CN111011621A - Preparation method of schizochytrium limacinum powder - Google Patents
Preparation method of schizochytrium limacinum powder Download PDFInfo
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Abstract
The invention discloses a preparation method of schizochytrium limacinum powder. Mixing schizochytrium limacinum thallus with wall materials, an emulsifier and an antioxidant, stirring, shearing and homogenizing to obtain a suspension; drying the obtained suspension to obtain schizochytrium limacinum powder with the water content within 5%; transferring the obtained dry powder into a coating machine, and spraying coating agent at 30-40 deg.C to obtain coating solution to obtain schizochytrium limacinum powder; the schizochytrium limacinum thallus is obtained by performing wall breaking pretreatment of acid treatment, alkali treatment or biological enzyme treatment on fermentation liquor obtained by fermenting schizochytrium limacinum strains. The prepared schizochytrium limacinum powder can be used for feeding animals, can prevent the schizochytrium limacinum powder from being degraded in rumens of the animals, and can be more easily absorbed by the animals after passing through the rumens, so that the milk of the animals is rich in DHA.
Description
Technical Field
The invention relates to the technical field of microorganisms, and particularly relates to a preparation method of schizochytrium limacinum powder.
Background
Schizochytrium limacinum powder is prepared with schizochytrium limacinum and through fermenting culture to obtain fermented liquid and drying the fermented liquid. Because the feed is rich in polyunsaturated fatty acid oil such as DHA and the like, the feed has great promotion effect on the growth of animals when added into animal feed.
DHA, an acronym of DocosaHexaenoic Acid (Docosa Hexaenoic Acid), commonly called as "brain gold", is a polyunsaturated fatty Acid very important for human body, plays an important role in the development of the nervous system and vision of infants, and also plays an important role in the heart, brain, eyes, immune system and the like of adults.
DHA has been widely used in infant formula, children's milk, and other dairy products. At present, DHA (docosahexaenoic acid) is mostly enhanced by adding DHA algal oil, but the DHA algal oil is directly added to bring fishy smell of different degrees to the product, and an antioxidant is needed to ensure the stability of the product. The focus in recent years has been to develop naturally occurring DHA milk (a product other than DHA fortified milk) because consumers may believe that naturally occurring DHA milk may be functionalized in a "zero-additive" manner, leading to the consumer demand of "clean labeling". The supplement of DHA for ruminants can obtain high-DHA milk, but the special digestion structure of the ruminants ensures that most of the DHA supplemented in an unprotected form is degraded by microorganisms in rumen, so that the DHA cannot be absorbed by the animals and secreted into milk, and also can generate certain toxicity to the microorganisms in the rumen, change the microflora of the rumen and reduce the degradation rate of cellulose in the rumen. Therefore, rumen protected DHA, which is able to pass through the rumen and reach where it is absorbed (small intestine), will be more likely to be secreted into milk by the animal.
CN 101999522A discloses a microalgae whole cell powder for high yield of DHA milk of mammals and a preparation method thereof, the microalgae whole cell powder is prepared from the following raw materials: the microalgae DHA cell paste is prepared from microalgae DHA cell paste, an emulsifier, an antioxidant, a filling material, a packing material, a dispersing agent and water. By feeding the microalgae whole-cell powder disclosed by the invention, the total content of docosahexaenoic acid in every 100ml of milk containing DHA can reach 6-12 mg. The microalgae whole-cell powder obtained by the invention has poor digestion effect in small intestine without pretreatment of thallus cells, so that the absorption effect is poor, and the DHA content in the obtained milk is low.
CN 109082381A discloses the application of schizochytrium limacinum and its preparation in improving DHA content in ruminant milk. The natural source milk with high DHA content is organic, safe, stable and easy to absorb, can be used as a safer and more effective way for people to take natural DHA, and can meet and meet the requirements of consumers, so that the schizochytrium limacinum and the schizochytrium limacinum powder disclosed by the invention have wide application in the fields of common food and livestock breeding. The schizochytrium limacinum powder is not protected by rumen, unsaturated fatty acid is easily degraded by microorganisms in rumen, DHA can not be absorbed and converted, normal metabolism of dairy cows is influenced, rumen bad fermentation is caused to aggravate gastrointestinal burden, and digestion speed of cellulose is greatly reduced, so that feed intake and production performance are influenced. The DHA content is 12.5 and 15.5mg/100g (free-ranging) after 60 days of the formal feeding period; 23.8 and 25.4mg/100g (captive).
CN 108419835A discloses a production method and application of pure natural DHA raw milk. The method for producing the pure natural DHA raw material milk only indicates that the schizochytrium limacinum powder is added into daily ration of the dairy cow, the daily ration added with the schizochytrium limacinum powder (200-250 g/day/head) is fed to the dairy cow in the lactation period so as to strengthen the DHA content ingested by the dairy cow, and the stable natural DHA raw material milk (the DHA content is about 8mg/100 g) is produced through the metabolic transformation of the dairy cow. However, this document describes the feeding situation and does not mention the preparation of algal flour.
The cell wall of schizochytrium consists of a number of compact scale layers that are pressed together, and cell wall anti-nutritional factors can be present, reducing or preventing the absorption of DHA by the intestines and stomach of the animal. Research results show that the main components of the schizochytrium limacinum cell wall are protein and carbohydrate, the content is 30% -43% and 21% -36%, respectively, and DHA exists in cell membranes and liposomes mainly in the form of neutral grease and is generally difficult to release. In order to reduce the influence of the anti-nutritional factors on the absorption degree of DHA in the schizochytrium limacinum powder, the cell wall must be firstly damaged, and the DHA is released. However, for ruminants such as dairy cows, if the schizochytrium limacinum powder is unprotected, unsaturated fatty acids in the schizochytrium limacinum powder are easily degraded by microorganisms in rumen, so that DHA is not absorbed and converted, normal metabolism of the dairy cows is affected, poor fermentation of the rumen is caused to aggravate gastrointestinal burden, and digestion speed of cellulose is greatly reduced, so that feed intake and production performance are affected.
CN 110074256A discloses a feed for improving the milk fat rate of ruminants and a preparation method thereof, and provides a preparation method of the thallus feed: the method comprises the following steps: 1) performing wall breaking treatment on the microbial thallus to obtain wall-broken microbial thallus; 2) and coating the wall-broken microbial cells with a tumor-gastric protective agent. The wall breaking method selected by the invention is sanding and homogenizing, which can completely break cells to form cell fragments, release grease, and can not keep the complete structure of the cells, and in addition, the tumor-treating gastric protective agent selected by the invention, such as shellac, acrylic resin II, acrylic resin III, polyvinyl alcohol acetate phthalate, hydroxypropyl methyl cellulose phthalate, Opadry or rhinacanthus belongs to enteric coating agents, is easy to dissolve or decompose in the rumen environment, and has poor rumen-passing protective effect.
Disclosure of Invention
The invention aims to provide schizochytrium powder which is prevented from being degraded in the rumen of an animal, but can be more easily absorbed by the animal after passing through the rumen, so that the milk of the animal is rich in DHA.
In order to achieve the aim, the invention provides a preparation method of schizochytrium limacinum powder, which is characterized by comprising the following steps,
embedding: mixing schizochytrium limacinum thallus with wall materials, an emulsifier and an antioxidant, stirring, shearing and homogenizing to obtain a suspension;
and (3) drying: drying the obtained suspension to obtain schizochytrium limacinum powder with the water content within 5%;
coating: transferring the obtained dry powder into a coating machine, and spraying coating agent at 30-40 deg.C to obtain coating solution to obtain schizochytrium limacinum powder;
the schizochytrium limacinum thallus is obtained by performing wall breaking pretreatment of acid treatment, alkali treatment or biological enzyme treatment on fermentation liquor obtained by fermenting schizochytrium limacinum strains.
Further, the Schizochytrium thallus is Schizochytrium sp.CGMCC No.6843 or Schizochytrium sp.ATCC No.20888 or Schizochytrium sp.ATCC No.20889 or Schizochytrium sp.ATCC No.28209 or Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381.
Further, the weight ratio of the thalli to the wall material, the emulsifier, the antioxidant and the coating agent in the schizochytrium limacinum powder is 150: (10-50): (0.5-5): (0.1-1): (5-30);
preferably, the weight ratio of the schizochytrium limacinum thallus to the wall material, the emulsifier, the antioxidant and the coating agent is 150: (10-50): (2-5): (0.25-1): (20-30);
more preferably, the weight ratio of the schizochytrium limacinum thallus to the wall material, the emulsifier, the antioxidant and the coating agent is 150: (25-50): (2-5): (0.5-1): (20-30).
Further, the wall material is at least one of starch sodium octenyl succinate, whey protein, soy protein isolate and sodium caseinate;
optionally, the emulsifier is at least one selected from monoglyceride, polyglyceryl fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, gum arabic, xanthan gum and sodium alginate.
Optionally, the antioxidant is at least one selected from butyl hydroxy anisole, dibutyl hydroxy toluene, propyl gallate, tert-butyl hydroquinone, tea polyphenols, vitamin E, L-ascorbic acid-6-palmitate, and herba Rosmarini officinalis.
Further, in the embedding step, the wall material: the mass-to-volume ratio of fermentation liquor after fermentation of schizochytrium limacinum strains is (10-50) kg:1000L, the mass-to-volume ratio of emulsifying agent to fermentation liquor after fermentation of schizochytrium limacinum strains is (0.5-5) kg:1000L, and the mass-to-volume ratio of antioxidant to fermentation liquor after fermentation of schizochytrium limacinum strains is (0.1-1.0) kg: 1000L;
optionally, the stirring time is 10-30 min, the shearing time is 10-30 min, and the process control temperature is 40-80 ℃.
Further, in the drying step, spray drying is adopted as a drying mode, the air inlet temperature of the spray drying is 160-200 ℃, and the air outlet temperature of the spray drying is 70-90 ℃;
further, the coating agent is polyacrylic resin IV or a composition formed by the polyacrylic resin IV and at least one of ethyl cellulose and hydrogenated vegetable oil; preferably, the weight ratio of the components of the polyacrylic resin IV is more than or equal to 50 percent;
the coating solution is obtained by dissolving a coating agent in ethanol.
Further, in the coating step, the coating machine is one of a centrifugal granulation coating machine or a fluidized bed coating machine;
further, the acid treatment is carried out at the temperature of 40-60 ℃ for 30-180 min, the pH value of the fermentation liquor is adjusted to 3-5 by adding acid, and the acid is one or more of hydrochloric acid, sulfuric acid or phosphoric acid; adding alkali after acid treatment to adjust the fermented liquid of the schizochytrium limacinum strain to be neutral;
the alkali treatment is carried out at the temperature of 40-60 ℃ for 30-180 min, the pH value of a fermentation liquid obtained after the fermentation of the schizochytrium limacinum strain is adjusted to 9-11 by adding alkali, and the alkali is one or more of sodium hydroxide, potassium hydroxide or ammonia water; adding acid after alkali treatment to adjust the fermented liquid of the schizochytrium limacinum strain to be neutral;
the biological enzyme treatment is carried out at the temperature of 40-60 ℃ for 1-3 h, the enzyme amount is (0.5-2.5) kg:1000L of fermentation liquor after the schizochytrium limacinum strain is fermented, and the enzyme is at least one of alkaline protease, neutral protease, papain, cellulase, β -glucanase and xylanase.
The invention also protects the schizochytrium limacinum powder prepared by the preparation method.
The invention aims to prepare rumen-protected schizochytrium limacinum powder which can be used for feeding ruminants to enable DHA in the schizochytrium limacinum powder to be successfully transferred to milk of the ruminants so as to obtain natural organic milk rich in DHA. The consumption of DHA-rich milk by humans results in human supplementation with DHA.
The invention has the beneficial effects that:
1. the schizochytrium limacinum powder prepared by the method is rich in DHA, and can obtain pure natural DHA milk products through the biotransformation capacity of the schizochytrium limacinum powder → animals → milk products;
2. the schizochytrium limacinum powder is subjected to wall breaking treatment in advance, so that the absorptivity of the schizochytrium limacinum powder in the true stomach and small intestine of a ruminant is greatly improved, and the DHA content of dairy products produced by the ruminant is improved;
3. the schizochytrium limacinum powder wall breaking treatment is obtained by controlling fermentation liquor pretreatment process parameters, the wall breaking condition is mild, and the operation steps are simple; compared with the existing additionally-arranged mechanical wall breaking step, the method can ensure that the microalgae cells are not completely broken, not only ensures the absorptivity of the schizochytrium limacinum powder, but also avoids the leakage of the DHA grease serving as an effective component, and reduces the difficulty of subsequent embedding, drying and coating;
4. the schizochytrium limacinum powder is embedded and coated, the coating layer can enable the schizochytrium limacinum powder to be kept stable in rumens (pH 5-7) and decomposed and released in the true stomach (pH 1-3), so that the schizochytrium limacinum powder can smoothly pass through the rumen and be digested and absorbed in the true stomach and small intestine, the influence on the rumen and the damage to effective molecule DHA are avoided, and the DHA content in ruminant milk is improved.
Drawings
FIG. 1 is a schematic process flow diagram of the present invention.
Detailed Description
Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.
A preparation method of schizochytrium limacinum powder is characterized by comprising the following steps,
embedding: mixing schizochytrium limacinum thallus with wall materials, an emulsifier and an antioxidant, stirring, shearing and homogenizing to obtain a suspension;
and (3) drying: drying the obtained suspension to obtain schizochytrium limacinum powder with the water content within 5%;
coating: transferring the obtained dry powder into a coating machine, and spraying coating agent at 30-40 deg.C to obtain coating solution to obtain schizochytrium limacinum powder;
the schizochytrium limacinum thallus is obtained by performing wall breaking pretreatment of acid treatment, alkali treatment or biological enzyme treatment on fermentation liquor obtained by fermenting schizochytrium limacinum strains.
Further, the Schizochytrium thallus is Schizochytrium sp.CGMCC No.6843 or Schizochytrium sp.ATCC No.20888 or Schizochytrium sp.ATCC No.20889 or Schizochytrium sp.ATCC No.28209 or Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381.
Further, the weight ratio of the thalli to the wall material, the emulsifier, the antioxidant and the coating agent in the schizochytrium limacinum powder is 150: (10-50): (0.5-5): (0.1-1): (5-30);
preferably, the weight ratio of the schizochytrium limacinum thallus to the wall material, the emulsifier, the antioxidant and the coating agent is 150: (10-50): (2-5): (0.25-1): (20-30);
more preferably, the weight ratio of the schizochytrium limacinum thallus to the wall material, the emulsifier, the antioxidant and the coating agent is 150: (25-50): (2-5): (0.5-1): (20-30).
Further, the wall material is at least one of starch sodium octenyl succinate, whey protein, soy protein isolate and sodium caseinate;
optionally, the emulsifier is at least one selected from monoglyceride, polyglyceryl fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, gum arabic, xanthan gum and sodium alginate.
Optionally, the antioxidant is at least one selected from butyl hydroxy anisole, dibutyl hydroxy toluene, propyl gallate, tert-butyl hydroquinone, tea polyphenols, vitamin E, L-ascorbic acid-6-palmitate, and herba Rosmarini officinalis.
Further, in the embedding step, the wall material: the mass-to-volume ratio of fermentation liquor after fermentation of schizochytrium limacinum strains is (10-50) kg:1000L, the mass-to-volume ratio of emulsifying agent to fermentation liquor after fermentation of schizochytrium limacinum strains is (0.5-5) kg:1000L, and the mass-to-volume ratio of antioxidant to fermentation liquor after fermentation of schizochytrium limacinum strains is (0.1-1.0) kg: 1000L;
optionally, the stirring time is 10-30 min, the shearing time is 10-30 min, and the process control temperature is 40-80 ℃.
Further, in the drying step, spray drying is adopted as a drying mode, the air inlet temperature of the spray drying is 160-200 ℃, and the air outlet temperature of the spray drying is 70-90 ℃;
further, the coating agent is polyacrylic resin IV or a composition formed by the polyacrylic resin IV and at least one of ethyl cellulose and hydrogenated vegetable oil; preferably, the weight ratio of the components of the polyacrylic resin IV is more than or equal to 50 percent;
the coating solution is obtained by dissolving a coating agent in ethanol.
Further, in the coating step, the coating machine is one of a centrifugal granulation coating machine or a fluidized bed coating machine;
further, the acid treatment is carried out at the temperature of 40-60 ℃ for 30-180 min, the pH value of the fermentation liquor is adjusted to 3-5 by adding acid, and the acid is one or more of hydrochloric acid, sulfuric acid or phosphoric acid; adding alkali after acid treatment to adjust the fermented liquid of the schizochytrium limacinum strain to be neutral;
the alkali treatment is carried out at the temperature of 40-60 ℃ for 30-180 min, the pH value of a fermentation liquid obtained after the fermentation of the schizochytrium limacinum strain is adjusted to 9-11 by adding alkali, and the alkali is one or more of sodium hydroxide, potassium hydroxide or ammonia water; adding acid after alkali treatment to adjust the fermented liquid of the schizochytrium limacinum strain to be neutral;
the biological enzyme treatment is carried out at the temperature of 40-60 ℃ for 1-3 h, the enzyme amount is (0.5-2.5) kg:1000L of fermentation liquor after the schizochytrium limacinum strain is fermented, and the enzyme is at least one of alkaline protease, neutral protease, papain, cellulase, β -glucanase and xylanase.
The detection basis is as follows:
DHA content: GB 26400 plus 2011 docosahexaenoic acid oil (fermentation method) which is a national standard food additive for food safety;
creaminess (fat content): GB5009.6-2016 determination of fat in food products in national standards of food safety.
Example 1: preparation of DHA fermentation broth
Inoculating the Schizochytrium sp.CGMCC No.6843 slant preserved strain into a 2L shake flask filled with 400mL seed culture medium, and culturing at 25 deg.C and 200rpm for 24h to complete strain activation culture. Inoculating the shake flask seed solution into a primary seed fermentation tank filled with the sterilized seed culture medium according to the inoculation amount of 0.5%, and culturing at 28 deg.C, ventilation amount of 1vvm and stirring speed of 50rpm for 30h to complete primary seed amplification culture. Inoculating the seed solution of the first-stage seed tank into a second-stage seed fermentation tank filled with a sterilized seed culture medium according to the inoculation amount of 5%, and culturing at 28 ℃, the ventilation volume of 1vvm and the stirring speed of 75rpm for 24h to complete the second-stage seed amplification culture. And (3) inoculating the seed solution of the secondary seed tank into a fermentation tank filled with a sterilized fermentation medium according to the inoculation amount of 3%, wherein the culture temperature is 28 ℃, the ventilation rate is 1vvm, the stirring speed is 100rpm, the glucose concentration is controlled at 5g/L by feeding a carbon source, a nitrogen source is supplemented by feeding, and the fermentation is terminated after 96h of culture to obtain DHA fermentation liquor with the microbial thallus content of 150 g/L.
The seed culture medium comprises the following components: 60g/L of glucose, 10g/L of yeast extract, 15g/L of sodium chloride, 4g/L of magnesium sulfate, 1g/L of calcium chloride, 0.5g/L of monopotassium phosphate, 0.2g/L of ammonium sulfate and 0.08g/L of sodium bicarbonate.
The fermentation medium comprises the following components: 60g/L of glucose, 10g/L of corn starch, 15g/L of sodium chloride, 4g/L of magnesium sulfate, 1g/L of calcium chloride, 0.5g/L of monopotassium phosphate, 0.2g/L of ammonium sulfate, 0.01g/L of zinc sulfate, 0.08g/L of sodium bicarbonate, 10.01mg/L of vitamin B and 60.01mg/L of vitamin B.
Example 2: preparation of schizochytrium limacinum powder A
Taking 10m of DHA fermentation broth obtained in example 13Adding caustic soda flakes to adjust the pH value to 10.0, heating to 50 ℃, stirring for 60min, and adding hydrochloric acid to adjust the pH value to be neutral. And then adding 50kg of starch sodium octenyl succinate, 50kg of whey protein, 50kg of monoglyceride, 0.5kg of butyl hydroxyanisole, 0.25kg of propyl gallate and 0.25kg of vitamin E, stirring at 40 ℃ for 30min, shearing at 2900r/min for 10min to obtain a stable suspension, then carrying out spray drying, wherein the air inlet temperature of the spray drying is 160-180 ℃, and the air outlet temperature is 70-80 ℃ to obtain dried bacterial powder.
150kg of polyacrylic resin IV and 50kg of ethyl cellulose are added into 1000L of ethanol, and the mixture is stirred and dissolved uniformly to obtain a coating solution. Transferring the dried bacterial powder into a coating machine, spraying coating liquid, and controlling the temperature of the spraying coating liquid to be 30 ℃; the schizochytrium limacinum powder A is 1760kg, and has the DHA content of 16.5 percent, the crude protein content of 23.5 percent, the fat content of 37.2 percent and the water content of 3.3 percent.
Example 3: preparation of schizochytrium limacinum powder B
Taking 10m of DHA fermentation broth obtained in example 13Adding 10kg of alkaline protease and 5kg of cellulase, and heating at 50 deg.C for 120min to break cell wall. And then adding 150kg of starch sodium octenyl succinate, 100kg of sodium caseinate, 20kg of polyglycerol fatty acid ester, 2.5kg of tert-butyl hydroquinone and 2.5kg of rosemary, heating to 60 ℃, stirring for 20min, shearing for 20min at 2900r/min to obtain a stable suspension, then performing spray drying, wherein the air inlet temperature of the spray drying is 170-190 ℃, and the air outlet temperature is 70-80 ℃ to obtain dried bacterial powder.
150kg of polyacrylic resin IV and 50kg of ethyl cellulose are added into 1000L of ethanol, and the mixture is stirred and dissolved uniformly to obtain a coating solution. Transferring the dried bacteria powder into a coating machine, spraying coating liquid, and controlling the temperature of the spraying coating liquid to be 40 ℃ to obtain schizochytrium limacinum powder B1980kg, wherein the DHA content by weight, the crude protein content by weight, the fat content by weight, 32.7% by weight and the water content by weight are 14.5%, 23.1%, 32.7% by weight and 3.8%.
Example 4 preparation of Schizochytrium powder C
Taking 10m of DHA fermentation broth obtained in example 13Adding hydrochloric acid to adjust pH to 3.5, heating to 60 deg.C, stirring for 90min, and adding caustic soda flakes to adjust pH to neutral. Then 200kg of whey protein, 50kg of isolated soy protein, 20kg of sucrose fatty acid ester, 5kg of gum arabic, 2.5kg of L-ascorbic acid-6-palmitate and 7.5kg of vitamin E are added, the mixture is heated to 80 ℃, stirred for 10min, sheared at 2900r/min for 20min to obtain stable suspension, and then spray-dried, the air inlet temperature of the spray-drying is 180-200 ℃, the air outlet temperature is 70-80 ℃ to obtain dried bacterial powder.
150kg of polyacrylic resin IV and 50kg of ethyl cellulose are added into 1000L of ethanol, and the mixture is stirred and dissolved uniformly to obtain a coating solution. Transferring the dried fungus powder into a coating machine, spraying a coating solution, and controlling the temperature of the spraying coating solution to be 35 ℃ to obtain schizochytrium limacinum powder C1800kg with the DHA content of 15.8 wt%, the crude protein content of 21.1 wt%, the fat content of 35.1 wt% and the water content of 3.7 wt%.
Example 5 preparation of Schizochytrium powder D
Taking 10m of DHA fermentation broth obtained in example 13Adding 10kg of alkaline protease and 5kg of cellulase, and heating at 50 deg.C for 120min to break cell wall. And then adding 200kg of starch sodium octenyl succinate, 300kg of whey protein, 15kg of sorbitan fatty acid ester, 5kg of xanthan gum, 2.5kg of tert-butyl hydroquinone and 2.5kg of tea polyphenol, heating to 60 ℃, stirring for 30min, shearing for 20min at 2900r/min to obtain stable suspension, then performing spray drying, wherein the air inlet temperature of the spray drying is 170-180 ℃, and the air outlet temperature is 70-80 ℃ to obtain dried bacterial powder.
Adding 300kg of polyacrylic resin IV into 1500L of ethanol, and stirring to dissolve uniformly to obtain a coating solution. Transferring the dried bacteria powder into a coating machine, spraying coating liquid, and controlling the temperature of the spraying coating liquid to be 30 ℃ to obtain schizochytrium limacinum powder D2120kg, wherein the DHA content by weight, the crude protein content by weight is 23.8%, the fat content by weight is 30.8%, and the water content by weight is 2.8%.
Example 6 preparation of Schizochytrium powder E
Taking 10m of DHA fermentation broth obtained in example 13Adding 10kg of alkaline protease and 5kg of cellulase, and heating at 50 deg.C for 120min to break cell wall. And then adding 150kg of starch sodium octenyl succinate, 100kg of whey protein, 3kg of polyglycerol fatty acid ester, 2kg of sodium alginate, 2kg of vitamin E and 0.5kg of rosemary, heating to 60 ℃, stirring for 30min, shearing for 30min at 2900r/min to obtain a stable suspension, then performing spray drying, wherein the air inlet temperature of the spray drying is 170-180 ℃, and the air outlet temperature is 70-80 ℃ to obtain dried bacterial powder.
Adding 40kg of polyacrylic resin IV and 10kg of hydrogenated vegetable oil into 500L of ethanol, and stirring and dissolving uniformly to obtain a coating solution. Transferring the dried bacteria powder into a coating machine, spraying coating liquid, and controlling the temperature of the spraying coating liquid to be 35 ℃ to obtain schizochytrium limacinum powder E1680kg, wherein the weight content of DHA is 16.8%, the weight content of crude protein is 22.3%, the weight content of fat is 37.5%, and the weight content of water is 2.5%.
Comparative example 1 preparation of Schizochytrium powder F (fermentation broth not subjected to wall breaking treatment)
Taking 10m of DHA fermentation broth obtained in example 13200kg of starch sodium octenyl succinate, 50kg of sodium caseinate, 20kg of polyglycerol fatty acid ester, 2.5kg of tert-butyl hydroquinone and 2.5kg of rosemary are added, the mixture is heated to 60 ℃ and stirred for 30min, the mixture is cut at 2900r/min for 30min to obtain stable suspension, then spray drying is carried out, the air inlet temperature of the spray drying is 170-180 ℃, the air outlet temperature is 70-80 ℃, and the dried mushroom powder is obtained.
150kg of polyacrylic resin IV and 50kg of ethyl cellulose are added into 1000L of ethanol, and the mixture is stirred and dissolved uniformly to obtain a coating solution. Transferring the dried fungus powder into a coating machine, spraying a coating solution, and controlling the temperature of the spraying coating solution to be 35 ℃ to obtain schizochytrium limacinum powder F1800kg with the DHA content of 15.4 wt%, the crude protein content of 22.8 wt%, the fat content of 34.5 wt% and the water content of 3.0 wt%.
Comparative example 2 preparation of Schizochytrium powder G (without coating treatment)
Taking 10m of DHA fermentation broth obtained in example 13Adding 10kg of alkaline protease and 5kg of cellulase, and heating at 50 deg.C for 120min to break cell wall. Then 200kg of starch sodium octenyl succinate, 50kg of sodium caseinate, 20kg of polyglycerol fatty acid ester, 2.5kg of tert-butyl hydroquinone and 2.5kg of rosemary are added, the mixture is heated to 60 ℃ and stirred for 30min, and the mixture is sheared for 30min at 2900r/min to obtain stable suspension, and then the suspension is subjected to spray drying, the air inlet temperature of the spray drying is 170-180 ℃, the air outlet temperature is 70-80 ℃, and dried bacterial powder, namely schizochytrium powder G1620kg, the DHA content of the powder is 17.1%, the weight content of crude protein is 20.5%, the weight content of fat is 38.9%, and the weight content of water is 3.2%.
Comparative example 3 preparation of Schizochytrium limacinum powder H (fermentation broth processed by sand mill for breaking cell wall, enteric coating agent for coating)
Taking 10m of DHA fermentation broth obtained in example 13The wall breaking treatment is carried out by adopting a sand mill, zirconium beads are used as grinding media, and the thalli flow out of the sand mill after being ground to obtain the fermentation liquor with the grain diameter of 1-10 mu m. Then adding 200kg of starch sodium octenyl succinate, 50kg of sodium caseinate, 20kg of polyglycerol fatty acid ester, 2.5kg of tert-butyl hydroquinone and 2.5kg of rosemary, heating to 60 ℃, stirring for 30min, shearing for 30min at 2900r/min to obtain stable suspension, then removing spray drying,and (3) spray drying, wherein the air inlet temperature is 170-180 ℃, and the air outlet temperature is 70-80 ℃, so as to obtain dried bacterial powder.
Dissolving hydroxypropyl methylcellulose phthalate 300kg with 80-90 deg.C purified water to obtain 20 wt% solution, and coating. Transferring the dried bacteria powder into a coating machine, spraying coating liquid, and controlling the temperature of the spraying coating liquid to be 35 ℃ to obtain schizochytrium limacinum powder H1820kg with the DHA content of 15.0%, the crude protein content of 21.1%, the fat content of 34.1% and the water content of 4.5%.
Example 7 cudweed powder rumen bypass test
Rumen bypass test was performed on schizochytrium limacinum powder A, B, C, F, G, H by nylon bag method.
Selecting adult healthy Holstein cattle, installing a permanent rumen fistula, and feeding conventional Total Mixed Ration (TMR) according to the normal nutritional requirement of the daily cow. The test cows were each in the morning 07: 00 and 17 pm: 00 feeding in equal amount.
A nylon sieve silk net with 300 meshes is selected to be made into nylon bags with the diameter of 12cm multiplied by 8 cm. The nylon bag is put into the rumen in advance for balancing for 72h, cleaned, dried to constant weight, weighed and recorded. Each bag is filled with 8g of sample, dried for 48h at 65 ℃ to constant weight and accurately weighed. The other end of the plastic hose is fixed on the fistula plug by a nylon rope. Taking out the nylon bags from the rumen of the fistula cattle after 0h, 2h, 6h, 12h, 24h, 36h and 48h respectively, slowly washing in clear water for 10min until the water flow is clean, drying in an oven at 65 ℃ for 48h to constant weight, and weighing. And calculating the dry matter and DHA degradation rate of the microalgae product according to the mass difference before and after digestion and the DHA content change. 3 cattle per sample, 1 for each cattle, and 2 nylon bags are put into each sample to be detected as parallel samples at each time point of each cattle.
Percent dry matter degradation%
DHA degradation rate%
The rumen bypass test results are shown in tables 1 and 2, the schizochytrium limacinum powder G has the highest degradation rate of dry matter and DHA, the schizochytrium limacinum powder H is the next highest, the schizochytrium limacinum powder A, B, C has a very low degradation rate, and the schizochytrium limacinum powder F has a slightly higher degradation rate than the schizochytrium limacinum powder A, B, C. Therefore, the schizochytrium limacinum powder (namely the schizochytrium limacinum powder A, B, C) obtained by the preparation process has the best rumen passing effect, the schizochytrium limacinum powder (namely the schizochytrium limacinum powder F) prepared by the non-wall-breaking process has lower degradation rate, and the schizochytrium limacinum powder (namely the schizochytrium limacinum powder H) obtained by wall-breaking treatment of a sand mill and coating of an enteric coating agent and the schizochytrium limacinum powder (namely the schizochytrium limacinum powder G) obtained by non-coating treatment have poorer rumen passing effects.
TABLE 1 rumen dry matter degradation Rate% for different microalgae products
TABLE 2 rumen DHA degradation Rate% for different microalgae products
Example 8 feeding test of schizochytrium limacinum
Healthy Holstein cows of 70 gestational age near to the mid-lactation period were selected and divided into 7 groups, the first 6 groups were experimental groups, and the 7 th group was a control group. The experimental groups 1, 2, 3, 4, 5 and 6 were fed with schizochytrium limacinum powder A, B, C, F, G, H, respectively, and the control group was fed with conventional Total Mixed Ration (TMR). Feeding 2 times daily (6: 30 in the morning and 6:30 in the evening), feeding 200 g/day of microalgae powder to each cow, and mixing Schizochytrium limacinum powder with TMR feed before feeding. In the pre-feeding period of 10 days, the adding amount of schizochytrium limacinum powder is gradually increased from 20 g/(first day) to 200 g/(first day), in the formal period of 2 months, the feeding amount of schizochytrium limacinum powder is 200 g/(first day). The milk samples of each cow are collected before the pre-test period, at the end of the pre-test period and in the official period for 7, 15, 30, 45 and 60 days respectively. Milking for 2 times every day, and collecting milk sample during milking of cow by using an automatic sampler, measuring milk fat percentage and DHA content, and recording daily milk yield of each cow.
TABLE 3 milk yield data sheet
TABLE 4 milk fat Rate data sheet
TABLE 5 data table of DHA content in milk
From the feeding results, the DHA content in the milk of the experimental groups 1, 2, 3, 4, 5 and 6 is obviously improved compared with that of the control group. The DHA content in the milk obtained by the experimental group 1, the experimental group 2 and the experimental group 3 (namely feeding schizochytrium limacinum powder A, B, C) is the highest, and compared with the control group, the milk yield and the milk fat rate have no abnormal change; the DHA content in the milk obtained by the experimental group 6 (namely feeding schizochytrium limacinum powder H) is not as high as that of the experimental group 1 and the experimental group 2, the milk yield and the milk fat rate are reduced in the later period of feeding, which indicates that the wall breaking degree is too violent, and the coating agent is not properly selected, so that a part of unsaturated fatty acid in the schizochytrium limacinum powder is degraded and damaged in the rumen, the absorption and conversion effects of the DHA are influenced, and the milk yield and the milk fat rate of the dairy cow are also influenced; the DHA content in the milk obtained by the experimental group 5 (namely feeding schizochytrium limacinum powder G) is low, and the milk yield and the milk fat rate are also obviously reduced in the later period of feeding, which indicates that the unsaturated fatty acid in the schizochytrium limacinum powder is easily degraded and damaged in the rumen without coating treatment, the absorption and conversion effects of the DHA are also influenced, and the milk yield and the milk fat rate are influenced; although the milk yield and the milk fat rate of the experimental group 4 (namely feeding schizochytrium limacinum powder F) are not affected, the DHA conversion rate is the lowest, which indicates that the absorption and conversion effects of DHA of the schizochytrium limacinum powder are poor without wall breaking treatment.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made in the above embodiments by those of ordinary skill in the art without departing from the principle and spirit of the present invention.
Claims (10)
1. A preparation method of schizochytrium limacinum powder is characterized by comprising the following steps,
embedding: mixing schizochytrium limacinum thallus with wall materials, an emulsifier and an antioxidant, stirring, shearing and homogenizing to obtain a suspension;
and (3) drying: drying the obtained suspension to obtain schizochytrium limacinum powder with the water content within 5%;
coating: transferring the obtained dry powder into a coating machine, and spraying coating agent at 30-40 deg.C to obtain coating solution to obtain schizochytrium limacinum powder;
the schizochytrium limacinum thallus is obtained by performing wall breaking pretreatment of acid treatment, alkali treatment or biological enzyme treatment on fermentation liquor obtained by fermenting schizochytrium limacinum strains.
2. The method for producing Schizochytrium limacinum powder according to claim 1, wherein the Schizochytrium limacinum thallus is Schizochytrium sp.CGMCC No.6843, Schizochytrium sp.ATCC No.20888, Schizochytrium sp.ATCC No.20889, Schizochytrium sp.ATCC No.28209, or Schizochytrium limacinum Honda et Yokochi ATCC MYA-1381.
3. The method for preparing schizochytrium limacinum powder as claimed in claim 1, wherein the weight ratio of thallus to wall material, emulsifier, antioxidant and coating agent in the schizochytrium limacinum powder is 150: (10-50): (0.5-5): (0.1-1): (5-30);
preferably, the weight ratio of the schizochytrium limacinum thallus to the wall material, the emulsifier, the antioxidant and the coating agent is 150: (10-50): (2-5): (0.25-1): (20-30);
more preferably, the weight ratio of the schizochytrium limacinum thallus to the wall material, the emulsifier, the antioxidant and the coating agent is 150: (25-50): (2-5): (0.5-1): (20-30).
4. The preparation method of schizochytrium limacinum powder as claimed in claim 1, wherein the wall material is at least one of starch sodium octenyl succinate, whey protein, isolated soy protein, and sodium caseinate;
optionally, the emulsifier is at least one selected from monoglyceride, polyglycerol fatty acid ester, sorbitan fatty acid ester, sucrose fatty acid ester, gum arabic, xanthan gum and sodium alginate;
optionally, the antioxidant is at least one selected from butyl hydroxy anisole, dibutyl hydroxy toluene, propyl gallate, tert-butyl hydroquinone, tea polyphenols, vitamin E, L-ascorbic acid-6-palmitate, and herba Rosmarini officinalis.
5. The method for preparing schizochytrium limacinum powder according to claim 1, wherein in the embedding step, the wall material: the mass-to-volume ratio of fermentation liquor after fermentation of schizochytrium limacinum strains is (10-50) kg:1000L, the mass-to-volume ratio of emulsifying agent to fermentation liquor after fermentation of schizochytrium limacinum strains is (0.5-5) kg:1000L, and the mass-to-volume ratio of antioxidant to fermentation liquor after fermentation of schizochytrium limacinum strains is (0.1-1.0) kg: 1000L;
optionally, the stirring time is 10-30 min, the shearing time is 10-30 min, and the process control temperature is 40-80 ℃.
6. The preparation method of schizochytrium limacinum powder as claimed in claim 1, wherein in the drying step, the drying mode is spray drying, the air inlet temperature of the spray drying is 160-200 ℃, and the air outlet temperature is 70-90 ℃.
7. The method for preparing schizochytrium limacinum powder according to claim 1, wherein the coating agent is polyacrylic resin IV or a composition of polyacrylic resin IV and at least one of ethyl cellulose and hydrogenated vegetable oil; preferably, the weight ratio of the components of the polyacrylic resin IV is more than or equal to 50 percent;
the coating solution is obtained by dissolving a coating agent in ethanol.
8. The method for preparing schizochytrium limacinum powder according to claim 1, wherein in the coating step, the coating machine is one of a centrifugal granulation coating machine and a fluidized bed coating machine.
9. The preparation method of schizochytrium limacinum powder as claimed in claim 1, wherein the acid treatment is carried out at 40-60 ℃ for 30-180 min, and the pH value of the fermentation liquid is adjusted to 3-5 by adding acid, wherein the acid is one or more of hydrochloric acid, sulfuric acid or phosphoric acid; adding alkali after acid treatment to adjust the fermented liquid of the schizochytrium limacinum strain to be neutral;
the alkali treatment is carried out at the temperature of 40-60 ℃ for 30-180 min, the pH value of a fermentation liquid obtained after the fermentation of the schizochytrium limacinum strain is adjusted to 9-11 by adding alkali, and the alkali is one or more of sodium hydroxide, potassium hydroxide or ammonia water; adding acid after alkali treatment to adjust the fermented liquid of the schizochytrium limacinum strain to be neutral;
the biological enzyme treatment is carried out at the temperature of 40-60 ℃ for 1-3 h, the enzyme amount is (0.5-2.5) kg:1000L of fermentation liquor after the schizochytrium limacinum strain is fermented, and the enzyme is at least one of alkaline protease, neutral protease, papain, cellulase, β -glucanase and xylanase.
10. Schizochytrium limacinum powder prepared by the preparation method of any one of claims 1 to 9.
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