CN107312732B - Probiotic feed additive - Google Patents
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- CN107312732B CN107312732B CN201710595231.5A CN201710595231A CN107312732B CN 107312732 B CN107312732 B CN 107312732B CN 201710595231 A CN201710595231 A CN 201710595231A CN 107312732 B CN107312732 B CN 107312732B
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- 239000006041 probiotic Substances 0.000 title claims abstract description 48
- 235000018291 probiotics Nutrition 0.000 title claims abstract description 48
- 239000003674 animal food additive Substances 0.000 title claims abstract description 40
- 230000000529 probiotic effect Effects 0.000 title claims abstract description 38
- 238000000855 fermentation Methods 0.000 claims abstract description 82
- 230000004151 fermentation Effects 0.000 claims abstract description 82
- 240000006024 Lactobacillus plantarum Species 0.000 claims abstract description 59
- 235000013965 Lactobacillus plantarum Nutrition 0.000 claims abstract description 59
- 229940072205 lactobacillus plantarum Drugs 0.000 claims abstract description 59
- 241000222175 Diutina rugosa Species 0.000 claims abstract description 47
- 241000509461 [Candida] ethanolica Species 0.000 claims abstract description 46
- 241000194040 Lactococcus garvieae Species 0.000 claims abstract description 40
- 235000020246 buffalo milk Nutrition 0.000 claims abstract description 24
- 239000000758 substrate Substances 0.000 claims abstract description 19
- 238000002156 mixing Methods 0.000 claims abstract description 11
- 238000009395 breeding Methods 0.000 claims abstract description 9
- 230000001488 breeding effect Effects 0.000 claims abstract description 9
- 239000007788 liquid Substances 0.000 claims description 51
- 241000894006 Bacteria Species 0.000 claims description 45
- 230000001580 bacterial effect Effects 0.000 claims description 27
- 239000000843 powder Substances 0.000 claims description 23
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 20
- 239000012153 distilled water Substances 0.000 claims description 18
- 239000001963 growth medium Substances 0.000 claims description 18
- 238000004321 preservation Methods 0.000 claims description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 18
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
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- 239000001888 Peptone Substances 0.000 claims description 10
- 108010080698 Peptones Proteins 0.000 claims description 10
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 10
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 10
- ZPWVASYFFYYZEW-UHFFFAOYSA-L dipotassium hydrogen phosphate Chemical compound [K+].[K+].OP([O-])([O-])=O ZPWVASYFFYYZEW-UHFFFAOYSA-L 0.000 claims description 10
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- 235000019797 dipotassium phosphate Nutrition 0.000 claims description 10
- 239000008103 glucose Substances 0.000 claims description 10
- 238000011081 inoculation Methods 0.000 claims description 10
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 10
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 10
- 229940099596 manganese sulfate Drugs 0.000 claims description 10
- 239000011702 manganese sulphate Substances 0.000 claims description 10
- 235000007079 manganese sulphate Nutrition 0.000 claims description 10
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 10
- 235000019319 peptone Nutrition 0.000 claims description 10
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 claims description 10
- 239000010802 sludge Substances 0.000 claims description 10
- 239000001632 sodium acetate Substances 0.000 claims description 10
- 235000017281 sodium acetate Nutrition 0.000 claims description 10
- 238000009631 Broth culture Methods 0.000 claims description 9
- 238000012360 testing method Methods 0.000 claims description 9
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- 239000007787 solid Substances 0.000 claims description 5
- 239000006228 supernatant Substances 0.000 claims description 5
- OYPRJOBELJOOCE-IGMARMGPSA-N Calcium-40 Chemical compound [40Ca] OYPRJOBELJOOCE-IGMARMGPSA-N 0.000 claims description 3
- 238000012258 culturing Methods 0.000 claims description 3
- 239000000203 mixture Substances 0.000 claims description 2
- 239000002609 medium Substances 0.000 claims 1
- 241001465754 Metazoa Species 0.000 abstract description 7
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- 239000003814 drug Substances 0.000 abstract description 6
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- 229920000136 polysorbate Polymers 0.000 description 3
- 244000144977 poultry Species 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 2
- 240000001046 Lactobacillus acidophilus Species 0.000 description 2
- 235000013956 Lactobacillus acidophilus Nutrition 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 229940039696 lactobacillus Drugs 0.000 description 2
- 229940039695 lactobacillus acidophilus Drugs 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 230000008520 organization Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000004460 silage Substances 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 241000186000 Bifidobacterium Species 0.000 description 1
- 241000030939 Bubalus bubalis Species 0.000 description 1
- 241000222120 Candida <Saccharomycetales> Species 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 241000282414 Homo sapiens Species 0.000 description 1
- 235000021052 average daily weight gain Nutrition 0.000 description 1
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- 230000015572 biosynthetic process Effects 0.000 description 1
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 208000010643 digestive system disease Diseases 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
- A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/10—Feeding-stuffs specially adapted for particular animals for ruminants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/80—Food processing, e.g. use of renewable energies or variable speed drives in handling, conveying or stacking
- Y02P60/87—Re-use of by-products of food processing for fodder production
Abstract
The invention relates to the technical field of feed additives and processing thereof, in particular to a probiotic feed additive, which is prepared by mixing and fermenting lactobacillus plantarum, candida rugosa, candida ethanolica, lactococcus garvieae and buffalo milk which are obtained by independent separation as fermentation substrates. Can obviously improve the intestinal microbial flora of animals, improve the immunity, reduce the use of antibiotic medicines, eliminate the medicine residue, be applied to production, shorten the feeding period, save the feed and further improve the breeding income.
Description
[ technical field ] A method for producing a semiconductor device
The invention relates to the technical field of feed additives and processing thereof, in particular to a probiotic feed additive.
[ background of the invention ]
Probiotics, which are defined as a microorganism having biological activity by the food and agricultural organization and the world health organization in the united nations, are in a wide variety and can be broadly classified into three major groups, i.e., lactobacillus, bifidobacterium and gram-positive coccus. A large number of researches prove that the probiotics have multiple effects of improving and preventing diarrhea, promoting the health of an intestinal digestive system, enhancing immunity, reducing serum cholesterol, preventing infection of a reproductive system and the like.
In recent years, the disadvantages caused by using a large amount of antibiotics as feed additives are more and more obvious, and the caused drug residues bring great influence to both human beings and animals. With the continuing ban of antibiotics in many countries, probiotics will be better developed as a replacement for antibiotics.
A microbial feed additive is a microbial preparation that replaces or balances one or more bacterial strains in an animal's ecosystem. After the lactobacillus acidophilus feed is used as feed to enter livestock and poultry bodies, the lactobacillus acidophilus feed can be rapidly propagated, the formation of dominant flora of lactobacillus in intestinal tracts of the livestock and poultry is promoted, the microecological balance of intestinal tracts is maintained, the growth of the livestock and poultry can be effectively promoted, and the consumption of the feed is reduced. At present, domestic reports on the application of probiotics to animal feed are that: for example, chinese patent CN 106906145 a discloses "a probiotic agent for fermenting silage and a production method" which utilizes probiotics to ferment silage to reduce animal digestive system diseases, but the fermented feed still has the problems of complex manufacturing process, inconvenient storage and carrying, and the like, and the method does not study the physical characteristics of milk-producing buffalo, so that a feed additive with simple process, convenient carrying and storage is produced to increase the synergistic effect between microorganisms, improve the breeding benefit, and improve the feeding efficiency of buffalo milk according to the physical condition of local buffalo milk.
[ summary of the invention ]
In view of the above, there is a need for a simple, portable, and storable feed additive that can increase the synergy between microbes, improve the breeding efficiency, and improve the feeding efficiency of buffalo milk for the body condition of local buffalo milk.
In order to achieve the purpose, the technical scheme adopted by the invention is as follows:
a probiotic feed additive contains viable bacteria of 2.6 × 10/g8CFU/g～9.3×108The probiotic is prepared by fermenting CFU/g, and the probiotic consists of lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica.
Further, the Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Y12 with the preservation number of CCTCC NO: M2016469; the Candida rugosa is Candida rugosa (Candida rugosa) Y7, and the preservation number is CCTCC M2016468; the Candida ethanolica is Candida ethanolica (Candidaethanolica) Y1 with the preservation number of CCTCC M2016467; are all separated from fresh buffalo milk.
Further, the method comprises the following steps:
(1) respectively inoculating lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica on an MRS slant culture medium for culturing at 37 ℃ for 24-48 h to respectively obtain lactococcus garvieae slant culture bacteria, lactobacillus plantarum slant culture bacteria, candida rugosa slant culture bacteria and candida ethanolica slant culture bacteria;
(2) respectively transferring the lactococcus garvieae slant culture bacteria, the lactobacillus plantarum slant culture bacteria, the candida rugosa slant culture bacteria and the candida ethanolica slant culture bacteria in the step (1) into a test tube filled with sterilized MRS broth culture medium to carry out shake cultivation, wherein the cultivation temperature is 37 ℃, and the cultivation time is 24 hours, so as to respectively obtain lactococcus garvieae seed liquid, lactobacillus plantarum seed liquid, candida rugosa seed liquid and candida ethanolica seed liquid;
(3) mixing the lactococcus garvieae seed liquid, the lactobacillus plantarum seed liquid, the candida rugosa seed liquid and the candida ethanolica seed liquid in the step (2) according to the proportion of 1: mixing the seed solution and the fermentation broth according to the mass ratio of 2:2:2 to obtain a mixed seed solution, and then inoculating the mixed seed solution and the fermentation broth into a fermentation tank filled with a fermentation substrate according to the inoculation amount of 2:25, wherein the fermentation temperature is 37 ℃, and the fermentation time is 24 hours to obtain an expanded seed bacterial solution;
(4) inoculating the seed bacterial liquid expanded in the step (3) into a fermentation tank filled with a fermentation substrate according to the inoculation amount of the seed bacterial liquid and the fermentation liquid in a mass ratio of 1-2:10, wherein the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the fermentation pH is 3.2-4.8, and cooling to room temperature to obtain mixed strain fermentation liquid;
(5) and (3) centrifuging the mixed strain fermentation liquor obtained in the step (4) by using a centrifuge with the rotation speed of 3000-8000rpm for 15-20min, centrifuging to remove supernatant to obtain semi-solid bacterial sludge, freezing the bacterial sludge in a refrigerator for 4h, drying in a vacuum freeze dryer, taking out, crushing and bagging to obtain the probiotic feed additive.
Further, the MRS slant culture medium of the step (1) comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14g/L of agar, tween-801 g/L and the balance of distilled water.
Further, the MRS broth culture medium of step (2) comprises the following components: 10.0g/L of peptone, 10.0g/L of meat extract, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.5g/L of cysteine, 0.8978 g/L of tween-801.0 g/L and the balance of distilled water, wherein the pH value is 6.2-6.4.
Further, the fermentation substrates in the steps (3) and (4) comprise the following components in percentage by mass: 25 to 41 percent of buffalo milk, 5 to 10 percent of cane sugar, 0.02 to 0.14 percent of yeast extract powder, 5 to 8 percent of agar, 0.07 to 1 percent of calcium carbonate and 40 to 60 percent of distilled water.
Furthermore, the viable count of the lactobacillus plantarum in the mixed strain fermentation liquor obtained in the step (4) is 2.6 multiplied by 108～5.3× 108CFU/g, the viable count of the lactococcus garvieae is 3.1 multiplied by 108～3.8×108CFU/g。
Further, the probiotic feed additive is applied to dairy buffalo breeding.
The sources of microbial strains in the probiotic feed additive are as follows:
1. the Lactobacillus plantarum of the present application is Lactobacillus plantarum (Lactobacillus plantarum) Y12:
the Lactobacillus plantarum is obtained by separating Lactobacillus plantarum Y12 from buffalo milk, the buffalo milk is obtained from a buffalo farm of Guangxi buffalo research institute, and is obtained by separating the buffalo milk by adopting an MRS medium plate coating method and an inclined plane scribing method, and the 16SrDNA strain identification is completed by a biological engineering (Shanghai) company. The physiological and biochemical experiments of bacteria were performed using the API 20A reagent strip from merriella, france. The strain is suitable for growth at 37-39 ℃, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2016469 in 2016, 9, 8 and 9 months.
2. Candida rugosa (Candida rugosa) Y7:
the Candida rugosa Y7 was isolated from buffalo milk obtained from the research institute of Buffalo, Guangxi, and isolated by the MRS medium plate coating method and slant streaking method, and the identification of 16SrDNA species was carried out by Biotech (Shanghai). The physiological and biochemical experiments of bacteria were performed using API 50CH reagent strips from Merrier, France. The strain is suitable for growth at the temperature of 30-35 ℃, and is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2016468 in 2016, 9, 8 and 9 months.
3. Candida ethanolica (candidathanolica) Y1 of the present application:
the Candida ethanolica Y1 was isolated from buffalo milk obtained from the research institute of Water buffalo, Guangxi, by applying MRS medium to plate and slant streaking, and the identification of 16SrDNA strain was carried out by Biotech (Shanghai). The physiological and biochemical experiments of bacteria were performed using API 50CH reagent strips from Merrier, France. The strain is suitable for growth at the temperature of 30-35 ℃, is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of M2016467 in 2016, 9, 8 and M
4. Lactococcus garvieae of the present application:
the above lactococcus garvieae is commercially available and purchased from the Chinese type culture Collection.
The invention has the following beneficial effects:
1. the probiotics used by the feed additive is obtained by separating, purifying and culturing from the fresh emulsion of local buffalo milk, has more specificity to buffalo bodies than the commercial microbial probiotics, can effectively improve the regulation and control of the probiotics to the buffalo bodies, can obviously improve the intestinal microbial flora of animals, improve the immunity, reduce the use of antibiotic medicines, eliminate medicine residues, be applied to production, shorten the feeding period and save feed, thereby improving the breeding income.
[ detailed description ] embodiments
All of the features disclosed in this specification, or all of the steps in any method or process so disclosed, may be combined in any combination, except combinations of features and/or steps that are mutually exclusive.
Any feature disclosed in this specification (including any accompanying claims, abstract) is intended to be incorporated in any specific embodiment, unless expressly stated otherwise, in such specific embodiment, each feature is an example of a generic series of equivalent or similar features.
Experiments show that the results of physiological and biochemical experiments of lactococcus garvieae (abbreviated as Grignard), Lactobacillus plantarum Y12 (abbreviated as Y12), Candida rugosa Y7 (abbreviated as Y7), and Candida ethanolica Y1 (abbreviated as Y1) are shown in Table 1:
TABLE 1
It was thus concluded that culture conditions of lactococcus garvieae, Lactobacillus plantarum Y12, Candida rugosa Y7, Candida ethanolica Y1, as shown in examples 1-3:
example 1:
a probiotic feed additive contains viable bacteria of 2.6 × 10/g8The probiotic is prepared by fermenting CFU/g, and the probiotic consists of lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica.
The Lactobacillus plantarum is Lactobacillus plantarum Y12 (Lactobacillus plantarum) with preservation number of CCTCC NO: M2016469; the Candida rugosa is Candida rugosa (Candida rugosa) Y7, and the preservation number is CCTCC M2016468; the Candida ethanolica is Candida ethanolica (Candida ethanolica) Y1 with the preservation number of CCTCC M2016467; are all separated from fresh buffalo milk.
The present embodiment also provides a preparation method for preparing a probiotic feed additive, the method comprising the steps of:
(1) respectively inoculating lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica on an MRS slant culture medium for culture, wherein the MRS slant culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14g/L of agar, 801g/L of Tween and the balance of distilled water, wherein the culture temperature is 37 ℃ and the culture time is 24-48 h, and lactococcus garvieae slant culture bacteria and lactobacillus plantarum slant culture bacteria are respectively obtained;
(2) respectively transferring the lactococcus garvieae slant culture bacteria, the lactobacillus plantarum slant culture bacteria, the candida rugosa slant culture bacteria and the candida ethanolica slant culture bacteria in the step (1) into an MRS broth culture medium test tube filled with sterilized bacteria for shake cultivation, wherein the MRS broth culture medium comprises the following components: 10.0g/L of peptone, 10.0g/L of meat extract, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.5g/L of cysteine, 0.8978 g/L of tween-801.0, the balance of distilled water, the pH value of 6.2, the culture temperature of 37 ℃ and the culture time of 24 hours, so as to respectively obtain a lactococcus garvieae seed solution and a lactobacillus plantarum seed solution;
(3) mixing the lactococcus garvieae seed liquid, the lactobacillus plantarum seed liquid, the candida rugosa seed liquid and the candida ethanolica seed liquid in the step (2) according to the proportion of 1: mixing the mixed seed solution according to the mass ratio of 2:2:2 to obtain a mixed seed solution, and then inoculating the mixed seed solution and the fermentation broth into a fermentation tank filled with a fermentation substrate according to the inoculation amount of 2:25, wherein the fermentation substrate comprises the following components in percentage by mass: 30% of buffalo milk, 5% of cane sugar, 0.02% of yeast extract powder, 5% of agar, 0.07% of calcium carbonate and 59.91% of distilled water, wherein the fermentation temperature is 37 ℃, and the fermentation time is 24 hours, so as to obtain expanded seed bacterial liquid;
(4) inoculating the seed bacterial liquid expanded in the step (3) into a fermentation tank filled with a fermentation substrate according to the inoculation amount of the seed bacterial liquid and the fermentation liquid with the mass ratio of 1.5:10, wherein the mass percentage of the fermentation substrate is as follows: 40% of buffalo milk, 10% of cane sugar, 0.14% of yeast extract powder, 8% of agar, 1% of calcium carbonate and 40.86% of distilled water, wherein the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the pH value of the fermentation is 3.2, and the mixed strain fermentation liquor is obtained after cooling to room temperature, wherein the viable count of lactobacillus plantarum in the mixed strain fermentation liquor is 2.6 multiplied by 108The viable count of the lactococcus garvieae is 3.1 × 108；
(5) And (3) centrifuging the mixed strain fermentation liquor obtained in the step (4) by using a centrifuge with the rotating speed of 3000 for 15min, centrifuging to remove supernatant to obtain semi-solid bacterial sludge, freezing the bacterial sludge in a refrigerator for 4h, drying in a vacuum freeze dryer, taking out, crushing and bagging to obtain the probiotic feed additive.
The feed additive of the embodiment is suitable for breeding dairy buffalos.
Example 2:
a probiotic feed additive contains viable bacteria of 9.3 × 10/g8The probiotic is prepared by fermenting CFU/g, and the probiotic consists of lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica.
The Lactobacillus plantarum is Lactobacillus plantarum Y12 (Lactobacillus plantarum) with preservation number of CCTCC NO: M2016469; the Candida rugosa is Candida rugosa (Candida rugosa) Y7, and the preservation number is CCTCC M2016468; the Candida ethanolica is Candida ethanolica (Candida ethanolica) Y1 with the preservation number of CCTCC M2016467; are all separated from fresh buffalo milk.
The present embodiment also provides a preparation method for preparing a probiotic feed additive, the method comprising the steps of:
(1) respectively inoculating lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica on an MRS slant culture medium for culture, wherein the MRS slant culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14g/L of agar, 801g/L of Tween and the balance of distilled water, wherein the culture temperature is 37 ℃ and the culture time is 24-48 h, and lactococcus garvieae slant culture bacteria and lactobacillus plantarum slant culture bacteria are respectively obtained;
(2) respectively transferring the lactococcus garvieae slant culture bacteria, the lactobacillus plantarum slant culture bacteria, the candida rugosa slant culture bacteria and the candida ethanolica slant culture bacteria in the step (1) into an MRS broth culture medium test tube filled with sterilized bacteria for shake cultivation, wherein the MRS broth culture medium comprises the following components: 10.0g/L of peptone, 10.0g/L of meat extract, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.5g/L of cysteine, 0.8978 g/L of tween-801.0, the balance of distilled water, the pH value of 6.4, the culture temperature of 37 ℃ and the culture time of 24 hours to respectively obtain a lactococcus garvieae seed solution and a lactobacillus plantarum seed solution;
(3) mixing the lactococcus garvieae seed liquid, the lactobacillus plantarum seed liquid, the candida rugosa seed liquid and the candida ethanolica seed liquid in the step (2) according to the proportion of 1: mixing the mixed seed solution according to the mass ratio of 2:2:2 to obtain a mixed seed solution, and then inoculating the mixed seed solution and the fermentation broth into a fermentation tank filled with a fermentation substrate according to the inoculation amount of 2:25, wherein the fermentation substrate comprises the following components in percentage by mass: 29.91 percent of buffalo milk, 5 percent of cane sugar, 0.02 percent of yeast extract powder, 5 percent of agar, 0.07 percent of calcium carbonate and 60 percent of distilled water, wherein the fermentation temperature is 37 ℃, and the fermentation time is 24 hours, so as to obtain expanded seed bacterial liquid;
(4) inoculating the seed bacterial liquid expanded in the step (3) into a fermentation tank filled with a fermentation substrate according to the inoculation amount of the seed bacterial liquid and the fermentation liquid in a mass ratio of 2:10, wherein the mass percentage of the fermentation substrate is as follows: 40.86 percent of buffalo milk, 10 percent of cane sugar, 0.14 percent of yeast extract powder, 8 percent of agar, 1 percent of calcium carbonate and 40 percent of distilled water, the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the pH value of the fermentation is 3.2 to 4.8, and the mixed strain fermentation liquor is obtained after cooling to the room temperature, wherein the viable count of the lactobacillus plantarum in the mixed strain fermentation liquor is 5.3 multiplied by 108CFU/g, the viable count of the lactococcus garvieae is 3.8 multiplied by 108CFU/g；
(5) And (3) centrifuging the mixed strain fermentation liquor obtained in the step (4) by using a centrifuge with the rotating speed of 8000rpm for 20min, centrifuging to remove supernatant to obtain semi-solid bacterial sludge, freezing the bacterial sludge in a refrigerator for 4h, drying in a vacuum freeze dryer, taking out, crushing and bagging to obtain the probiotic feed additive.
The feed additive of the embodiment is suitable for breeding dairy buffalos.
Example 3:
a probiotic feed additive contains viable bacteria of 6.3 × 10/g8The probiotic is prepared by fermenting CFU/g, and the probiotic consists of lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica.
The Lactobacillus plantarum is Lactobacillus plantarum Y12 (Lactobacillus plantarum) with preservation number of CCTCC NO: M2016469; the Candida rugosa is Candida rugosa (Candida rugosa) Y7, and the preservation number is CCTCC M2016468; the Candida ethanolica is Candida ethanolica (Candida ethanolica) Y1 with the preservation number of CCTCC M2016467; are all separated from fresh buffalo milk.
The present embodiment also provides a preparation method for preparing a probiotic feed additive, the method comprising the steps of:
(1) respectively inoculating lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica on an MRS slant culture medium for culture, wherein the MRS slant culture medium comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14g/L of agar, 801g/L of Tween and the balance of distilled water, wherein the culture temperature is 37 ℃ and the culture time is 24-48 h, and lactococcus garvieae slant culture bacteria and lactobacillus plantarum slant culture bacteria are respectively obtained;
(2) respectively transferring the lactococcus garvieae slant culture bacteria, the lactobacillus plantarum slant culture bacteria, the candida rugosa slant culture bacteria and the candida ethanolica slant culture bacteria in the step (1) into an MRS broth culture medium test tube filled with sterilized bacteria for shake cultivation, wherein the MRS broth culture medium comprises the following components: 10.0g/L of peptone, 10.0g/L of meat extract, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.5g/L of cysteine, 0.8978 g/L of tween-801.0, the balance of distilled water, the pH value of 6.3, the culture temperature of 37 ℃ and the culture time of 24 hours, so as to respectively obtain lactococcus garvieae seed liquid and lactobacillus plantarum seed liquid;
(3) mixing the lactococcus garvieae seed liquid, the lactobacillus plantarum seed liquid, the candida rugosa seed liquid and the candida ethanolica seed liquid in the step (2) according to the proportion of 1: mixing the mixed seed solution according to the mass ratio of 2:2:2 to obtain a mixed seed solution, and then inoculating the mixed seed solution and the fermentation broth into a fermentation tank filled with a fermentation substrate according to the inoculation amount of 2:25, wherein the fermentation substrate comprises the following components in percentage by mass: 40% of buffalo milk, 8% of cane sugar, 0.12% of yeast extract powder, 7% of agar, 0.08% of calcium carbonate and 44.8% of distilled water, wherein the fermentation temperature is 37 ℃ and the fermentation time is 24 hours, so that expanded seed bacterial liquid is obtained;
(4) inoculating the seed bacterial liquid expanded in the step (3) into a fermentation tank filled with fermentation substrate according to the inoculation amount of the seed bacterial liquid and the fermentation liquid with the mass ratio of 1:10Wherein the fermentation substrate comprises the following components in percentage by mass: 25% of buffalo milk, 9% of cane sugar, 0.10% of yeast extract powder, 6% of agar, 0.9% of calcium carbonate and 59% of distilled water, wherein the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the pH value of the fermentation is 4.1, and the mixed strain fermentation liquor is obtained by cooling to room temperature, wherein the viable count of lactobacillus plantarum in the mixed strain fermentation liquor is 4.3 multiplied by 108CFU/g, the viable count of the lactococcus garvieae is 3.5 multiplied by 108CFU/g；
(5) And (4) centrifuging the mixed strain fermentation liquor obtained in the step (4) by using a centrifuge with the rotating speed of 5000rpm for 17min, centrifuging to remove supernatant to obtain semi-solid bacterial sludge, freezing the bacterial sludge in a refrigerator for 4h, drying in a vacuum freeze dryer, taking out, crushing and bagging to obtain the probiotic feed additive.
The feed additive of the embodiment is suitable for breeding dairy buffalos.
According to group 1:
the other preparation method and the formula are completely the same as the example 1, but the commercial EM bacterial liquid is used for replacing the probiotic fermentation agent.
Control group 2:
the other preparation method and formulation are the same as those in example 1, but commercially available Lactobacillus plantarum Y12, Candida rugosa Y7, and Candida ethanolica Y1 were replaced by commercially available Candida plantarum, Candida rugosa, and Candida ethanolica.
Feeding experiment:
guangxi buffalo institute species cattle farm, experimental design: the test time is from 10 months 7 days in 2015 to 7 days in 2015, a random pairing principle is adopted in the test, the test cows are three-grade mixed milk buffalo (local buffalo x nii-lai-phenanthrene buffalo x mora buffalo), 60 milk buffalos are randomly divided into an experimental group and a control group, 10 milk buffalos in each group, and the feeding test is carried out according to the following feeding formula:
experimental group 1: adding 20g of the probiotic feed additive of example 1 to 4kg of basal ration;
experimental group 2: adding 20g of the probiotic feed additive of example 2 to 4kg of basal ration;
experimental group 3: adding 20g of the probiotic feed additive of example 3 to 4kg of basal ration;
experimental group 4: adding 20g of probiotic feed additive of control group 1 to 4kg of basal diet;
experimental group 5: adding 20g of probiotic feed additive of control group 2 to 4kg of basal diet;
control group: basal diet
After feeding for 10 days, calculating the increase of the dry matters of the excrement of each group of the milk buffaloes; after feeding for 30 days, calculating the milk yield of each group; after feeding for 60 days, calculating the daily average weight gain of each group; the appearance of the milk buffalo was observed and recorded, and the specific experimental results are shown in table 2: : stool dry matter increase calculation formula:
milk yield calculation formula:
daily average weight gain calculation formula:
TABLE 2
As can be seen from the above table, the increase of dry matter in the experimental groups 1-3 is obviously lower than that in the experimental groups 4-5 and the control group, the increase of milk production is obviously higher than that in the experimental groups 4-5 and the control group, and the average daily weight gain is obviously higher than that in the experimental groups 4-5 and the control group; the incidence rate of diarrhea is obviously lower than that of the experimental group 4-5 and the control group; the application proves that the feed additive prepared by the method can obviously improve the milk yield of the dairy cow, the daily average weight gain and the dry matter content reduction of the excrement, and can improve the application of the effective substances; meanwhile, the incidence rate of diarrhea is reduced, and the intestinal tract can be effectively improved.
In conclusion, the feed additive produced by the method can obviously improve the intestinal microbial flora of animals, improve the immunity, reduce the use of antibiotic medicines, eliminate the medicine residues, shorten the feeding period and save the feed when being applied to production, thereby improving the culture income.
The above examples merely represent some embodiments of the present invention, which are described in more detail and in more detail, but are not to be construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the appended claims.
Claims (7)
1. The probiotic feed additive is characterized in that the number of viable bacteria per gram of the probiotic feed additive is 2.6 multiplied by 108CFU/g～9.3×108CFU/g probiotics is prepared by fermentation, and the probiotics consists of lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica;
the Lactobacillus plantarum is Lactobacillus plantarum (Lactobacillus plantarum) Y12 with the preservation number of CCTCCNO M2016469; the Candida rugosa is Candida rugosa (Candida rugosa) Y7, and the preservation number is CCTCC M2016468; the Candida ethanolica is Candida ethanolica (Candida ethanolica) Y1 with the preservation number of CCTCC M2016467; are all separated from fresh buffalo milk.
2. A method for preparing the probiotic feed additive of claim 1, characterized in that it comprises the steps of:
(1) respectively inoculating lactococcus garvieae, lactobacillus plantarum, candida rugosa and candida ethanolica on an MRS slant culture medium for culturing at 37 ℃ for 24-48 h to respectively obtain lactococcus garvieae slant culture bacteria, lactobacillus plantarum slant culture bacteria, candida rugosa slant culture bacteria and candida ethanolica slant culture bacteria;
(2) respectively transferring the lactococcus garvieae slant culture bacteria, the lactobacillus plantarum slant culture bacteria, the candida rugosa slant culture bacteria and the candida ethanolica slant culture bacteria in the step (1) into a test tube filled with sterilized MRS broth culture medium to carry out shake bed culture, wherein the culture temperature is 37 ℃, and the culture time is 24 hours, so as to respectively obtain lactococcus garvieae seed liquid, lactobacillus plantarum seed liquid, candida rugosa seed liquid and candida ethanolica seed liquid;
(3) mixing the lactococcus garvieae seed liquid, the lactobacillus plantarum seed liquid, the candida rugosa seed liquid and the candida ethanolica seed liquid in the step (2) according to the proportion of 1: mixing the seed solution and the fermentation broth according to the mass ratio of 2:2:2 to obtain a mixed seed solution, and then inoculating the mixed seed solution and the fermentation broth into a fermentation tank filled with a fermentation substrate according to the inoculation amount of 2:25, wherein the fermentation temperature is 37 ℃, and the fermentation time is 24 hours to obtain an expanded seed bacterial solution;
(4) inoculating the expanded seed bacterial liquid obtained in the step (3) into a fermentation tank filled with a fermentation substrate according to the inoculation amount of the seed bacterial liquid and the fermentation liquid in a mass ratio of 1-2:10, wherein the fermentation temperature is 37 ℃, the fermentation time is 24 hours, the fermentation pH is 3.2-4.8, and cooling to room temperature to obtain mixed strain fermentation liquid;
(5) and (3) centrifuging the mixed strain fermentation liquor obtained in the step (4) by using a centrifuge with the rotation speed of 3000-8000rpm for 15-20min, centrifuging to remove supernatant to obtain semi-solid bacterial sludge, freezing the bacterial sludge in a refrigerator for 4h, drying in a vacuum freeze dryer, taking out, crushing and bagging to obtain the probiotic feed additive.
3. The preparation method according to claim 2, wherein the MRS slant medium of step (1) comprises the following components: 10.0g/L of peptone, 8.0g/L of beef powder, 4.0g/L of yeast powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.2g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 14g/L of agar, tween-801 g/L and the balance of distilled water.
4. The method according to claim 2, wherein the MRS broth of step (2) comprises the following components: 10.0g/L of peptone, 10.0g/L of meat extract, 5.0g/L of yeast extract powder, 20.0g/L of glucose, 2.0g/L of dipotassium phosphate, 2.0g/L of diammonium hydrogen citrate, 5.0g/L of sodium acetate, 0.58g/L of magnesium sulfate, 0.25g/L of manganese sulfate, 0.5g/L of cysteine, 0.8978 g/L of tween-801.0, the balance of distilled water, and the pH value of the mixture is 6.2-6.4.
5. The preparation method according to claim 2, wherein the fermentation substrate in the steps (3) and (4) comprises the following components in percentage by mass: 25 to 41 percent of buffalo milk, 5 to 10 percent of cane sugar, 0.02 to 0.14 percent of yeast extract powder, 5 to 8 percent of agar, 0.07 to 1 percent of calcium carbonate and 40 to 60 percent of distilled water.
6. The method according to claim 2, wherein the viable count of Lactobacillus plantarum in the mixed strain broth of step (4) is 2.6X 108～5.3×108CFU/g, the viable count of the lactococcus garvieae is 3.1 multiplied by 108～3.8×108CFU/g。
7. The probiotic feed additive according to claim 1, wherein the probiotic feed additive is applied to dairy buffalo breeding.
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| CN110063407B (en) * | 2019-04-26 | 2022-01-14 | 闽南师范大学 | Composite probiotic liquid, preparation method thereof and composite probiotic fermented feed prepared from same |
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| CN112094777B (en) * | 2020-09-24 | 2022-05-13 | 青岛普罗百世生物科技有限公司 | Lactobacillus plantarum and application thereof in Laoshan milk goat feed |
| CN113749179A (en) * | 2021-09-10 | 2021-12-07 | 南京工业大学 | Application of curdlan in preparation of feed additive |
| CN115197885B (en) * | 2022-09-13 | 2022-12-09 | 中国农业科学院北京畜牧兽医研究所 | Lactococcus garvieae LGM15, microbial inoculum and application |
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