CN114557403A - Preparation method and application of sulfated polysaccharide feed additive for high-fat low-protein feed - Google Patents
Preparation method and application of sulfated polysaccharide feed additive for high-fat low-protein feed Download PDFInfo
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- CN114557403A CN114557403A CN202111614082.5A CN202111614082A CN114557403A CN 114557403 A CN114557403 A CN 114557403A CN 202111614082 A CN202111614082 A CN 202111614082A CN 114557403 A CN114557403 A CN 114557403A
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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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/10—Culture of aquatic animals of fish
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- A23K—FODDER
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- 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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- A23K—FODDER
- A23K10/00—Animal feeding-stuffs
- A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
- A23K10/14—Pretreatment of feeding-stuffs with enzymes
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- 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
- A23K20/189—Enzymes
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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
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
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Abstract
The invention discloses a preparation method and application of a sulfate-based polysaccharide feed additive for high-fat low-protein feed. According to the invention, raw materials are pretreated by solid state fermentation, and xylanase, algin lyase, mannase and pectinase are subjected to compound enzymolysis, so that enzymolysis can be carried out at a higher substrate concentration, the enzymolysis rate is greatly improved, the product yield is improved, and the sulfate-based polysaccharide with low molecular weight and good solubility is produced by using an environment-friendly and efficient process means. The sulfated polysaccharide can improve the survival rate and the growth rate of the trachinotus ovatus under the condition of high-fat low-protein feeding, reduce and repair the body damage of the trachinotus ovatus caused by the feeding of the high-fat low-protein feed to a certain extent.
Description
Technical Field
The invention relates to a preparation method for preparing sulfated polysaccharides by compound enzymolysis, in particular to a method for preparing a sulfated polysaccharides feed additive for high-fat low-protein feed by performing compound enzymolysis on eucheuma by xylanase, algin lyase, mannase and pectinase.
Background
The fish has short digestive tract, low digestive enzyme activity, low utilization rate of feed protein and high ammonia nitrogen content in excrement. The accumulation of excrement can also cause the content of ammonia nitrogen in the water body to be too high, restrict the growth of fishes and cause fish diseases. In order to reduce the content of nitrogen and phosphorus in the water environment and prevent fish outbreak diseases, strategies of reducing the protein level of feed and properly improving the fat level are generally adopted under the condition of ensuring the balanced nutrition of the feed in the actual culture process, and the culture strategy can achieve the purposes of promoting the growth of fish bodies and protecting culture water areas. However, when the feed fat level is too high, the fish body fat accumulation is excessive, the anti-stress capability is reduced, tissues such as liver and pancreas and the like are damaged, the fish body grows slowly, the feed coefficient is increased, hyperlipidemia and nutritional fatty liver are caused in severe cases, a large amount of death occurs in high-temperature seasons, and great loss is caused to production.
Sulfated polysaccharides, also known as sulfated derivatives of polysaccharides, are a class of multifunctional substances formed by substituting sulfate groups for a portion of the hydroxyl groups of monosaccharide molecules in the polysaccharide structure, and can be obtained by natural preparation or structural modification of sulfation. The sulfated polysaccharide is widely present in seaweed plants, has various physiological activities, has obvious effects on reducing blood fat, blood pressure, oxidation resistance and the like, and has no toxic or side effect. The sulfated polysaccharide is an excellent functional polysaccharide, and can play an important role in fish culture as an excellent feed additive.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: overcomes the defects of the prior art, and provides a preparation method and related application of a sulfated polysaccharide feed additive for high-fat low-protein feed prepared by compound enzymolysis.
The technical scheme adopted by the invention for solving the technical problems is as follows: a preparation method for preparing sulfate polysaccharide by compound enzymolysis comprises the following steps:
step 1: synergistic fermentation of Eucheuma Gelatinosum Murr enzyme: putting the eucheuma powder into a homogenizer, adding rice hulls accounting for 10% of the weight of the eucheuma powder, adding water according to the feed-liquid ratio of 1:1.5, dissolving alginate lyase in advance by using fermentation water, wherein the enzyme activity of the alginate lyase is 25000-.
Wherein the fermentation strain is trichoderma reesei.
Step 2: enzymolysis of xylanase: adding water into the materials according to the ratio of the materials to the liquid of about 1:10, and carrying out enzymolysis by using xylanase, wherein the enzyme activity is 30000-;
and step 3: compound enzymolysis: adjusting the pH value of the enzymolysis liquid to 3.5 after the enzymolysis in the step 3 is finished by hydrochloric acid, and then carrying out enzymolysis by using mannase and pectinase, wherein the enzyme activity of the mannase is 40000-50000U/g, the enzyme addition amount is 300-350U/g Eucheuma substrates, the enzyme activity of the pectinase is 25000-30000U/g, the enzyme addition amount is 200-250U/g Eucheuma substrates, the enzymolysis temperature is 45-60 ℃, and the action time is 2 h;
and 4, step 4: enzyme deactivation: heating the enzymolysis solution to 95 deg.C, and keeping for 15min to inactivate enzyme;
and 5: plate-frame filtration: after enzyme deactivation, performing plate-and-frame filtration on the enzymatic hydrolysate to remove insoluble residues;
step 6: alcohol precipitation: adding ethanol into the enzymolysis liquid prepared in the step 3 according to 10% of the volume of the enzymolysis liquid to settle macromolecular saccharides, and then centrifuging to remove enzymolysis residues;
and 7: and 6, filtering the enzymatic hydrolysate by using a filter membrane to remove impurities and bacteria, wherein the pore size of the filter membrane is 0.45-0.95 mu m.
And 8: concentrating the enzymolysis liquid: centrifuging to remove impurities, and concentrating the supernatant to solid content not less than 25%;
and step 9: spray drying: the inlet temperature is 170 ℃ and 190 ℃, the outlet temperature is 90-110 ℃, and the flow rate is 8-12mL/min, so as to obtain the sulfated polysaccharide dry powder.
The application method of the sulfate polysaccharide prepared by compound enzymolysis is characterized by comprising the following steps: the application method of the sulfated polysaccharide for high-fat low-protein feed comprises the following steps:
the method comprises the following steps: adding the sulfated polysaccharides into the feed according to the proportion of 1-2%, mixing uniformly, processing and preparing according to the required feed form, and directly feeding.
The method 2 comprises the following steps: the sulfated polysaccharide is dissolved into the daily drinking water of animals for use according to the proportion of 1 to 2 percent.
Compared with the prior art, the invention has the beneficial effects that: according to the invention, through high-temperature homogenization and viscosity reduction, and then, compound enzymolysis by using four enzymes of xylanase, algin lyase, mannase and pectinase, the purposes of reducing the viscosity of a reaction substrate, improving the concentration of the reaction substrate and greatly improving the enzymolysis rate are achieved, and sulfate-based polysaccharide with low molecular weight and good solubility is produced and produced by using an environment-friendly and efficient technological means. Has the advantages of mild reaction conditions, simple operation and low cost.
Description of the drawings:
FIG. 1 shows the effect of sulfated polysaccharides on fatness of trachinotus ovatus which is a high-fat and low-protein diet
FIG. 2 shows the effect of sulfated polysaccharides on the survival rate of trachinotus ovatus in high-fat and low-protein diet
FIG. 3 shows the effect of sulfated polysaccharides on the liver body ratio of high-fat and low-protein diet trachinotus ovatus
FIG. 4 shows the effect of sulfated polysaccharides on the body ratio of high-fat and low-protein diet trachinotus ovatus
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making any creative effort based on the embodiments in the present invention, belong to the protection scope of the present invention.
Example 1:
1) adding 10g rice hull into 100g Eucheuma Gelatinosum powder, adding 165mL fermentation water (adding alginate lyase into water according to the amount of 300U/g Eucheuma Gelatinosum powder substrate), performing solid state fermentation at 30 ℃ for 48h, and stirring every 12 h.
Wherein the zymocyte is Trichoderma reesei.
2) After fermentation, adding 1L of water into the material, adding xylanase according to the amount of 300U/g Eucheuma Gelatinosum powder substrate, and performing enzymolysis at 50 deg.C for 3h
3) Adjusting pH of the enzymolysis solution to 3.5 with hydrochloric acid, adding mannase according to 300U/g Eucheuma Gelatinosum powder substrate, adding pectase according to 250U/g Eucheuma Gelatinosum powder substrate, performing enzymolysis, and performing enzymolysis at 50 deg.C for 3 hr.
4) Heating the enzymolysis solution to 95 deg.C, and keeping for 15min to inactivate enzyme.
5) After enzyme deactivation, the enzymolysis liquid is filtered by double-layer filter paper to remove insoluble impurities
6) Adding 100mL of ethanol into the enzymolysis solution according to 10% of the volume of the enzymolysis solution, standing at room temperature of 25 ℃ for 15min to settle macromolecular saccharides, and centrifuging at 8000rpm/min by using a centrifuge for 20min to remove insoluble residues.
7) And (3) carrying out suction filtration on the centrifuged enzymolysis liquid by using a glass core funnel to remove thallus residues, wherein the aperture of the glass core funnel is 0.65 mu m.
8) Concentrating the supernatant to about 500-600 mL by rotary evaporation, determining the solid content in the concentrated solution until the solid content in the solution is about 25%,
9) and (3) carrying out spray drying on the concentrated solution obtained in the step (8) to obtain the sulfated polysaccharide. Wherein the air inlet temperature of spray drying is 170-.
The method comprises the following steps: adding the sulfated polysaccharides into the feed according to the proportion of 1-2%, mixing uniformly, processing and preparing according to the required feed form, and directly feeding.
The method 2 comprises the following steps: the sulfated polysaccharide is dissolved into the daily drinking water of animals for use according to the proportion of 1 to 2 percent.
Example 2
1) Adding 0.75kg rice hull into 7.5kg Eucheuma Gelatinosum powder, adding 12.5L fermentation water (adding alginate lyase into water at the amount of 300U/g Eucheuma Gelatinosum powder substrate), solid-state fermenting at 30 deg.C for 48 hr, and stirring every 12 hr.
Wherein the zymocyte is Trichoderma reesei.
2) After fermentation, adding 80L of water into the material, adding xylanase according to the amount of 300U/g Eucheuma Gelatinosum powder substrate, and performing enzymolysis at 50 deg.C for 3h
3) Adjusting pH of the enzymolysis solution to 3.5 with hydrochloric acid, adding mannase according to the amount of 250U/g Eucheuma Gelatinosum powder substrate, adding pectase according to the amount of 250U/g Eucheuma Gelatinosum powder substrate, performing enzymolysis, and performing enzymolysis at 50 deg.C for 3 hr.
4) After enzymolysis, steam is introduced into the interlayer of the fermentation tank, and the temperature is raised to 90 ℃ and maintained for 15min for enzyme deactivation.
5) After enzyme deactivation, filtering the enzymolysis solution by using a 300-mesh plate frame to remove insoluble impurities
6) Adding 5L ethanol into the enzymolysis solution according to 10% of the volume of the enzymolysis solution, standing at room temperature for 15min to allow macromolecular saccharide substances to settle, and centrifuging with a tubular centrifuge to remove insoluble residues.
Wherein the rotation speed of the tubular centrifuge is 10000rpm/min
7) The centrifuged enzymatic hydrolysate was filtered through a 0.45 μm filter.
8) The supernatant was concentrated by rotary evaporation to a solid content of 23%.
9) And (3) spray-drying the concentrated solution obtained in the step (8) to obtain the sulfated polysaccharides. Wherein the air inlet temperature of spray drying is 170-.
Example 3:
the preparation method of the invention has the advantages of the preparation of sulfated polysaccharides and the influence on high-fat diet trachinotus ovatus.
Preparation of sulfated polysaccharides according to the technique of the invention according to example 2
Adding 3kg of rice hull into 30kg of eucheuma powder, adding 50L of fermentation water (adding algin lyase into water according to the amount of 300U/g of eucheuma powder substrate), inoculating trichoderma reesei into the material, performing solid state fermentation at 30 ℃ for 48h, and stirring every 12 h. After fermentation, adding 300L of water into the material, adding xylanase according to the amount of 300U/g eucheuma powder substrate, starting a stirring paddle of a fermentation tank, rotating at 50r/min, and performing enzymolysis at 50 ℃ for 3 h. Adjusting pH of the enzymolysis solution to 3.5 with hydrochloric acid, adding mannase according to the amount of 250U/g Eucheuma Gelatinosum powder substrate, adding pectase according to the amount of 250U/g Eucheuma Gelatinosum powder substrate, performing enzymolysis, and performing enzymolysis at 50 deg.C for 3 hr. Heating the enzymolysis solution to 95 deg.C, and keeping for 15min to inactivate enzyme. And after enzyme deactivation, filtering the enzymolysis liquid by using a 300-mesh plate frame to remove insoluble impurities. Adding 20L ethanol into the enzymolysis solution according to 10% of the volume of the enzymolysis solution, standing at room temperature of 25 deg.C for 15min to allow macromolecular saccharide substance to settle, and centrifuging with tubular centrifuge at 10000rpm to remove insoluble residue. Filtering the centrifuged enzymolysis solution by using a filter membrane with the aperture of 0.45 mu m for sterilization. And (3) performing rotary evaporation and concentration on the supernatant to about 80-100L, measuring the content of solid matters in the concentrated solution until the content of the solid matters in the solution is about 25%, and performing spray drying to obtain the sulfate-based polysaccharide. Wherein the air inlet temperature of spray drying is 170-. The analysis of the sulfated polysaccharide components is shown in Table 1.
According to the experimental requirements, basic ration of trachinotus ovatus is taken as a main feed ingredient, and red fish meal is taken as a main protein source. The positive control group uses the following feeds: the basic diet of trachinotus ovatus and the red fish meal are adopted, the content of the fish meal in the feed is 25%, the content of the total protein in the feed is 45%, and the content of the total fat in the feed is 12%. The negative control group used the feed: the basic diet of trachinotus ovatus and the red fish meal are adopted, the content of the fish meal in the feed is 25%, the content of the total protein in the feed is 40%, and the content of the total fat in the feed is 15%. The feed used by the test group is prepared by replacing basic ration of trachinotus ovatus with sulfate polysaccharide with different contents on the basis of a negative control group. The total protein content of the feed of the test group is 40 percent, the total fat content of the feed is 15 percent, and the sulfate polysaccharide is used for replacing 0.5 percent of basic ration of the trachinotus ovatus in the test group 1; test group 2 used sulfated polysaccharide instead of 1% of basic ration of trachinotus ovatus; experimental group 3 used sulphated polysaccharide instead of 2% of basic diet of trachinotus ovatus. The raw materials and nutritional composition of the experimental feed are shown in table 2.
According to the experimental requirements, basic ration of trachinotus ovatus is taken as a main feed ingredient, and red fish meal is taken as a main protein source. In the positive control group, the total protein content in the feed was set to 45% (25% fish meal added) and the total fat content was set to 12%. In the negative control group, the total protein content in the feed was set to 40% (25% fish meal was added) and the total fat content was set to 16%. The test groups 1, 2, 3 added different levels (0.5%, 1%, 2%) of sulfated polysaccharides on the basis of high-fat low-protein diet.
Selecting 450 trachinotus ovatus tails with uniform size, healthy physique and initial weight of 16 +/-0.5 g, randomly dividing into 5 groups, setting 3 repeats in each group, feeding trachinotus ovatus with 30 repeats in a net cage, and carrying out culture test for 8 weeks. Feeding 8-10% of the feed for 1 time at the ratio of 07:00, 11:00, 17:00 and 21:00 respectively; feeding for 1h, observing ingestion condition, and properly adjusting feeding amount according to weather, water quality and other conditions. Fasted for 24h after the culture experiment was finished, counted in duplicate and weighed. Calculating the relative growth indexes of the trachinotus ovatus, namely the Survival Rate (SR), the weight gain rate (WG), the Feed Coefficient (FCR), the fullness (CF), the liver-body ratio (LBW) and the viscera-body ratio (VBW) as follows:
survival (survival rate, SR,%) 100x terminal/initial mantissa;
weight gain ratio (WG,%) 100x (final weight average-initial weight average)/initial weight average;
feed Conversion Rate (FCR) feed dry weight/(end weight-initial weight).
Fullness factor (CF) ═ end body weight/end body length 3
Liver to body ratio (LBW) × 100%
Visceral volume ratio (VBW) × 100%
As shown in table 3, fig. 1, compared with the normal feed feeding of the positive control group, the high-fat low-protein feed of the negative control group resulted in the significant increase of the food intake and the feed coefficient of the test fish, but reduced the weight gain rate of the test fish and significantly reduced the weight of the test fish. In contrast, in the case of the test fish of the test group 2, the weight gain rate and the weight loss were significantly higher than those of the negative control group, even slightly higher than those of the positive control group, when 1% sulfated polysaccharide was added to the feed. The high-fat low-protein feed of the negative control group simultaneously causes the fatness of the test fish to be reduced, while the fatness of the test fish of the test groups 2 and 3 is improved under the condition of adding 1 percent and 2 percent of sulfated polysaccharide, and is better than that of the test fish of the positive control group, as shown in figure 1.
In addition, as shown in fig. 2, 3 and 4, the negative control group containing high-fat and low-protein feed resulted in a significant decrease in survival rate of the test fish and a significant increase in liver body ratio and visceral body ratio of the test fish. And after the test group 2 adds 1% of sulfated polysaccharide into the feed, the liver body ratio and the viscera body ratio of the test fish can be reduced to a certain extent, the survival rate of the test fish can be effectively improved, and the survival rate of the test fish in the test group 2 is not different from that in the positive control group.
In addition, the liver morphology of the trachinotus ovatus in the experimental group is observed, the liver of the trachinotus ovatus in the positive control group is normal bright red, and the liver color of the trachinotus ovatus in the negative control group is changed from the red color of the positive control group to white color. The test group 1 added with 0.5% of sulfated polysaccharide in the feed can gradually restore the liver color of the test fish to pink, while the test group 2 and the test group 3 added with 1% and 2% of sulfated polysaccharide can make the liver of the test fish turn into normal red again.
The results show that the high-fat low-protein feed causes damage to the test fish, the utilization rate of the test fish to the test feed is reduced, the phenomena of large liver and high liver fat content are presented, and the feeding mode can cause intestinal injury and reduced digestion capacity of the test fish, so that the liver injury is caused; and 1% of sulfuric acid-based polysaccharide is added, so that the weight gain rate of the test fish can be obviously improved, the feed coefficient, the liver ratio and the viscera ratio are reduced to a certain extent, and the survival rate of the test fish is ensured, which shows that the damage can be repaired to a certain extent by adding the sulfuric acid-based polysaccharide, and the effect of protecting the intestinal tracts and the liver of the trachinotus ovatus is achieved under the condition of using high-fat low-protein feed.
TABLE 1 sulfated polysaccharide product composition analysis (%)
Table 2 basal feed composition and nutritional level (%)
TABLE 3 Effect of sulfated polysaccharides on growth of high-fat low-protein diet Trachinotus ovatus
Claims (2)
1. A preparation method of a sulfated polysaccharide feed additive for high-fat low-protein feed is characterized by comprising the following steps:
step 1: synergistic fermentation of Eucheuma Gelatinosum Murr: putting the eucheuma powder into a homogenizer, adding rice hulls accounting for 10% of the weight of the eucheuma powder, adding water according to the material-liquid ratio of 1:1.5, dissolving alginate lyase in advance by using fermentation water, wherein the enzyme activity of the alginate lyase is 25000-;
wherein the fermentation strain is trichoderma reesei;
step 2: enzymolysis of xylanase: adding water into the materials according to the ratio of the materials to the liquid of about 1:10, and carrying out enzymolysis by using xylanase, wherein the enzyme activity is 30000-;
and step 3: compound enzymolysis: adjusting the pH value of the enzymolysis liquid to 3.5 after the enzymolysis in the step 3 is finished by hydrochloric acid, and then carrying out enzymolysis by using mannase and pectinase, wherein the enzyme activity of the mannase is 40000-50000U/g, the enzyme addition amount is 300-350U/g Eucheuma substrates, the enzyme activity of the pectinase is 25000-30000U/g, the enzyme addition amount is 200-250U/g Eucheuma substrates, the enzymolysis temperature is 45-60 ℃, and the action time is 2 h;
and 4, step 4: enzyme deactivation: heating the enzymolysis solution to 95 deg.C, and keeping for 15min to inactivate enzyme;
and 5: plate-frame filtration: after enzyme deactivation, performing plate-and-frame filtration on the enzymatic hydrolysate to remove insoluble residues;
step 6: alcohol precipitation: adding ethanol into the enzymolysis liquid prepared in the step 3 according to 10% of the volume of the enzymolysis liquid to settle macromolecular saccharides, and then centrifuging to remove enzymolysis residues;
and 7: step 6, filtering the enzymatic hydrolysate by using a filter membrane to remove impurities and bacteria, wherein the pore size of the filter membrane is 0.45-0.95 mu m;
and 8: concentrating the enzymolysis liquid: centrifuging to remove impurities, and concentrating the supernatant to solid content not less than 25%;
and step 9: spray drying: the inlet temperature is 170 ℃ and 190 ℃, the outlet temperature is 90-110 ℃, and the flow rate is 8-12mL/min, so as to obtain the sulfated polysaccharide dry powder.
2. A method for preparing the sulphated polysaccharide feed additive for high-fat low-protein feed according to claim 1, wherein: the application method of the sulfated polysaccharide for high-fat low-protein feed comprises the following steps:
the method comprises the following steps: adding the sulfated polysaccharides into the feed according to the proportion of 1-2%, uniformly mixing, processing and preparing according to the required feed form, and directly feeding;
the method 2 comprises the following steps: the sulfated polysaccharides are dissolved into the daily drinking water of animals according to the proportion of 1-2% for use.
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