complete after 12 hours under the conditions that the water body is static and the water flow is disturbed; the fermentation material group is slightly deficient in granularity appearance, but still maintains complete granularity, and the normal feeding of the penaeus vannamei when the penaeus vannamei is fed by the fermentation material group is not influenced.

Example 8 application study of predigested feed

1. Material

The carp required by the experiment is obtained from a fry farm and is temporarily cultured in standard water circulation for 1 week. After 24h of hunger, selecting 120 tails (85.9 +/-0.6 g) of healthy and uniform juvenile fish, weighing in batches, and randomly distributing the healthy and uniform juvenile fish into 20 culture tanks according to the density of 6 tail fish in each tank, wherein the specifications of the culture tanks are as follows: 45X 45 cm.

Preparing a basic feed (the following mass percentages): 15% of wheat flour; 32% of soybean meal; 10% of cottonseed meal; 10% of corn protein powder; 11% of rice bran; 15% of chicken powder; 3% of soybean oil; 2.5 percent of calcium dihydrogen phosphate; 1.5 percent of premix (Beijing New road combined aquatic product science and technology limited, 1 percent of compound premix feed for aquatic animals).

The basal feed was made into pellets by the method of preparation of the normal feed of example 7.

Preparing fermented feed: adding 5g of the carefree mate B into 1kg of basic group feed, adding water to adjust the final water content of the feed to 32%, slightly mixing uniformly, transferring into a breathing bag, draining air, sealing, and fermenting at 37 ℃ for 72 h. After fermentation, the mixture is made into particles with the diameter of 0.42mm by a small noodle making machine and dried in a constant temperature oven at 90 ℃.

2. Feeding scheme (the following percentages are all mass percent)

The experiments were divided into 5 groups of 4 replicates each. The control group was fed basal diet, the treatment group used fermented feed in place of basal diet at 5%, 10%, 15%, 20% of the feeding amount, respectively, and the experiment employed a Completely Random Design (CRD) as follows:

in the control group, 100% of basic feed is added, and 0% of fermented feed is added; treatment group 1, adding 95% of basal feed and 5% of fermented feed; 90% of basic feed and 10% of fermented feed are added in the treatment group 2; treatment group 3, adding 85% of basic feed and 15% of fermented feed; and in the treatment group 4, 80% of basic feed is added, and 20% of fermented feed is added. The test period was 3 times daily (7:00, 12:00, 18: 00). Feeding 2% of the initial body weight for 6 days, 3% for 7 days, and 6% of the initial body weight for 15 days.

3. Cultivation environment and water quality detection

The culture system adopts a commercial fish circulating water culture system. The volume of the culture tank is 90L, the culture water temperature is 26.0 ℃, the culture period is 4 weeks, water is not changed during the culture period, and the water quality is detected every day.

The experimental results are as follows:

after 4 weeks of cultivation, the water quality of the fish tank is as follows: dissolved Oxygen (DO) is 6mg/L, ammonia nitrogen content is 0.5mg/L, and nitrite concentration is 0.05 mg/L.

4. Measurement of growth Performance and feed utilization efficiency

The experimental fish were weighed every two weeks, starved for 24 hours and weighed for each tank and the survival rate (SR,%), weight gain (WG,%), feed Factor (FCR) were calculated as 12% water for both basal and fermented feed, to evaluate the effect of feed additives on feeding performance. The calculation formula is as follows:

survival rate (SR,%) 100% × number of fish at end of experiment/number of fish at start of experiment;

weight gain (WG,%) 100% × (final body weight (g) -initial body weight (g))/initial body weight (g);

the Feed Coefficient (FCR) is [ basal feed intake/(100-5.52 +12) + fermented feed intake/(100-31.93 +12) ]/fish body weight gain.

The experimental results are as follows:

as shown in table 1 and fig. 13, at 2 weeks of cultivation, there was no significant difference in survival rate between the control group and each treatment group; the weight gain rate of the treatment group 4 was significantly higher than that of the control group; the feed factors of treatment group 3 and treatment group 4 were significantly reduced compared to the control group. Therefore, the feed has no influence on the survival rate of experimental fish, can obviously improve the weight gain rate of the fish, reduces the feed coefficient, and has higher feed conversion rate.

TABLE 1 feeding 2 weeks growth Performance parameters

In the table, IBW is the average initial weight; FBW is the average terminal weight; the same superscript letters between groups indicate no significant difference (p > 0.05), and the different superscript letters between groups indicate significant difference (p < 0.05, p < 0.01, or p < 0.001).

<110> institute of feed of Chinese academy of agricultural sciences

<120> a predigested feed for improving the health and growth of herbivorous and omnivorous aquatic animals

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 1020

<212> DNA

<213> Saccharomyces cerevisiae GCC-1

<400> 1

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tgcttaaaat ctcgaccctt tggaagagat gtatttatta gataaaaaat caatgtcttc 180

ggactctttg atgattcata ataacttttc gaatcgcatg gccttgtgct ggcgatggtt 240

cattcaaatt tctgccctat caactttcga tggtaggata gtggcctacc atggtttcaa 300

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ccaaggaagg cagcaggcgc gcaaattacc caatcctaat tcagggaggt agtgacaata 420

aataacgata cagggcccat tcgggtcttg taattggaat gagtacaatg taaatacctt 480

aacgaggaac aattggaggg caagtctggt gccagcagcc gcggtaattc cagctccaat 540

agcgtatatt aaagttgttg cagttaaaaa gctcgtagtt gaactttggg cccggttggc 600

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gtccttgtgg ctcttggcga accgggactt ttactttgaa aaaattagag tgttcaaagc 720

aggcgtattg ctcgaatata ttagcatgga ataatagaat aggacgtttg gttctatttt 780

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tgtcagaggt gaaattcttg gatttattga agactaacta ctgcgaaagc atttgccaag 900

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<213> Lactobacillus rhamnosus GCC-3

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ggttgcgctc gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg 420

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caagacctgg taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg 540

cgggcccccg tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc 600

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gtggggatga cgtcaaatca tcatgcccct tatgacctgg gctacacacg tgctacaatg 1200

gacagaacaa agggcagcga aaccgcgagg ttaagccaat cccacaaatc tgttctcagt 1260

tcggatcgca gtctgcaact cgactgcgtg aagctggaat cgctagtaat cgcggatcag 1320

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Claims

1. The preservation number of the Bacillus subtilis HGCC-1 is CGMCC No. 21820.

2. A microbial composition comprises Lactobacillus rhamnosus, Bacillus subtilis, Saccharomyces cerevisiae and whale bacillus Sovieae;

3. A leaven comprises active ingredients including Lactobacillus rhamnosus, Bacillus subtilis, Saccharomyces cerevisiae and whale bacillus Sovieae;

specifically, in each kilogram of the leavening agent, the number of the live bacteria of the lactobacillus rhamnosus, the bacillus subtilis and the saccharomyces cerevisiae is 8000 +/-800 hundred million cfu, 140000 +/-14000 hundred million cfu and 8000 +/-800 hundred million cfu respectively; the number of the whale bacillus sojae is 10000 (+ -1000) hundred million cells.

4. A starter culture according to claim 3 wherein: the starter also comprises a carrier;

in the leavening agent, the mass percentage content of the carrier can be 26-35%;

the lactobacillus rhamnosus is lactobacillus rhamnosus GCC-3;

the bacillus subtilis is bacillus subtilis HGCC-1;

the saccharomyces cerevisiae is saccharomyces cerevisiae GCC-1;

the whale funeralis is whale funeralis XMX-1.

5. A predigested feed comprising a basal feed and the starter culture of claim 3 or 4; the basic feed is prepared from the following raw materials in percentage by mass: 15 +/-1% of wheat flour; 32 +/-1% of soybean meal; 10% +/-1% of cottonseed meal; 10 plus or minus 1 percent of corn protein powder; 11 +/-1% of rice bran; 15 +/-1% of chicken powder; 3% of soybean oil; 2.5 percent of calcium dihydrogen phosphate; 1.5 percent of premix;

6. The method for producing a predigested feed according to claim 6, which comprises the following steps (1) or (2):

the method (1) comprises the following steps: mixing the basal feed and the starter in claim 5, adjusting the water content to 30-35 wt%, and fermenting to obtain the predigested feed;

the method (2) comprises the following steps: preparing the base feed and the leaven as defined in claim 5 into pellets, adjusting the moisture content to 30-35 wt%, and fermenting to obtain the predigested feed.

7. The method of claim 6, wherein: the method (1) also comprises the steps of granulating and drying the materials after fermentation;

specifically, the drying temperature is 90 +/-5 ℃;

in the method (2), the step of drying is also included after the fermentation; specifically, the drying temperature is 90 +/-5 ℃.

8. The production method according to claim 7, characterized in that: the fermentation temperature is 20-37 ℃; the fermentation time is 48-72 h.

9. Use of the predigested feed according to claim 5 for the cultivation of herbivorous and omnivorous aquatic animals.