CN109757610B - Special feed additive for brown croakers - Google Patents
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- CN109757610B CN109757610B CN201811374998.6A CN201811374998A CN109757610B CN 109757610 B CN109757610 B CN 109757610B CN 201811374998 A CN201811374998 A CN 201811374998A CN 109757610 B CN109757610 B CN 109757610B
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Abstract
The invention provides a special feed compound additive for miichthys miiuy, belonging to the technical field of miichthys miiuy breeding and comprising (I) an oligosaccharide composition; and/or (II) a carboxylic acid ester; and (III) a protease complex; the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum. The invention also provides a special feed for brown croaker, which contains the basic feed and the special feed additive for brown croaker. The beneficial effects are that: the special feed additive for brown croakers is helpful for improving intestinal flora of brown croakers, promoting intestinal villus regeneration, improving intestinal absorption efficiency, maintaining gastrointestinal health and/or reducing the demand on antibiotics, reducing abuse of antibiotics in the breeding process and obviously reducing morbidity and mortality of brown croakers.
Description
Technical Field
The invention belongs to the technical field of miichthys miiuy breeding, and particularly relates to a special feed compound additive for miichthys miiuy.
Background
Miichthys miiuy is in family of Pteriformes, family of Shishoyu, and is in body color of silver gray. Most produced in the eastern sea. Miichthys miiuy generally has a length of 45-55 cm per tail body and a weight of 500-1000 g, and the maximum amount is hundreds of jin. Distributed in Bohai sea, yellow sea, east sea, south sea and other water areas. The body of the Miichthys miiuy is prolonged and flat on the side, the back and the abdomen are shallow and arc-shaped, the large individual can reach more than 10 kilograms, the mouth is large and slightly inclined, the body is hackled scales, the scales are small, the surface layer is rough, the head is round, the jaw holes are 4, the central jaw hole and the inner jaw hole are arranged in a square shape, and no jaw whisker exists. The upper jaw external teeth and the lower jaw internal teeth are enlarged and are in a canine shape, the inner side small teeth form a banded group, the eye ring is large, and the eye mask has high transparency and is red and bright. The body is blue-grey-brown, the abdomen is grey-white, 2 dorsal fins are connected together, and the middle is deeply carved. The upper edge of the fin spine is black, and the center of the fin ray part is provided with a longitudinal black stripe; yellow at the base of pectoral fin and black at the edge; hip fin 2 spine; the tail fin is wedge-shaped, the base of the tail fin is yellow, and the edge of the tail fin is slightly lighter in color.
Miichthys miiuy is sweet, salty and mild in nature, has the effects of nourishing blood, stopping bleeding, tonifying kidney, controlling nocturnal emission and diminishing inflammation, and is effective in treating aplastic anemia, hematemesis, kidney deficiency, spermatorrhea, pain and swelling, nameless sores, mastitis and the like. The brown croaker has delicious meat taste, is one of important marine economic fishes and is also a fish with higher economic value, thereby being deeply loved by consumers and breeders. At present, because the immunity and the anti-stress capability of the croakers cultured by the method for artificially culturing the croakers at high density are relatively reduced, pathological changes are easy to occur, the death of large pieces of croakers is caused, and the economy of the breeding industry is seriously influenced. In the aquaculture industry, antibiotics are often used for preventing and treating fish diseases in order to improve the culture yield and benefits, and some antibiotics are also used for promoting the growth of fishes and improving the utilization rate of fish feed. However, the abuse of antibiotics brings deep hidden dangers for the healthy growth of aquatic animals and the edible safety of human beings, and the abuse of antibiotics cannot be ignored at present.
Disclosure of Invention
The invention aims to provide the special feed additive for brown croakers, which is helpful for improving intestinal flora of brown croakers, promoting intestinal villus regeneration, improving intestinal absorption efficiency, maintaining gastrointestinal health and/or reducing the requirement on antibiotics, reducing abuse of antibiotics in the breeding process and obviously reducing morbidity and mortality of brown croakers.
The technical scheme adopted by the invention for realizing the purpose is as follows:
the invention provides a special feed compound additive for miichthys miiuy, which comprises the following components:
(I) an oligosaccharide composition; and/or
(II) a carboxylic acid ester; and
(III) a protease complex; the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum. The special feed additive for brown croakers is beneficial to improving intestinal microbial flora of brown croakers, promoting regeneration of intestinal villus of brown croakers, improving intestinal absorption of target nutrient substances, maintaining gastrointestinal health and/or reducing the requirement on antibiotics, reducing abuse of the antibiotics in the breeding process, also being capable of remarkably reducing the feed-meat ratio in breeding of brown croakers and shortening the breeding period.
The oligosaccharide composition (I) has the following distribution of glycosidic bonds: having not less than 8 mol% alpha-1, 6 glycosidic linkages, and not less than 8 mol% beta-2, 1 glycosidic linkages, and/or not less than 8 mol% beta-1, 3 glycosidic linkages, and wherein at least 8% by dry weight of the oligosaccharide composition has a degree of polymerization of 2-6.
The oligosaccharide composition (I) comprises: isomaltose-oligosaccharide, isomaltose-inulin disaccharide-oligosaccharide, inulin ditacharide-oligosaccharide, chondroitin ditacharide-oligosaccharide, hyaluronic ditacharide-isomaltose-oligosaccharide, chondroitin ditacharide-isomaltose-oligosaccharide, lactose-hyaluronic ditacharide-oligosaccharide, isomaltose-lactose-oligosaccharide, lactose-inulin disaccharide-oligosaccharide, inulin disaccharide-isomaltose-oligosaccharide. The oligosaccharide composition applied to the feed additive or feed special for the miichthys miiuy has beneficial health effect, is beneficial to reducing fish mortality, improving intestinal microbial flora, improving nutrient absorption, maintaining gastrointestinal health and/or reducing the demand on antibiotics, and reduces abuse of antibiotics in the culture process.
The (II) carboxylic acid ester is selected from:
a lactyl-carboxylic acid ester according to formula 1,
formula 1: r1-COO-[-CH(CH3)-COO]n-R2
Or/and Ba, Be, Mn (II), Ca, Na, NH4Or a Co (II) salt of (I),
the glycolyl carboxylate according to formula 2,
formula 2: r is1-COO-[-CH2-COO]n-R2
Or/and Ba, Be, Mn (II), Ca, Na, NH4Or a Co (II) salt,
in the above formula, R1Selected from chlorophenyl or bromophenyl, R2Is selected from C2～C10An alkyl or alkenyl chain, and n represents an integer having a value of 1 to 10; the carboxylate product is applied to special feed or feed additive for brown croakers, can obviously enhance antibacterial activity of brown croakers, has beneficial effects on preventing and treating intestinal infection of brown croakers, is beneficial to improving growth rate of brown croakers, reducing feed-meat ratio and breeding cycle, and increasing economic benefit.
The (III) protease complex is a protease complex obtained by culturing Dryophora lanuginosa and comprising an effective protease mixture in a proportion of more than 75% of the activity of the protease complex, wherein one protease of the effective protease mixture has an isoelectric point of about 7.4 and the other protease has an isoelectric point of about 8.3; the protease complex is helpful for promoting the regeneration of intestinal villi of fishes such as miichthys miiuy and greatly improving the intestinal absorption of target nutrient substances, thereby remarkably reducing the feed-meat ratio of miichthys miiuy during breeding and shortening the breeding period, and in addition, the protease complex is helpful for strengthening the immune system of the miichthys miiuy and can obviously reduce the morbidity, especially the mortality of the miichthys miiuy caused by bacterial pollution.
Preferably, the protease having an isoelectric point of about 7.4 in the protease complex (III) has a specific activity of 82000 to 90000 Anson units/mg protein.
Preferably, the protease having an isoelectric point of about 8.3 in the protease complex (III) has a specific activity of 20000 to 25000 Anson units/mg protein.
Preferably, the (III) protease complex is used in the special feed for Miichthys miiuy as a supplement of a feed additive, and the feed additive is in a form of powder, liquid or any other form suitable for being mixed with a feed composition.
Preferably, in order to obtain the (III) protease complex, the strain of Trichosporon lanuginosus is cultured and the fermentation broth is filtered, then the extraction of the protease complex by ultrafiltration and isoelectric focusing is performed, and finally the obtained protease complex is freeze-dried and pulverized, specifically comprising:
fermentation: selecting a single colony with a transparent ring from a primary screening culture medium plate (the protease produced by the strain can decompose the substrate protein in the culture medium, so that the transparent ring phenomenon appears around the colony), and streaking and separating; transferring the purified strain to a fermentation shake flask containing a fermentation enzyme production culture medium, ventilating in sterile air with the volume of 0.3/volume of the culture medium/minute, fermenting at constant temperature for 4-6 days under the conditions that the pH of the fermentation enzyme production culture medium is 7.2-7.5, the temperature is 40-45 ℃ and the rotating speed is 150-200 r/min, and centrifuging to obtain a supernatant;
and (3) ultrafiltration: ultrafiltration is carried out by means of a membrane having a cut-off threshold of 50kDa, the ultrafiltration being carried out for 3-10 hours;
isoelectric focusing: controlling the pH value of the solution to be 7.4 +/-0.1 and 8.3 +/-0.1 respectively to perform isoelectric focusing, namely obtaining most of protease with the isoelectric point (pI) of about 7.4 and less protease with the isoelectric point (pI) of about 8.3; freeze drying and crushing to obtain the protease compound.
Further preferably, the enzyme production medium for fermentation comprises the following components in parts by weight: corn cob xylan of 180-240 meshes 0.18-0.2%, threitol 0.012-0.014%, tryptone 1.8-3%, yeast extract powder 0.3-0.5%, sodium nitrate 0.1-0.2%, KH2PO40.5～0.6％、K2HPO40.15～0.2％、MgSO40.03-0.05% and natural pH; the carbon source with special particle size in the fermentation enzyme production culture medium is beneficial to the production of high enzyme activity protease, the carbon source with proper particle size can balance the growth and the propagation of the microorganism and the dissolved oxygen in the shake flask, and can also avoid that carbon source particles are too fine to form suspension with higher viscosity with other nutrient substances and further influence the production of bacterial strain metabolites, thereby reducing the enzyme production level; in addition, the corn cob xylan and the threitol which are added into the fermentation enzyme production medium in a special proportion have an induction stimulation effect on the production of the high-enzyme activity protease, and can greatly accelerate the secretion and precipitation of the hyphomycete lanuginosus strain on the high-activity protease with the isoelectric point of about 7.4, so that the proportion of the protease with the isoelectric point of about 7.4 is remarkably increased on the basis of maintaining the content of the whole protease compound, and the activity of the protease compound is integrally increased.
The invention also provides the special feed for brown croakers, which contains the basic feed and the special feed additive for brown croakers,
the basal feed comprises:
31-35 wt% protein;
22-24 wt% fat;
0.06-0.09 wt% of nucleotides and/or nucleosides;
2.3 to 5.0 wt.% digestible sulphur-containing amino acid;
0.3 to 0.5 wt% of total available calcium;
0.32 to 0.4 wt% total available phosphorus;
0.3 to 0.35 wt% of sodium.
The special feed additive for brown croakers comprises:
(I) an oligosaccharide composition; and/or
(II) a carboxylic acid ester; and
(III) a protease complex; the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum.
The invention has the beneficial effects that:
1) the oligosaccharide composition is applied to the feed additive or feed special for the miichthys miiuy to feed the miichthys miiuy, so that the beneficial health effect is achieved, the fish mortality is reduced, the intestinal microbial flora is improved, the nutrient absorption is improved, the gastrointestinal health is maintained, the requirement on antibiotics is reduced, and the abuse of the antibiotics in the culture process is reduced;
2) the carboxylate product is applied to the special feed or feed additive for the brown croakers, can obviously enhance the antibacterial activity of the brown croakers, has beneficial effects on preventing and treating intestinal infection of the brown croakers, is beneficial to improving the growth rate of the brown croakers, reducing the feed-meat ratio and the culture period and increasing the economic benefit;
3) the protease compound is helpful for promoting the regeneration of intestinal villi of fishes such as miichthys miiuy and greatly improving the intestinal absorption of target nutrient substances, thereby obviously reducing the feed-meat ratio of the miichthys miiuy during breeding and shortening the breeding period, and in addition, the protease compound is helpful for strengthening the immune system of the miichthys miiuy and can obviously reduce the morbidity, especially the mortality of the miichthys miiuy caused by bacterial pollution;
4) when a protease compound is obtained by fermentation, a carbon source with a special particle size is added into a fermentation enzyme production culture medium to help the production of high-enzyme-activity protease, the carbon source with a proper particle size can balance the growth and the propagation of microorganisms with the dissolved oxygen in a shake flask, and the phenomenon that carbon source particles are too fine to form a suspension with higher viscosity with other nutrient substances and further influence the production of bacterial strain metabolites is avoided, so that the enzyme production level is reduced;
5) the addition of the corncob xylan and the threitol in a special ratio in the fermentation enzyme-producing culture medium has an induction stimulation effect on the production of the high-activity protease, and can greatly accelerate the secretion and precipitation of the high-activity protease with the isoelectric point of 7.4 by the strain of the hyphomycetes lanuginosus, so that the proportion of the high-activity protease is remarkably increased on the basis of maintaining the content of the whole protease complex, and the activity of the protease complex is integrally increased.
The technical scheme is adopted to provide the special feed compound additive for miichthys miiuy, so that the defects of the prior art are overcome, the design is reasonable, and the operation is convenient.
Detailed Description
The present invention is further described in detail with reference to the following examples:
example 1:
a special feed additive for Miichthys miiuy comprises:
(I) an oligosaccharide composition; and/or
(II) a carboxylic acid ester; and
(III) a protease complex; the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum. The special feed additive for brown croakers is beneficial to improving intestinal microbial flora of brown croakers, promoting regeneration of intestinal villus of brown croakers, improving intestinal absorption of target nutrient substances, maintaining gastrointestinal health and/or reducing the requirement on antibiotics, reducing abuse of the antibiotics in the breeding process, also being capable of remarkably reducing the feed-meat ratio in breeding of brown croakers and shortening the breeding period.
The oligosaccharide composition (I) has the following distribution of glycosidic bonds: having 8 mol% alpha-1, 6 glycosidic linkages and 8 mol% beta-2, 1 glycosidic linkages, and 8 mol% beta-1, 3 glycosidic linkages, and wherein the degree of polymerization of the 8 dry weight% oligosaccharide composition is 2.
The oligosaccharide composition (I) comprises: isomalto-and isomalto-inuiino-oligosaccharides and inulino-diutano-and inulino-isomalto-oligosaccharides. The oligosaccharide composition applied to the feed additive or feed special for the miichthys miiuy has beneficial health effect, is beneficial to reducing fish mortality, improving intestinal microbial flora, improving nutrient absorption, maintaining gastrointestinal health and/or reducing the demand on antibiotics, and reduces abuse of antibiotics in the culture process.
The (II) carboxylic acid ester includes: a lactyl-carboxylic acid ester according to formula 1,
formula 1: r1-COO-[-CH(CH3)-COO]2-R2And Ba salts thereof, glycolyl carboxylate according to formula 2,
formula 2: r1-COO-[-CH2-COO]3-R2And Ca salts, in the above formula, R1Is chlorophenyl, R of formula 12Is C2Alkyl chain, R of formula 22Is C4An alkyl chain; the carboxylate product is applied to special feed or feed additive for brown croakers, can obviously enhance antibacterial activity of brown croakers, has beneficial effects on preventing and treating intestinal infection of brown croakers, is beneficial to improving growth rate of brown croakers, reducing feed-meat ratio and breeding cycle, and increasing economic benefit.
The (III) protease complex is a protease complex obtained by culturing Dryophora lanuginosa and comprising an effective protease mixture in a proportion of 76% of the activity of the protease complex, wherein one protease has an isoelectric point of 7.4 and the other protease has an isoelectric point of 8.3; the protease complex is helpful for promoting the regeneration of intestinal villi of fishes such as miichthys miiuy and greatly improving the intestinal absorption of target nutrient substances, thereby remarkably reducing the feed-meat ratio of miichthys miiuy during breeding and shortening the breeding period, and in addition, the protease complex is helpful for strengthening the immune system of the miichthys miiuy and can obviously reduce the morbidity, especially the mortality of the miichthys miiuy caused by bacterial pollution.
The protease having isoelectric point of 7.4 in the protease complex of (III) has specific activity of 82000 Anson units/mg protein.
The protease with isoelectric point of 8.3 in the protease complex (III) has specific activity of 20000 Anson units/mg protein.
The (III) protease complex is used in special feed for Miichthys miiuy as a supplement of feed additives, and the feed additives are mixed with feed compositions in a powder form.
In order to obtain the (III) protease complex, the strain of Trichosporon lanuginosum is cultured and the fermentation broth is filtered, then the extraction of the protease complex by ultrafiltration and isoelectric focusing is performed, and finally the obtained protease complex is lyophilized and ground, specifically including:
fermentation: selecting a single colony with a transparent ring from a primary screening culture medium plate, carrying out streaking separation, transferring the purified strain to a fermentation shake flask containing a fermentation enzyme production culture medium, ventilating in sterile air with the volume of 0.3/culture medium volume/min, fermenting for 4 days at constant temperature under the conditions that the pH of the fermentation enzyme production culture medium is 7.2, the temperature is 40 ℃ and the rotating speed is 150r/min, and centrifuging to obtain a supernatant;
and (3) ultrafiltration: ultrafiltration was carried out by means of a membrane with a cut-off threshold of 50kDa, ultrafiltration being carried out for 3 hours;
isoelectric focusing: controlling the pH value of the solution to be 7.3 and 8.2 respectively to perform isoelectric focusing, namely obtaining most of protease with the isoelectric point (pI) of about 7.4 and less protease with the isoelectric point (pI) of about 8.3; freeze drying and crushing to obtain the protease compound.
The fermentation enzyme-producing culture medium comprises the following substances in parts by weight: corn cob xylan of 180 meshes 0.18%, threitol 0.012%, tryptone 1.8%, yeast extract powder 0.3%, sodium nitrate 0.1%, KH2PO40.5％、K2HPO40.15％、MgSO40.03%, natural pH; the carbon source with special particle size in the fermentation enzyme production culture medium is beneficial to the production of high enzyme activity protease, the carbon source with proper particle size can balance the growth and the propagation of the microorganism and the dissolved oxygen in the shake flask, and can also prevent carbon source particles from being too fine to form suspension with higher viscosity with other nutrient substances and further influence the production of bacterial strain metabolites, thereby reducing the enzyme production level; in addition, the fermentation enzyme production culture medium is added with the corn cob xylan and threitol in special proportion to the high enzyme activity proteaseThe produced high-activity protease has an induced stimulation effect, and can greatly accelerate the secretion and precipitation of the high-activity protease with the isoelectric point of 7.4 by the strain of the hyphomycetes lanuginosus, so that the proportion of the protease with the isoelectric point of 7.4 is remarkably improved on the basis of maintaining the content of the whole protease complex, and the activity of the protease complex is integrally improved.
Comparative example 1:
comparative example 1 is substantially the same as example 1 except that: the fermentation enzyme-producing medium does not contain threitol.
Example 2:
an oligosaccharide composition (I) having the following distribution of glycosidic linkages: having 15 mol% alpha-1, 6 glycosidic linkages and 12 mol% beta-2, 1 glycosidic linkages and wherein 10% by dry weight of the oligosaccharide composition has a degree of polymerization of 6.
The oligosaccharide composition (I) comprises: isomalto-lactose-oligosaccharides, lactose-inulinobiose-oligosaccharides and inulinobiose-isomalto-oligosaccharides. The oligosaccharide composition applied to the feed additive or feed special for the miichthys miiuy has beneficial health effect, is beneficial to reducing fish mortality, improving intestinal microbial flora, improving nutrient absorption, maintaining gastrointestinal health and/or reducing the demand on antibiotics, and reduces abuse of antibiotics in the culture process.
A carboxylic acid ester (II) is: a lactylcarboxylic acid ester according to formula 1,
formula 1: r1-COO-[-CH(CH3)-COO]2-R2And Na salts thereof, glycolyl carboxylate according to formula 2,
formula 2: r1-COO-[-CH2-COO]4-R2And NH thereof4Salt; in the above formula, R of formula 11Is chlorophenyl, R of formula 12Is C3Alkenyl chain, R of formula 21Is bromophenyl, R of formula 22Is C4An alkyl chain; the carboxylic ester product is applied to special feed or feed additive for brown croaker, can remarkably enhance antibacterial activity of brown croaker, has beneficial effect in preventing and treating intestinal infection of brown croaker, and is helpful for improving growth rate of brown croaker, reducing feed-meat ratio and culture period, and increasing economic efficiencyIt is beneficial to.
A protease complex comprising an effective protease mixture obtained by culturing a strain of hyphomyces lanuginosus in a proportion of 82% of the activity of the protease complex, wherein one protease has an isoelectric point of 7.4 and the other protease has an isoelectric point of 8.3; the protease complex is helpful for promoting the regeneration of intestinal villi of fishes such as miichthys miiuy and greatly improving the intestinal absorption of target nutrient substances, thereby remarkably reducing the feed-meat ratio of miichthys miiuy during breeding and shortening the breeding period, and in addition, the protease complex is helpful for strengthening the immune system of the miichthys miiuy and can obviously reduce the morbidity, especially the mortality of the miichthys miiuy caused by bacterial pollution.
The protease having an isoelectric point of about 7.4 in the protease complex of (III) has a specific activity of 85000 Anson units/mg protein.
The protease having an isoelectric point of about 8.3 in the protease complex (III) has a specific activity of 22000 Anson units/mg protein.
The (III) protease compound is used in special feed for miichthys miiuy as a supplement of feed additives, and the feed additives are mixed with feed compositions in a powder form.
In order to obtain the (III) protease complex, the strain of Trichosporon lanuginosus is cultured and the fermentation broth is filtered, then the extraction of the protease complex by ultrafiltration and isoelectric focusing is undertaken, and finally the obtained protease complex is freeze-dried and comminuted, including in particular:
1, fermentation: selecting a single colony with a transparent ring from the primary screening culture medium flat plate, and carrying out streaking separation; transferring the purified strain to a fermentation shake flask containing a fermentation enzyme production culture medium, ventilating in sterile air of 0.3 volume/culture medium volume/minute, fermenting at constant temperature for 6d under the conditions that the pH of the fermentation enzyme production culture medium is 7.5, the temperature is 45 ℃ and the rotating speed is 200r/min, and centrifuging to obtain a supernatant;
2, ultrafiltration: ultrafiltration is carried out by means of a membrane having a cut-off threshold of 50kDa, ultrafiltration being carried out for 10 hours;
3 isoelectric focusing: controlling the pH value of the solution to be 7.5 and 8.4 respectively to perform isoelectric focusing, so as to obtain most of protease with isoelectric point (pI) of about 7.4 and less of protease with isoelectric point (pI) of about 8.3; freeze drying and crushing to obtain the protease compound.
The fermentation enzyme production culture medium comprises the following substances in parts by weight: corn cob xylan of 240 meshes 0.2%, threitol 0.014%, tryptone 3%, yeast extract powder 0.5%, sodium nitrate 0.2%, KH2PO40.6％、K2HPO40.2％、MgSO40.05%, natural pH; the carbon source with special particle size in the fermentation enzyme production culture medium is beneficial to the production of high enzyme activity protease, the carbon source with proper particle size can balance the growth and the propagation of the microorganism and the dissolved oxygen in the shake flask, and can also avoid that carbon source particles are too fine to form suspension with higher viscosity with other nutrient substances and further influence the production of bacterial strain metabolites, thereby reducing the enzyme production level; in addition, the corn cob xylan and the threitol which are added into the fermentation enzyme production medium in a special proportion have an induction stimulation effect on the production of the high-enzyme activity protease, and can greatly accelerate the secretion and precipitation of the hyphomycete lanuginosus strain on the high-activity protease with the isoelectric point of about 7.4, so that the proportion of the protease with the isoelectric point of about 7.4 is remarkably increased on the basis of maintaining the content of the whole protease compound, and the activity of the protease compound is integrally increased.
A special feed additive for Miichthys miiuy comprises the following components:
(ii) a carboxylate, and (iii) a protease complex; the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum. The special feed additive for brown croakers is beneficial to improving intestinal microbial flora of brown croakers, promoting regeneration of intestinal villus of brown croakers, improving intestinal absorption of target nutrient substances, maintaining gastrointestinal health and/or reducing the requirement on antibiotics, reducing abuse of the antibiotics in the breeding process, also being capable of remarkably reducing the feed-meat ratio in breeding of brown croakers and shortening the breeding period.
Comparative example 2:
comparative example 2 is substantially the same as example 2 except that: the fermentation enzyme-producing culture medium does not contain 240-mesh corncob xylan.
Example 3:
a special feed additive for Miichthys miiuy comprises:
(I) an oligosaccharide composition; and (II) a carboxylic acid ester; and (III) a protease complex; the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum. The special feed additive for brown croakers is beneficial to improving intestinal microbial flora of brown croakers, promoting regeneration of intestinal villus of brown croakers, improving intestinal absorption of target nutrient substances, maintaining gastrointestinal health and/or reducing the requirement on antibiotics, reducing abuse of the antibiotics in the breeding process, also being capable of remarkably reducing the feed-meat ratio in breeding of brown croakers and shortening the breeding period.
The oligosaccharide composition (I) has the following distribution of glycosidic bonds: having 20 mol% alpha-1, 6 glycosidic linkages, and 20 mol% beta-2, 1 glycosidic linkages, and 20 mol% beta-1, 3 glycosidic linkages, and wherein 13% by dry weight of the oligosaccharide composition has a degree of polymerization of 4.
The oligosaccharide composition (I) comprises: isomalto-oligosaccharide, isomalto-inulin disaccharide-oligosaccharide, inulin ditacharide-oligosaccharide, chondroitin ditacharide-oligosaccharide, hyaluronan ditacharide-isomalto-oligosaccharide, chondroitin ditacharide-isomalto-oligosaccharide, lactose-hyaluronan ditacharide-oligosaccharide, isomalto-lactose-oligosaccharide, lactose-inulin disaccharide-oligosaccharide and inulinbiose-isomalto-oligosaccharide. The oligosaccharide composition applied to the feed additive or feed special for the miichthys miiuy has beneficial health effect, is beneficial to reducing fish mortality, improving intestinal microbial flora, improving nutrient absorption, maintaining gastrointestinal health and/or reducing the demand on antibiotics, and reduces abuse of antibiotics in the culture process.
The (II) carboxylic acid ester is selected from: a lactyl-carboxylic acid ester according to formula 1,
formula 1: r1-COO-[-CH(CH3)-COO]3-R2And Mn (ii) salts thereof, glycolyl carboxylates according to formula 2, formula 2: r1-COO-[-CH2-COO]6-R2And Co (II) salts thereof, in the above formulae, R of formulae 1 and 21Is bromophenyl, R of formula 12Is C5Alkyl, R of formula 22Is C3An alkenyl chain; the carboxylate product is applied to special feed or feed additive for brown croakers, can obviously enhance antibacterial activity of brown croakers, has beneficial effects on preventing and treating intestinal infection of brown croakers, is beneficial to improving growth rate of brown croakers, reducing feed-meat ratio and breeding cycle, and increasing economic benefit.
The (III) protease complex is obtained by culturing Dryophora lanuginosa and comprises an effective protease mixture, wherein the effective protease mixture accounts for 85% of the activity of the protease complex, one protease in the effective protease mixture has an isoelectric point of 7.4, and the other protease has an isoelectric point of 8.3; the protease compound is helpful for promoting the regeneration of intestinal villus of fishes such as brown croakers and greatly improving the intestinal absorption of target nutrient substances, so that the feed-meat ratio of the brown croakers during breeding is remarkably reduced, the breeding period is shortened, and in addition, the protease compound is helpful for strengthening the immune system of the brown croakers, so that the morbidity, especially the mortality of the brown croakers caused by bacterial pollution can be remarkably reduced.
The protease having an isoelectric point of about 7.4 in the protease complex (III) has a specific activity of 88000 Anson units/mg protein.
The protease having an isoelectric point of about 8.3 in the protease complex of (III) has a specific activity of 24000 Anson units/mg protein.
The (III) protease complex is used in special feed for Miichthys miiuy as a supplement of feed additives, and the feed additives are mixed with feed compositions in a powder form.
In order to obtain the (III) protease complex, the strain of Trichosporon lanuginosus is cultured and the fermentation broth is filtered, then the extraction of the protease complex by ultrafiltration and isoelectric focusing is undertaken, and finally the obtained protease complex is freeze-dried and comminuted, including in particular:
1, fermentation: selecting a single colony with a transparent ring from a primary screening culture medium plate (the protease produced by the strain can decompose the substrate protein in the culture medium, so that the transparent ring phenomenon appears around the colony), and streaking and separating; transferring the purified strain to a fermentation shake flask containing a fermentation enzyme-producing culture medium, ventilating in sterile air of 0.3 volume/culture medium volume/minute, fermenting at constant temperature for 5d under the conditions that the pH of the fermentation enzyme-producing culture medium is 7.4, the temperature is 42 ℃ and the rotating speed is 180r/min, and centrifuging to obtain a supernatant;
2, ultrafiltration: ultrafiltration was carried out by means of a membrane with a cut-off threshold of 50kDa, ultrafiltration being carried out for 5 hours;
3 isoelectric focusing: controlling the pH value of the solution to be 7.4 and 8.3 respectively to perform isoelectric focusing, namely obtaining most of protease with the isoelectric point (pI) of about 7.4 and less protease with the isoelectric point (pI) of about 8.3; freeze drying and crushing to obtain the protease compound.
The fermentation enzyme production culture medium comprises the following substances in parts by weight: corn cob xylan of 200 meshes 0.18%, threitol 0.012%, tryptone 2%, yeast extract powder 0.4%, sodium nitrate 0.15%, KH2PO40.55％、K2HPO40.18％、MgSO40.04%, natural pH; the carbon source with special particle size in the fermentation enzyme production culture medium is beneficial to the production of high enzyme activity protease, the carbon source with proper particle size can balance the growth and the propagation of the microorganism and the dissolved oxygen in the shake flask, and can also avoid that carbon source particles are too fine to form suspension with higher viscosity with other nutrient substances and further influence the production of bacterial strain metabolites, thereby reducing the enzyme production level; in addition, the addition of the corncob xylan and the threitol with special proportion in the fermentation enzyme production culture medium has an induced stimulation effect on the production of high-enzyme activity protease, and can greatly accelerate the application of the strain of the hyphomycete lanuginosus to high-activity protein with the isoelectric point of about 7.4The secretion and precipitation of the enzyme remarkably increase the proportion of the protease having an isoelectric point of about 7.4 while maintaining the content of the protease complex as a whole, thereby improving the activity of the protease complex as a whole.
Comparative example 3:
comparative example 3 is substantially the same as example 3 except that: the fermentation enzyme-producing culture medium does not contain 200-mesh corncob xylan and threitol.
Experimental example:
1. the ratio and specific activity of each of the proteases having isoelectric points of about 7.4 and about 8.3 in the protease complexes (III) of examples 1 to 3 and comparative examples 1 to 3 were measured, and the statistics are shown in Table 1.
TABLE 1 ratio and specific Activity of proteases of examples 1 to 3 and comparative examples 1 to 3
As is clear from Table 1, in preferred embodiments 1 to 3 of the present invention, the ratio of the highly active protease having a pI of about 7.4 is high, and the activity is stable, so that the overall enzymatic activity of the protease (III) complex is greatly improved.
2. The special feed for brown croakers contains a base feed and a special feed compound additive for brown croakers, wherein the base feed comprises: 32 wt% protein, 22 wt% fat; 0.08 wt% nucleotide, 4 wt% digestible sulphur containing amino acid, 0.4 wt% total available calcium, 0.35 wt% total available phosphorus and 0.3 wt% sodium.
The special feed additive for brown croakers is selected from examples 1-3 and comparative examples 1-3.
The special feed for the 6 kinds of miichthys miiuy is respectively fed to the cultivated miichthys miiuy, other factors are completely consistent, the average body length, the average body weight and the death rate of the miichthys miiuy are respectively measured after the miichthys miiuy is fed for 4 months, and the statistical results are shown in table 2.
TABLE 2 statistics of Miichthys miiuy breeding conditions
| Measurement items | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 |
| Average body length (cm) | 40.5 | 41.6 | 43.0 | 35.5 | 34.2 | 33.8 |
| Average body weight (g) | 855.1 | 860.9 | 905.5 | 705.8 | 710.6 | 700.2 |
| Mortality (%) | 0.5％ | 1.0％ | 0.5％ | 3.5％ | 4.0％ | 4.5％ |
Table 2 shows that the average body length, average body weight and survival rate of brown croakers are significantly superior to those of the comparative examples, which indicates that the special feed additive for brown croakers can effectively improve the growth rate of brown croakers and reduce the feed-meat ratio and the breeding period, and is an efficient and economical special feed additive for brown croakers.
Conventional techniques in the above embodiments are known to those skilled in the art, and therefore, will not be described in detail herein.
The above embodiments are merely illustrative, and not restrictive, and those skilled in the art can make various changes and modifications without departing from the spirit and scope of the invention. Therefore, all equivalent technical solutions also belong to the scope of the present invention, and the protection scope of the present invention should be defined by the claims.
Claims (8)
1. The special feed additive for brown croakers is characterized by comprising:
(I) an oligosaccharide composition; and
(II) a carboxylic acid ester; and
(III) a protease complex;
the (III) protease complex is a protease complex comprising an effective protease mixture obtained by culturing Trichosporon lanuginosum;
the preparation process of the (III) protease complex comprises the following steps:
fermentation: selecting a single colony of the hyphomycetes lanuginosus strain with a transparent circle from a primary screening culture medium plate for streaking separation; transferring the purified strain to a fermentation shake flask containing a fermentation enzyme-producing culture medium for fermentation, and centrifuging to obtain a supernatant;
and (3) ultrafiltration: ultrafiltration is performed by means of a membrane having a cut-off threshold of 50 kDa;
isoelectric focusing: controlling the pH value of the solution to be 7.4 +/-0.1 and 8.3 +/-0.1 respectively to perform isoelectric focusing, so as to obtain protease with the isoelectric point of 7.4 and protease with the isoelectric point of 8.3; freeze-drying and crushing to obtain (III) protease compound;
the fermentation enzyme production culture medium comprises the following substances in parts by weight: corn cob xylan of 180-240 meshes 0.18-0.2%, threitol 0.012-0.014%, tryptone 1.8-3%, yeast extract powder 0.3-0.5%, sodium nitrate 0.1-0.2%, KH2PO40.5～0.6％、K2HPO40.15～0.2％、MgSO40.03-0.05% and natural pH.
2. The special feed additive for miichthys miiuy as claimed in claim 1, which comprises: the oligosaccharide composition (I) has the following distribution of glycosidic bonds: has the advantages of
Not less than 8 mol% of alpha-1, 6 glucosidic bonds, and
not less than 8 mol% of beta-2, 1-glycosidic linkages, and/or
Not less than 8 mol% of beta-1, 3 glycosidic linkages, and
the oligosaccharide composition (I) has a degree of polymerization of 2 to 6 in at least 8% by dry weight of the oligosaccharide composition.
3. The feed additive specially used for miichthys miiuy according to claim 1 or 2, which comprises: the oligosaccharide composition (I) comprises: isomalto-oligosaccharides, isomalto-inulin di-oligo-saccharides, inulino-oligosaccharides, chondroitin di-oligo-saccharides, hyaluronan-oligosaccharides, hyaluronan-isomalto-oligosaccharides, chondroitin di-isomalto-oligosaccharides, lactose-hyaluronan-oligosaccharides, isomalto-lactose-oligosaccharides, lactose-inulino-di-oligosaccharides, inulino-isomalto-oligosaccharides.
4. The special feed additive for miichthys miiuy as claimed in claim 1, which comprises: the (II) carboxylic acid ester is selected from:
a lactyl-carboxylic acid ester according to formula 1,
formula 1: r1-COO-[-CH(CH3)-COO]n-R2
Or/and Ba, Be, Mn (II), Ca, Na, NH4Or a Co (II) salt,
a glycolyl carboxylate according to formula 2,
formula 2: r1-COO-[-CH2-COO]n-R2
Or/and Ba, Be, Mn (II), Ca, Na, NH4Or a Co (II) salt,
in the above formula, R1Selected from chlorophenyl or bromophenyl, R2Is selected from C2～C10An alkyl or alkenyl chain, and n represents an integer having a value of 1 to 10.
5. The special feed additive for miichthys miiuy as claimed in claim 1, which comprises: (iii) the proportion of the effective protease mixture in the protease complex, of which proteases one protease has an isoelectric point of 7.4 and the other protease has an isoelectric point of 8.3, exceeds 75% of the activity of the protease complex.
6. The special feed additive for miichthys miiuy as claimed in claim 5, which comprises the following components: the protease with the isoelectric point of 7.4 in the protease complex (III) has the specific activity of 82000-90000 Anson units/mg protein, and the protease with the isoelectric point of 8.3 in the protease complex (III) has the specific activity of 20000-25000 Anson units/mg protein.
7. The feed additive specially used for miichthys miiuy according to claim 5 or 6, which comprises: the (III) protease complex is used in the special feed for miichthys miiuy as a supplement of a feed additive, and the feed additive is in a form of powder, liquid or any other form suitable for being mixed with a feed composition.
8. A feed for Miichthys miiuy, which comprises the feed additive for Miichthys miiuy of any one of claims 1 to 7, characterized by comprising a base feed and the feed additive for Miichthys miiuy of any one of claims 1 to 7.
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|---|---|---|---|---|
| CA2200344A1 (en) * | 1994-10-13 | 1996-04-25 | Anne-Cathrine Samuelsson | Animal feed of higher nutritive value, method for production thereof and use of a polyethylene glycol compound |
| CN101326958A (en) * | 2008-07-22 | 2008-12-24 | 浙江大学 | Peptide feedstuff additive for regulating immunity function of sea water fish |
| CN102362635A (en) * | 2011-10-18 | 2012-02-29 | 珠海市世海饲料有限公司 | High-quality compound feed with low nitrogen and phosphorus emission for Lateolabrax |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CA2200344A1 (en) * | 1994-10-13 | 1996-04-25 | Anne-Cathrine Samuelsson | Animal feed of higher nutritive value, method for production thereof and use of a polyethylene glycol compound |
| CN101326958A (en) * | 2008-07-22 | 2008-12-24 | 浙江大学 | Peptide feedstuff additive for regulating immunity function of sea water fish |
| CN102362635A (en) * | 2011-10-18 | 2012-02-29 | 珠海市世海饲料有限公司 | High-quality compound feed with low nitrogen and phosphorus emission for Lateolabrax |
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| Title |
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| 饲料中菌酶协同作用的研究与应用进展;李旺等;《动物营养学报》(第08期);全文 * |
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