CN112640987A - Hair loss preventing dog food and preparation method thereof - Google Patents
Hair loss preventing dog food and preparation method thereof Download PDFInfo
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Classifications
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- 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
- A23K10/12—Animal feeding-stuffs obtained by microbiological or biochemical processes by fermentation of natural products, e.g. of vegetable material, animal waste material or biomass
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- 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
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/20—Animal feeding-stuffs from material of animal origin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
- A23K10/37—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms from waste material
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/158—Fatty acids; Fats; Products containing oils or fats
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/163—Sugars; Polysaccharides
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K40/00—Shaping or working-up of animal feeding-stuffs
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- 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
- A23K50/40—Feeding-stuffs specially adapted for particular animals for carnivorous animals, e.g. cats or dogs
- A23K50/42—Dry feed
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- 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
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- 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
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Abstract
The invention discloses a hair loss preventing dog food and a preparation method thereof, and the hair loss preventing dog food comprises a bone biscuit which is prepared by performing compound fermentation on corncobs, mature crude chaetomium globosum sporocarp powder, minced pork, shortening, lactobacillus plantarum of white granulated sugar and water and selenium-rich yeast. The in vivo fungal immunomodulatory protein of Flammulina velutipes is recombined into crude filamentous fungi by genetic engineering means to obtain crude filamentous fungi with high fungal immunomodulatory protein expression. Inoculating the mycelium of the crude-chaetomium globosum containing the fungal immunomodulatory protein gene to corncobs for culturing, and stopping the fermentation of the corncobs when the activities of lignin peroxidase, laccase and xylanase are high and the cellulase is low so as to keep the content of the corncob cellulose. Fermenting pork paste by using lactobacillus plantarum, and adding the crude filamentous fungi powder. During secondary fermentation, crude chaetomium fortunei powder and lactobacillus plantarum fermented pork are added, the pork and selenium-enriched yeast are fermented together, and then corncobs are wrapped to prepare the dog food which can be eaten by dogs and has high immunity.
Description
Technical Field
The invention relates to the technical field of food, in particular to a hair loss preventing dog food and a preparation method thereof.
Background
Immune globulin (representing an important index of humoral immunity function of body, playing a role of immunoregulation in vivo, developed products have been used for treating immune-related diseases, and has a ' Y ' -shaped structure composed of 2 heavy chains and 2 light chains, and the top variable region is the main active region, Fungal Immunomodulatory Protein (FIP) is a small molecular protein which is separated from fungi, has a structure extremely similar to that of the heavy chain variable region and has immunoregulation function, 7 of which are currently found, wherein the immunomodulatory protein derived from Jinxue has immunoregulation function through research, has biological activities of activating lymphocytes to produce cytokines, preventing and treating asthma, rhinitis, eczema and the like, and inhibiting cancer cell growth, but the natural yield extracted from swallow's sporophore is very low (the highest yield is less than 88mg/Kg) at present, which proves that the expression level of the immune globulin in the body is low, therefore, the protein is produced by variety improvement or gene recombination technology and has important significance.
Dog hair loss generally has two reasons, and skin diseases or low autoimmunity are caused. The immunity of the dog can be improved, and the dog can be prevented from non-natural depilation. In addition, dog food which can be eaten by dogs really is limited, and the cost is high.
Therefore, the anti-hair-loss dog food with low design cost and containing the immunoglobulin and the preparation method thereof are necessary.
Disclosure of Invention
The invention aims to provide hair loss preventing dog food and a preparation method thereof, and aims to solve the problems in the background art.
In order to solve the technical problems, the invention provides the following technical scheme: a hair loss preventing dog food comprises corncob, mature crude chaetomium globosum sporocarp powder, minced pork, shortening, lactobacillus plantarum of white granulated sugar and water and selenium-rich yeast, and is a bone-type biscuit through compound fermentation.
According to the technical scheme, the corncobs are the corncobs which are fermented and degraded for 7-10 days by the crude fiber pore fungi.
According to the technical scheme, the mature crude chaetomium fortune sporophore powder is a genetic engineering mature crude chaetomium fortune sporophore powder containing flammulina velutipes fungus immunomodulatory protein.
A preparation method of the hair loss preventing dog food comprises the following specific steps:
the method comprises the following steps: mixing low gluten flour with appropriate amount of water and selenium-rich yeast, stirring to obtain dough, standing in a fermenting box at 28-32 deg.C and humidity of 60% for fermenting for 3-4 hr to obtain primary fermented dough; the selenium-enriched yeast can improve good protein selenium for dogs and improve the free radical resistance;
step two: taking minced pork, adding mature crude filamentous fungi sporophore powder, and carrying out anaerobic stirring fermentation for 2-4h by using lactobacillus plantarum at 30 ℃ to obtain lactic acid fermented minced pork; the crude filamentous fungi powder is rich in fungal polysaccharide, can promote the growth of lactobacillus plantarum, save fermentation time, improve the taste and quality of pork and is beneficial to dog digestion;
step three: adding the lactic acid fermentation minced pork, shortening, white granulated sugar and mature crude chaetomium fortune sporophore powder obtained in the step two into the primary fermentation dough obtained in the step one, uniformly stirring, standing in a fermentation box with the temperature of 35 ℃ and the humidity of 70%, and fermenting for 3-6 hours to obtain biscuit molding dough; the lactobacillus plantarum can obviously improve the content of reducing sugar in dough and inhibit the generation of acrylamide;
step four: pressing the fermented dough to 2mm thickness with a tabletting machine, wrapping the corn cob, making into bone shape, baking at 180 deg.C and 160 deg.C for 15min, and cooling to obtain dog food biscuit.
According to the technical scheme, the mass ratio of the materials in the third step is as follows: 3-5 parts of lactic acid fermentation minced pork, 0.5-1.5 parts of shortening, 0.1-0.5 part of white granulated sugar, 0.2-0.8 part of mature crude capillary fungus sporophore powder and 10-25 parts of primary fermentation dough.
According to the technical scheme, the preparation of the mature crude chaetomium fortune sporophore powder comprises the following steps: obtaining a Fungus Immunomodulatory Protein (FIP) cDNA sequence from needle mushroom bodies by an RT-PCR method, and adding a His tag at the C end of FIP to facilitate the purification of protein; the double restriction enzymes EcoR I and BamHI are used for enzyme digestion, T4DNA ligase is used for connection, a plant binary expression vector p139035S-FIP is constructed, and the recombinant plasmid is transferred into Agrobacterium tumefaciens EHA 05.
According to the technical scheme, the agrobacterium tumefaciens EHA05 is detected by gel electrophoresis, and the size of a band is found to be consistent with that of a target gene, so that the recombinant plasmid is transferred into the agrobacterium tumefaciens EHA 05; taking crude filamentous fungi mycelium pellets as a receptor material, controlling the bacterial liquid concentration OD600 of Agrobacterium tumefaciens EHA05 to be 0.5, controlling the infection time to be 20-40min, and co-culturing for 72h on a co-culture medium with 200 mu mol/L of phthalein syringone.
According to the technical scheme, the result of PCR and Southern hybridization of the genetic engineering crude capillary indicates that the immunoglobulin gene is integrated into the genome of the crude capillary, and the existence of the gene is still detected after subculture for five times on a PDA solid culture medium without selective pressure, which indicates that the gene can be stably inherited in the transgenic crude capillary, and the crude capillary mycelium with high fungal immunomodulatory protein expression is obtained.
According to the technical scheme, the genetic engineering chaetomium globosum is subjected to amplification culture: 3-5 parts of sawdust, 2-3 parts of rice hull, 10-15 parts of corn flour, 0.5-1 part of xylose, 0.8-2 parts of yeast extract powder, 0.5-0.8 part of lime and 0.5-1.2 parts of gypsum in mass ratio, and 40-70 parts of water are mixed, stirred uniformly, wrapped outside the corn cob, cultured for 9-12 days, transferred to 60 parts of sawdust and cultured for 15-20 days continuously, and the sporocarp is cultured.
According to the technical scheme, the corncobs are fermented and cut into 10-15cm long, and are pasteurized for later use; after the mature genetic engineering crude capillary fungus sporocarp is cleaned, dried at low temperature and ground into powder for standby.
Culturing the crude Firmiana sinensis on corncob meal, detecting the activities of lignin peroxidase and lysine-manganese peroxidase after 1 day, wherein the activity of the lignin peroxidase reaches the highest value on the 5 th day, and the activity of the lysine-manganese peroxidase reaches the maximum value on the 6 th day. The laccase enzyme activity is relatively low, and reaches the maximum value at day 7. From this, it is known that lignin peroxidase and lysine-manganese peroxidase exhibit high activities in the first 7 days, and are extracellular enzymes produced by c. Along with the growth of the crude filamentous fungi, the xylanase activity gradually increases and reaches a maximum value on day 10. The enzyme activity of the cellulase is extremely low at the initial stage of culture, and reaches the maximum value when the cellulase is cultured to the 16 th day. So the corn cob which grows for 9-12 days is selected.
Compared with the prior art, the invention has the following beneficial effects: in the invention, the raw materials are mixed,
(1) a fungus immune regulatory protein (FIP) cDNA sequence is obtained from needle mushroom bodies by an RT-PCR method, a plant binary expression vector p139035S-FIP is constructed by utilizing restriction enzymes EcoR I and BamHI enzyme to carry out double enzyme digestion and T4DNA ligase connection, and the recombinant plasmid is transferred into agrobacterium tumefaciens EHA 05. The crude filamentous fungi mycelium pellet is used as a receptor material, and is transferred into a fungal immunomodulatory protein gene by an agrobacterium tumefaciens mediated method to obtain the crude filamentous fungi mycelium with high fungal immunomodulatory protein expression, and the highest expression level in the pileus is found by culture tests. The flammulina velutipes immunomodulatory protein is one of fungal immunomodulatory proteins, has the effects of promoting the activity of lymphocytes and inducing the generation of interleukin-2 and gamma-interferon, can improve the autoimmunity of dogs and avoid hair loss caused by insufficient autoimmunity;
(2) the mycelium of the crude chaetomium globosum containing the fungal immunomodulatory protein gene is inoculated to corncobs for culture, lignin peroxidase, lysine-manganese peroxidase, laccase, cellulase and xylanase are secreted in the growth process of the crude chaetomium globosum, lignin in the corncobs can be degraded, and part of cellulose and hemicellulose can be degraded at the same time. According to the action time difference of different enzymes, the corncob fermentation is stopped when the activities of lignin peroxidase, laccase and xylanase are high and the cellulase is low so as to keep the content of corncob cellulose, the hardness is favorable for dog to eat, the corncob dog with extremely low contents of lignin and hemicellulose can eat, and the cellulose can promote dog digestion; the corncob is rich in cellulose, starch and various amino acids, and can improve the protein content of the crude linter fungi;
(3) cleaning mature crude Inonotus obliquus fruiting body, lyophilizing, and grinding into powder. The lactobacillus plantarum is used for fermenting pork, the crude-pore-fungus powder is added, and fungus polysaccharide contained in the crude-pore-fungus powder can promote the growth of lactobacillus plantarum, so that the fermentation time is saved, the taste and quality of the pork are improved, and dog digestion is facilitated. The selenium-enriched yeast is used for fermenting dough, and crude filamentous fungi powder and pork fermented by lactobacillus plantarum are added during secondary fermentation and fermented together with the selenium-enriched yeast. The lactobacillus plantarum can obviously improve the content of reducing sugar in the dough and inhibit the generation of acrylamide. Sterilizing the fermented corncobs, wrapping the compound fermented dough outside the corncobs, making the corncobs into bone shapes, baking the corncobs at high temperature, and making the dog food which can be gnawed by dogs and has high immunity.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The invention provides the technical scheme that: a hair loss preventing dog food comprises corncob, mature crude chaetomium globosum sporocarp powder, minced pork, shortening, lactobacillus plantarum of white granulated sugar and water and selenium-rich yeast, and is a bone-type biscuit through compound fermentation.
According to the technical scheme, the corncob is degraded by fermentation of the crude fiber pore fungus for 7-10 days.
According to the technical scheme, the mature crude chaetomium globosum sporophore powder is a genetic engineering mature crude chaetomium globosum sporophore powder containing flammulina velutipes fungal immunomodulatory protein.
A preparation method of the hair loss preventing dog food comprises the following specific steps:
the method comprises the following steps: mixing low gluten flour with appropriate amount of water and selenium-rich yeast, stirring to obtain dough, standing in a fermenting box at 28-32 deg.C and humidity of 60% for fermenting for 3-4 hr to obtain primary fermented dough;
step two: taking minced pork, adding mature crude filamentous fungi sporophore powder, and carrying out anaerobic stirring fermentation for 2-4h by using lactobacillus plantarum at 30 ℃ to obtain lactic acid fermented minced pork;
step three: adding the lactic acid fermentation minced pork, shortening, white granulated sugar and mature crude chaetomium fortune sporophore powder obtained in the step two into the primary fermentation dough obtained in the step one, uniformly stirring, standing in a fermentation box with the temperature of 35 ℃ and the humidity of 70%, and fermenting for 3-6 hours to obtain biscuit molding dough;
step four: pressing the fermented dough to 2mm thickness with a tabletting machine, wrapping the corn cob, making into bone shape, baking at 180 deg.C and 160 deg.C for 15min, and cooling to obtain dog food biscuit.
According to the technical scheme, the three steps comprise 3-5 parts of lactic acid fermentation minced pork, 0.5-1.5 parts of shortening, 0.1-0.5 part of white granulated sugar, 0.2-0.8 part of mature crude chaetomium globosum sporocarp powder and 10-25 parts of primary fermentation dough.
According to the technical scheme, the mature crude Inonotus pileus sporophore powder is prepared by obtaining a Fungus Immunomodulatory Protein (FIP) cDNA sequence from needle mushroom bodies through an RT-PCR method, and a His tag is added to the C end of FIP, so that protein purification is facilitated; the double restriction enzymes EcoR I and BamHI are used for enzyme digestion, T4DNA ligase is used for connection, a plant binary expression vector p139035S-FIP is constructed, and the recombinant plasmid is transferred into Agrobacterium tumefaciens EHA 05.
According to the technical scheme, the size of a band is consistent with that of a target gene by gel electrophoresis detection of the agrobacterium tumefaciens EHA05, which indicates that the recombinant plasmid is transferred into the agrobacterium tumefaciens EHA 05; taking crude filamentous fungi mycelium pellets as a receptor material, controlling the bacterial liquid concentration OD600 of Agrobacterium tumefaciens EHA05 to be 0.5, controlling the infection time to be 20-40min, and co-culturing for 72h on a co-culture medium with 200 mu mol/L of phthalein syringone.
According to the technical scheme, PCR and Southern hybridization results of the genetically engineered crude capillary fungus show that immunoglobulin genes are integrated into the genome of the crude capillary fungus, and the existence of the genes is still detected after subculture for five times on a PDA solid culture medium without selective pressure, which shows that the genes can be stably inherited in transgenic crude capillary fungus, and crude capillary fungus mycelium with high fungal immunomodulatory protein expression is obtained.
According to the technical scheme, the genetic engineering chaetomium globosum is subjected to enlarged culture, the mass ratio of a culture medium to wood chips is 3-5 parts, the mass ratio of rice hulls is 2-3 parts, the mass ratio of corn flour is 10-15 parts, the mass ratio of xylose is 0.5-1 part, the mass ratio of yeast extract powder is 0.8-2 parts, the mass ratio of lime is 0.5-0.8 part, the mass ratio of gypsum is 0.5-1.2 parts, the mixture is uniformly mixed and stirred with 40-70 parts of water, the mixture is wrapped outside a corn cob and cultured for 9-12 days, the mixture is transferred to 60 parts of wood chips and continuously cultured for 15-20 days.
According to the technical scheme, the corncobs are fermented and made into corncobs with the length of 10-15cm, and are pasteurized for later use; after the mature genetic engineering crude capillary fungus sporocarp is cleaned, dried at low temperature and ground into powder for standby.
Example 1
A hair loss preventing dog food comprises corncob, mature crude chaetomium globosum sporocarp powder, minced pork, shortening, lactobacillus plantarum of white granulated sugar and water and selenium-rich yeast, and is a bone-type biscuit through compound fermentation.
According to the technical scheme, the corncobs are fermented and degraded by the crude fiber pore fungi for 8 days.
According to the technical scheme, the mature crude chaetomium globosum sporophore powder is a genetic engineering mature crude chaetomium globosum sporophore powder containing flammulina velutipes fungal immunomodulatory protein.
A preparation method of the hair loss preventing dog food comprises the following specific steps:
the method comprises the following steps: adding proper amount of water and selenium-enriched yeast into low-gluten flour, stirring to obtain dough, standing in a proofing box with temperature of 32 ℃ and humidity of 60%, and fermenting for 3h to obtain primary fermented dough;
step two: taking minced pork, adding mature crude filamentous fungi sporophore powder, and carrying out anaerobic stirring fermentation for 3h by using lactobacillus plantarum at 30 ℃ to obtain lactic acid fermented minced pork;
step three: adding the lactic acid fermentation minced pork, shortening, white granulated sugar and mature crude chaetomium fortune sporophore powder obtained in the step two into the primary fermentation dough obtained in the step one, uniformly stirring, standing in a fermentation box with the temperature of 35 ℃ and the humidity of 70%, and fermenting for 4 hours to obtain biscuit molding dough;
step four: pressing the fermented dough to 2mm thickness with a tabletting machine, wrapping the corn cob, making into bone shape, baking at 180 deg.C and 160 deg.C for 15min, and cooling to obtain dog food biscuit.
According to the technical scheme, the three steps comprise 5 parts of lactic acid fermentation minced pork, 1.2 parts of shortening, 0.2 part of white granulated sugar, 0.5 part of mature crude capillary fungus sporophore powder and 25 parts of primary fermentation dough.
According to the technical scheme, the mature crude Inonotus pileus sporophore powder is prepared by obtaining a Fungus Immunomodulatory Protein (FIP) cDNA sequence from needle mushroom bodies through an RT-PCR method, and a His tag is added to the C end of FIP, so that protein purification is facilitated; the double restriction enzymes EcoR I and BamHI are used for enzyme digestion, T4DNA ligase is used for connection, a plant binary expression vector p139035S-FIP is constructed, and the recombinant plasmid is transferred into Agrobacterium tumefaciens EHA 05.
According to the technical scheme, the size of a band is consistent with that of a target gene by gel electrophoresis detection of the agrobacterium tumefaciens EHA05, which indicates that the recombinant plasmid is transferred into the agrobacterium tumefaciens EHA 05; taking crude filamentous fungi mycelium pellets as a receptor material, controlling the bacterial liquid concentration OD600 of Agrobacterium tumefaciens EHA05 to be 0.5, infecting for 40min, and co-culturing for 72h on a co-culture medium with phthalein syringone of 200 mu mol/L.
According to the technical scheme, PCR and Southern hybridization results of the genetically engineered crude capillary fungus show that immunoglobulin genes are integrated into the genome of the crude capillary fungus, and the existence of the genes is still detected after subculture for five times on a PDA solid culture medium without selective pressure, which shows that the genes can be stably inherited in transgenic crude capillary fungus, and crude capillary fungus mycelium with high fungal immunomodulatory protein expression is obtained.
According to the technical scheme, the genetic engineering chaetomium globosum is subjected to enlarged culture, the mass ratio of a culture medium to wood chips is 5 parts, the mass ratio of rice hulls is 3 parts, the mass ratio of corn flour is 15 parts, the mass ratio of xylose is 1 part, the mass ratio of yeast extract powder is 1.2 parts, the mass ratio of lime is 0.6 part, the mass ratio of gypsum is 0.8 part, the mixture is uniformly mixed and stirred with 60 parts of water, the mixture is wrapped outside a corn cob and cultured for 10 days, the mixture is transferred to 60 parts of wood chips to be continuously cultured for 18.
According to the technical scheme, the corncobs are fermented and made into corncobs with the length of 15cm, and are pasteurized for later use; after the mature genetic engineering crude capillary fungus sporocarp is cleaned, dried at low temperature and ground into powder for standby.
Example 2
A hair loss preventing dog food comprises corncob, mature crude chaetomium globosum sporocarp powder, minced pork, shortening, lactobacillus plantarum of white granulated sugar and water and selenium-rich yeast, and is a bone-type biscuit through compound fermentation.
According to the technical scheme, the corncobs are the corncobs which are fermented and degraded for 9 days by the crude fiber pore fungi.
According to the technical scheme, the mature crude chaetomium globosum sporophore powder is a genetic engineering mature crude chaetomium globosum sporophore powder containing flammulina velutipes fungal immunomodulatory protein.
A preparation method of the hair loss preventing dog food comprises the following specific steps:
the method comprises the following steps: adding proper amount of water and selenium-enriched yeast into low-gluten flour, stirring to obtain dough, standing in a proofing box with 30 deg.C and humidity of 60%, and fermenting for 4 hr to obtain primary fermented dough;
step two: taking minced pork, adding mature crude filamentous fungi sporophore powder, and carrying out anaerobic stirring fermentation for 3h by using lactobacillus plantarum at 30 ℃ to obtain lactic acid fermented minced pork;
step three: adding the lactic acid fermentation minced pork, shortening, white granulated sugar and mature crude chaetomium fortune sporophore powder obtained in the step two into the primary fermentation dough obtained in the step one, uniformly stirring, standing in a fermentation box with the temperature of 35 ℃ and the humidity of 70%, and fermenting for 5 hours to obtain biscuit molding dough;
step four: pressing the fermented dough to 2mm thickness with a tabletting machine, wrapping the corn cob, making into bone shape, baking at 180 deg.C and 160 deg.C for 15min, and cooling to obtain dog food biscuit.
According to the technical scheme, the three steps comprise 3 parts of lactic acid fermentation minced pork, 0.55 part of shortening, 0.1 part of white granulated sugar, 0.5 part of mature crude capillary fungus sporophore powder and 20 parts of one-time fermentation dough.
According to the technical scheme, the mature crude Inonotus pileus sporophore powder is prepared by obtaining a Fungus Immunomodulatory Protein (FIP) cDNA sequence from needle mushroom bodies through an RT-PCR method, and a His tag is added to the C end of FIP, so that protein purification is facilitated; the double restriction enzymes EcoR I and BamHI are used for enzyme digestion, T4DNA ligase is used for connection, a plant binary expression vector p139035S-FIP is constructed, and the recombinant plasmid is transferred into Agrobacterium tumefaciens EHA 05.
According to the technical scheme, the size of a band is consistent with that of a target gene by gel electrophoresis detection of the agrobacterium tumefaciens EHA05, which indicates that the recombinant plasmid is transferred into the agrobacterium tumefaciens EHA 05; taking crude filamentous fungi mycelium pellets as a receptor material, controlling the bacterial liquid concentration OD600 of Agrobacterium tumefaciens EHA05 to be 0.5, infecting for 30min, and co-culturing for 72h on a co-culture medium with phthalein syringone of 200 mu mol/L.
According to the technical scheme, PCR and Southern hybridization results of the genetically engineered crude capillary fungus show that immunoglobulin genes are integrated into the genome of the crude capillary fungus, and the existence of the genes is still detected after subculture for five times on a PDA solid culture medium without selective pressure, which shows that the genes can be stably inherited in transgenic crude capillary fungus, and crude capillary fungus mycelium with high fungal immunomodulatory protein expression is obtained.
According to the technical scheme, the genetic engineering chaetomium globosum is subjected to enlarged culture, the mass ratio of a culture medium to wood chips is 3 parts, the mass ratio of rice hulls is 2 parts, the mass ratio of corn flour is 10 parts, the mass ratio of xylose is 0.5 part, the mass ratio of yeast extract powder is 0.8 part, the mass ratio of lime is 0.5 part, the mass ratio of gypsum is 0.5 part, the mixture is uniformly mixed and stirred with 40 parts of water, the mixture is wrapped outside a corn cob, the mixture is cultured for 11 days, the mixture is transferred to 60 parts of wood chips to be continuously.
According to the technical scheme, the corncobs are fermented and made into corncobs with the length of 12cm, and are pasteurized for later use; after the mature genetic engineering crude capillary fungus sporocarp is cleaned, dried at low temperature and ground into powder for standby.
In order to verify the actual effect of the product, the following tests are specially designed:
test 1 immunoglobulin content test
Taking 1g of crude Inonotus fuliginosus powder, carrying out ultrasonic crushing, carrying out pure crude extraction by 90% ethanol, centrifuging and collecting supernate.
Purification using a Ni column: firstly, using a Binding Buffer balance column, slowly flowing the collection liquid through a Ni column, using the Binding Buffer balance column again, finally using an Elution Buffer to elute the immunoglobulin for collection, and desalting the collected immunoglobulin by using an ultrafiltration tube to obtain the purified target protein. The calculation shows that the content of the crude capillary fungus immunoglobulin is 206mg/Kg, which is obviously higher than the content of 88mg/Kg of the immunoglobulin in the needle mushroom.
Experiment 2 evaluation of the influence of composite fermented dog food on digestibility
The apparent digestibility of dog food was determined by the total manure method and analyzed by Duncan, the results of which are shown in table 1.
| Group of | N | Digestibility (100%) |
| Composite fermented dog food | 10 | 86.16±0.53 |
| Control common fermented dog food | 10 | 78.35±0.62 |
TABLE 1 Duncan comparison of apparent digestibility of different groups of dog foods
According to the detection result, the apparent digestibility of the composite fermented dog food is 86.16 +/-0.53, which is obviously higher than that of a control group, and the digestibility is higher, and is improved compared with the digestibility of common fermented dog food.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. The hair loss preventing dog food is characterized in that: the bone biscuit comprises corncobs, mature crude chaetomium globosum sporocarp powder, minced pork, shortening, lactobacillus plantarum of white granulated sugar and water and selenium-enriched yeast which are subjected to compound fermentation together.
2. The hair loss prevention dog food as claimed in claim 1, wherein the hair loss prevention dog food comprises: the corncob is the corncob degraded by the fermentation of the crude fiber pore fungus for 7-10 days.
3. The hair loss prevention dog food as claimed in claim 1, wherein the hair loss prevention dog food comprises: the mature crude chaetomium globosum sporophore powder is gene engineering mature crude chaetomium globosum sporophore powder containing flammulina velutipes fungal immunomodulatory protein.
4. A preparation method of the hair loss preventing dog food comprises the following specific steps:
the method comprises the following steps: mixing low gluten flour with appropriate amount of water and selenium-rich yeast, stirring to obtain dough, standing in a fermenting box at 28-32 deg.C and humidity of 60% for fermenting for 3-4 hr to obtain primary fermented dough;
step two: taking minced pork, adding mature crude filamentous fungi sporophore powder, and carrying out anaerobic stirring fermentation for 2-4h by using lactobacillus plantarum at 30 ℃ to obtain lactic acid fermented minced pork;
step three: adding the lactic acid fermentation minced pork, shortening, white granulated sugar and mature crude chaetomium fortune sporophore powder obtained in the step two into the primary fermentation dough obtained in the step one, uniformly stirring, standing in a fermentation box with the temperature of 35 ℃ and the humidity of 70%, and fermenting for 3-6 hours to obtain biscuit molding dough;
step four: pressing the fermented dough to 2mm thickness with a tabletting machine, wrapping the corn cob, making into bone shape, baking at 180 deg.C and 160 deg.C for 15min, and cooling to obtain dog food biscuit.
5. The preparation method of the hair loss preventing dog food according to claim 4, characterized by comprising the following steps: the mass ratio of the materials in the third step is as follows: 3-5 parts of lactic acid fermentation minced pork, 0.5-1.5 parts of shortening, 0.1-0.5 part of white granulated sugar, 0.2-0.8 part of mature crude capillary fungus sporophore powder and 10-25 parts of primary fermentation dough.
6. The preparation method of the hair loss preventing dog food according to claim 4, characterized by comprising the following steps: the preparation of the mature crude chaetomium sporophore powder comprises the following steps: obtaining a Fungus Immunomodulatory Protein (FIP) cDNA sequence from needle mushroom bodies by an RT-PCR method, and adding a His tag at the C end of FIP to facilitate the purification of protein; the double restriction enzymes EcoR I and BamHI are used for enzyme digestion, T4DNA ligase is used for connection, a plant binary expression vector p139035S-FIP is constructed, and the recombinant plasmid is transferred into Agrobacterium tumefaciens EHA 05.
7. The preparation method of the hair loss preventing dog food according to claim 6, characterized by comprising the following steps: the agrobacterium tumefaciens EHA05 is detected by gel electrophoresis, and the size of a band is found to be consistent with that of a target gene, which indicates that the recombinant plasmid is transferred into the agrobacterium tumefaciens EHA 05; taking crude filamentous fungi mycelium pellets as a receptor material, controlling the bacterial liquid concentration OD600 of Agrobacterium tumefaciens EHA05 to be 0.5, controlling the infection time to be 20-40min, and co-culturing for 72h on a co-culture medium with 200 mu mol/L of phthalein syringone.
8. The preparation method of the hair loss preventing dog food according to claim 7, characterized by comprising the following steps: the result of PCR and Southern hybridization of the genetic engineering crude capillary indicates that immunoglobulin genes are integrated into the genome of the crude capillary, and the existence of the genes is still detected after subculture for five times on a PDA solid culture medium without selective pressure, which indicates that the genes can be stably inherited in transgenic crude capillary, and the crude capillary mycelium with high fungal immunomodulatory protein expression is obtained.
9. The preparation method of the hair loss preventing dog food according to claim 8, characterized by comprising the following steps: the genetic engineering crude capillary fungus is subjected to amplification culture: 3-5 parts of sawdust, 2-3 parts of rice hull, 10-15 parts of corn flour, 0.5-1 part of xylose, 0.8-2 parts of yeast extract powder, 0.5-0.8 part of lime and 0.5-1.2 parts of gypsum in mass ratio, and 40-70 parts of water are mixed, stirred uniformly, wrapped outside the corn cob, cultured for 9-12 days, transferred to 60 parts of sawdust and cultured for 15-20 days continuously, and the sporocarp is cultured.
10. The preparation method of the hair loss preventing dog food according to claim 9, characterized by comprising the following steps: the corncobs are fermented and cut into 10-15cm long, and are pasteurized for later use; after the mature genetic engineering crude capillary fungus sporocarp is cleaned, dried at low temperature and ground into powder for standby.
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