CN109077194B - Application of schizochytrium limacinum and schizochytrium limacinum preparation in improving egg quality - Google Patents
Application of schizochytrium limacinum and schizochytrium limacinum preparation in improving egg quality Download PDFInfo
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Abstract
The invention discloses schizochytrium limacinum and application of a schizochytrium limacinum preparation in improving egg quality. The preservation number of the schizochytrium limacinum in the China general microbiological culture Collection center is CGMCC No. 13746. The schizochytrium limacinum powder prepared by the schizochytrium limacinum can improve the DHA content in eggs and can reduce the cholesterol content in the eggs. The natural-source egg 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 ingest natural DHA, can meet and meet the requirements of consumers, and has wide application value.
Description
Technical Field
The invention relates to the field of biotechnology, and discloses application of schizochytrium limacinum and a schizochytrium limacinum preparation in improving egg quality.
Background
DHA (docosahexaenoic acid) belongs to omega-3 series polyunsaturated fatty acids (omega-3 PUFAs), is an important component substance of human cell membranes and nervous tissues, has important physiological functions of strengthening brain, improving intelligence, promoting development of optic nerves, preventing and treating senile dementia and the like, and plays an important role in promoting growth and development of infants, preventing cardiovascular diseases, inhibiting and treating certain cancers, ensuring normal work of nervous systems and the like.
With the improvement of living standard and consumption level and the continuous and deep research of DHA physiological function, the DHA intake problem of different groups of people is more and more concerned. According to the Chinese DHA food condition survey completed by the Chinese food society, the intake of DHA directly from food by Chinese people per day is only about 40mg, and the DHA is in a severe hunger state. It is well known that DHA is obtained from diet, and especially dairy products are common, and the annual demand of milk products such as cow milk and goat milk rich in high-quality DHA is increasing, so that the intake of high-quality DHA from milk products is a trend.
The DHA content in the common milk is extremely low, and the daily requirements of people are difficult to meet, but the exogenous addition of DHA in the milk product has the problems of more required materials, higher consumption of production cost of enterprises, easy DHA price reduction, decomposition or generation of peculiar smell and the like in the adding process. The DHA content in milk and muscle tissues can be improved by properly increasing the intake of polyunsaturated fatty acids such as DHA in the daily ration of the ruminant, but the special digestion structure of the ruminant enables most of the polyunsaturated fatty acids such as DHA to be converted into saturated fatty acids after entering the rumen, thereby greatly reducing the utilization rate of the polyunsaturated fatty acids such as DHA. The commercial fatty acid rumen protection technology mainly comprises coating, hydrogenation, calcification and the like, so that the treatment technology is difficult, the production cost is high, and the defects of reduction of daily feed intake, reduction of digestion and absorption rate, generation of milk fat reduction syndrome, even toxic and side effects and the like exist. Therefore, the problem of how to increase the content of DHA in the milk of mammals, especially ruminants, and further utilize the transformation of the ruminant organism to produce natural organic milk rich in DHA is an urgent need to be solved at present.
In addition, by increasing the content of DHA in the eggs, the source of DHA can be enriched, and the application field and consumption range of DHA are greatly expanded.
Disclosure of Invention
The technical problem to be solved by the invention is how to improve the quality of animal products.
In order to solve the technical problems, the invention firstly provides any one of the following applications of Schizochytrium limacinum (Schizochytrium limacinum) or a preparation thereof:
A1) application in improving the quality of animal products;
A2) application in preparing substances for improving quality of animal products.
The Schizochytrium limacinum (Schizochytrium limacinum) can be Schizochytrium limacinum HS01, and the preservation number of the Schizochytrium limacinum HS01 in the China general microbiological culture Collection center of the culture Collection of microorganisms is CGMCC No. 13746.
The active ingredient of the formulation may be the Schizochytrium limacinum.
In the above application, the animal may be poultry. The animal may further be a chicken. The chicken is further Beijing white chicken, Hailan brown laying hen or Hailan pink shell chicken.
In the above application, the animal product may be an egg produced by the animal.
In the above application, the improving the quality of the animal product may be improving the DHA content of the animal product and/or reducing the cholesterol content of the animal product.
In the above application, the preparation can be prepared according to a method (which is referred to as a preparation method of the schizochytrium limacinum preparation) comprising the following steps: culturing the Schizochytrium limacinum (Schizochytrium limacinum) to obtain a fermentation broth; and preparing the preparation by using the fermentation liquor.
In the application, the Schizochytrium limacinum (Schizochytrium limacinum) can be cultured by using a fermentation culture medium, the fermentation culture medium consists of a solvent and a solute, the solvent is water, and the solute and the concentration thereof are respectively 60-150 g/L of glucose, 8-25 g/L of yeast extract and 3-8 g/L, Na g/L of yeast powder2SO4 5～20g/L、KCl 0.5～1.5g/L、MgSO41.0～3.0g/L、K2SO4 0.5～2.5g/L、KH2PO4 1.0～2.0g/L、(NH4)2SO4 2.0～5.0g/L、CaCl2 0.5～2.5g/L、CuSO4 0.001～0.02g/L、ZnSO40.001-0.02 g/L, biotin 0.001-0.06 g/L, starch 0.1-10 g/L and albumen powder 0-20 g/L, and the pH value is 4.5-6.5.
The starch can be corn starch or sodium starch octenyl succinate, and the protein powder can be pea protein powder or whey protein powder. The pH of the fermentation medium may specifically be 6.
The solutes and their concentrations in the fermentation medium may be specifically n1) or n2) or n3) or n4) as follows:
n1) glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na g2SO4 5g/L、KCl 0.5g/L、MgSO41.0g/L、K2SO4 0.5g/L、KH2PO4 1.0g/L、(NH4)2SO4 2.0g/L、CaCl2 0.5g/L、CuSO4 0.001g/L、ZnSO40.001g/L, biotin 0.001g/L and corn starch 0.1 g/L;
n2) glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na g2SO4 20g/L、KCl 1.5g/L、MgSO43.0g/L、K2SO4 2.5g/L、KH2PO4 2.0g/L、(NH4)2SO4 5.0g/L、CaCl2 2.5g/L、CuSO4 0.02g/L、ZnSO40.02g/L, 0.06g/L biotin, 10g/L corn starch and 20g/L pea protein powder;
n3) glucose 60g/L, yeast extract 8g/L, yeast powder 3g/L, Na g2SO4 5g/L、KCl 0.5g/L、MgSO41.0g/L、K2SO4 0.5g/L、KH2PO4 1.0g/L、(NH4)2SO4 2.0g/L、CaCl2 0.5g/L、CuSO4 0.001g/L、ZnSO40.001g/L, biotin 0.001g/L and starch sodium octenyl succinate 0.1 g/L;
n4) glucose 150g/L, yeast extract 25g/L, yeast powder 8g/L, Na g2SO4 20g/L、KCl 1.5g/L、MgSO43.0g/L、K2SO4 2.5g/L、KH2PO4 2.0g/L、(NH4)2SO4 5.0g/L、CaCl2 2.5g/L、CuSO4 0.02g/L、ZnSO40.02g/L, biotin 0.06g/L, sodium starch octenyl succinate 10g/L and whey protein powder 20 g/L.
In the above application, the preparation prepared by using the fermentation broth may include: drying the fermentation liquor to obtain the preparation.
The method can also comprise adding an antioxidant into the fermentation liquor after the fermentation liquor is obtained, and then drying to obtain the schizochytrium limacinum powder.
The antioxidant may be an oil soluble antioxidant and/or a water soluble antioxidant. The oil-soluble antioxidant can be rosemary, natural mixed tocopherol, tea polyphenol and/or ascorbyl palmitate. The water soluble antioxidant may be phytic acid, ascorbic acid and/or erythorbic acid.
When the antioxidant consists of a plurality of different specific antioxidants, the proportion of the components is not required, and can be adjusted according to specific requirements.
The antioxidant is a mixed antioxidant composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid. The mixture ratio of the substances in the mixed antioxidant can be p1), p2), p3) or p4) as follows:
p1) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 20:2:10:10: 2;
p2) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 40:3:20:20: 4;
p3) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 60:2:40:30: 6;
p4) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 80:2:40:40: 8.
The drying may be spray drying or drum drying or freeze drying.
The above method may further comprise washing the Schizochytrium limacinum (Schizochytrium limacinum) in the fermentation broth. The method may further comprise adding the antioxidant to the washed Schizochytrium limacinum (Schizochytrium limacinum) and drying.
The dissolved oxygen amount for the cultivation may be 0 to 80% (e.g., 10 to 80%). The temperature of the culture can be 20-30 ℃. The culture time can be 72-120 h.
The preparation method of the schizochytrium limacinum preparation also belongs to the protection scope of the invention.
Any of the following products is also within the scope of the present invention:
y1) medium for culturing the Schizochytrium limacinum (Schizochytrium limacinum), being the fermentation medium;
y2) the formulation.
In order to solve the above technical problems, the present invention also provides a method for improving the quality of an animal product, comprising: feeding animal Schizochytrium limacinum (Schizochytrium limacinum) or a preparation thereof to achieve an improvement in the quality of the animal product.
The Schizochytrium limacinum (Schizochytrium limacinum) may be Schizochytrium HS 01.
The active ingredient of the preparation is the Schizochytrium limacinum (Schizochytrium limacinum).
In the above method, the animal may be poultry. The animal may further be a chicken. The chicken is further Beijing white chicken, Hailan brown laying hen or Hailan pink shell chicken.
In the above method, the animal product may be an egg produced by the animal.
In the above method, the improving the quality of the animal product may be improving the DHA content of the animal product and/or reducing the cholesterol content of the animal product.
In the above method, the preparation can be prepared by the preparation method of the schizochytrium limacinum preparation.
In the present invention, the formulation may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier can be a mineral material, a plant material or a high molecular compound; the mineral material may be at least one of clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth; the plant material may be at least one of corn flour, bean flour and starch; the high molecular compound may be polyvinyl alcohol and/or polyglycol. The liquid carrier can be an organic solvent, vegetable oil, mineral oil, or water; the organic solvent may be decane and/or dodecane. In the microbial inoculum, the active ingredient may be present in the form of cultured living cells, a fermentation broth of living cells, a filtrate of a cell culture, or a mixture of cells and a filtrate. The composition can be prepared into various dosage forms, such as liquid, emulsion, suspending agent, powder, granules, wettable powder or water dispersible granules. Specifically, the schizochytrium preparation can be schizochytrium powder.
The Schizochytrium limacinum powder prepared by the Schizochytrium limacinum can improve the DHA content in animal products and can reduce the cholesterol content in eggs. The natural-source high-DHA-content egg 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 have wide application value.
Biological material preservation instructions
Classification nomenclature of biological materials: schizochytrium limacinum
Strain number of biological material: HS01
Deposit name of biological material: china general microbiological culture Collection center
The preservation unit of the biological material is abbreviated as: CGMCC (China general microbiological culture Collection center)
Deposit unit address of biological material: west road No.1, north west of the township, beijing, ministry of sciences, china, institute of microbiology, zip code: 100101
Preservation date of biological material: 3 and 10 months in 2017
Accession number to the collection of biological materials: CGMCC No.13746
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.
Screening a liquid culture medium: 50g of glucose and 15g of yeast powder are dissolved in 1L of mixed solution (formed by mixing 1 volume of natural seawater and 1 volume of distilled water), and the pH value is natural.
Screening the plates: the screened solid medium at about 55 ℃ is poured into a petri dish and cooled to obtain a solid plate.
Fermentation medium: mixing glucose 60g, glutamic acid or sodium glutamate 10g, corn steep liquor dry powder 10g, and NaSO4 14g、KCl 0.5g、MgSO4 2.0g、K2SO4 1.0g、KH2PO4 1.0g、(NH4)2SO41.0g and CaCl20.5g of this solution was dissolved in 1L of distilled water, and the pH was adjusted to 6.0.
Wort agar medium: dissolving 150g of malt extract powder in 1L of mixed solution (prepared by mixing 1 volume of natural seawater and 1 volume of distilled water), wherein the pH value is natural; agar powder was then added to a concentration of 15g/100mL to obtain a medium.
The natural mixed tocopherol is a product of ADM company, and the product number is MTS-90; rosemary is a product of Guangzhou division of the Korea trade (Shanghai) of Kenai Europe, Inc., with a product number of ROSEMARY 41-19-58; the tea polyphenol is a product of biological engineering limited company of Fujianlikuan, and the product number is TP-98; the isoascorbic acid is a product of Zhengzhou Tuoyang experiment Limited company, and the cargo number is more than or equal to 98 percent; the phytic acid is a product of VargyVer bioengineering company Limited in Laiyang city.
Example 1 isolation and identification of Schizothorax (HS) 01
Separation of first, fission pot HS01
1. The inventor collects schizonts at multiple places of mangrove forest in Yuanxiao county of Zhangzhou city in Fujian province, and mixes the schizonts to obtain a mixed solution; inoculating 0.5mL of the mixed solution to 5mL of a screening liquid culture medium, and then culturing at 25 ℃ and 200rpm/min for 2d to obtain a culture solution.
2. And (3) uniformly coating the culture bacterial liquid obtained in the step (1) on a screening plate, and performing static culture at 25 ℃ for 2d to generate a single bacterial colony.
3. After the step 2 is completed, single colonies are respectively selected and inoculated to 5mL of fermentation culture medium, and then cultured for 2d at 25 ℃ and 200rpm/min to obtain culture bacteria liquid.
4. And (4) centrifuging the culture solution obtained in the step (3) at 4 ℃ and 2000rpm for 5min, and collecting thalli.
5. Taking 1.0-2.0 g of the thalli to a measuring cylinder with a plug (the specification is 100mL), firstly adding 15mL of HCl aqueous solution with the concentration of 8.3mol/L, covering a cover, and hydrolyzing in a water bath at 70-80 ℃ for 50-60 min (during the hydrolysis, placing the measuring cylinder with the plug on a vortex mixer for 1 time every 10 min); after cooling to room temperature, firstly adding 10mL of 95% (v/v) ethanol aqueous solution, sufficiently and uniformly shaking, then adding 20mL of anhydrous ether, sufficiently shaking for extraction for 1-2 min, finally adding 20mL of petroleum ether, sufficiently shaking for extraction for 1-2 min, standing for layering, placing the upper organic phase in a glass weighing dish (dried and weighed), placing the glass weighing dish on a boiling water bath in a ventilation cabinet to sufficiently evaporate the organic phase completely (sufficiently and completely volatilize completely), and obtaining the liquid phase as the grease.
6. And (3) taking the grease extracted in the step (5), detecting the DHA content according to the national food safety standard GB 26400-2011, and detecting the composition and the content of fatty acid according to the method of AOAC 996.06.
And selecting strains with higher DHA content, and repeatedly purifying for 24 times. The screened schizochytrium strain is named as schizochytrium HS 01.
The schizochytrium HS01 was inoculated monoclonally to the fermentation medium for 12 serial passages and the DHA content was determined as described above. The result shows that the stability of DHA produced by schizochytrium HS01 is good.
II, identification of schizochytrium HS01
1. Morphological identification
And inoculating schizochytrium HS01 to a wort agar culture medium, performing dark culture at 25 ℃, observing the morphology of a bacterial colony after 5 days, and analyzing and observing the morphological characteristics of the thallus by a high-resolution transmission electron microscope.
The result shows that the colony diameter of the schizochytrium HS01 is 2-4.3 mm, the schizochytrium HS01 is white (light orange at the later stage), and the edge is irregular; the thallus is proliferated in a fission mode, the cell wall is thin, spherical, colorless or light orange, transparent and 4.5-15.5 mu m in size, and zoospores and an exoplasmic reticulum are not seen.
2. 18s rDNA sequence homology analysis
The partial sequence of the 18s rDNA of the schizochytrium HS01 is shown as the sequence 1 in the sequence table.
The partial sequence of the 18s rDNA of the schizochytrium HS01 is shown as the sequence 2 in the sequence table.
Combining the above identification results, schizochytrium limacinum HS01 was schizochytrium (Schizochytrium limacinum).
Third, preservation of Schizothorax (HS 01)
Schizothorax limacinum (Schizoochytrium limacinum) HS01 has been deposited in China general microbiological culture Collection center (CGMCC, address: No. 3 Hospital No.1, Xilu, Beijing area of the republic of Beijing) in 10.03.2017 at month and month, and the deposit number is CGMCC No. 13746.
Example 2 preparation of Schizochytrium limacinum powder
The procedure for the preparation of schizochytrium limacinum powder using schizochytrium HS01 of example 1 was as follows, the experiment was repeated three times and the results averaged:
first, preparation of culture medium
The shake flask culture medium 1 consists of a solute and a solvent, wherein the solvent is water, and the solute and the concentration thereof are respectively 60g/L glucose and 5g/L yeast extract; the shake flask culture medium 2 consists of a solute and a solvent, wherein the solvent is water, and the concentration of the solute are respectively 150g/L glucose and 25g/L yeast extract.
The seed culture medium 1 comprises solute and solvent, wherein the solvent is water, and the solute and the concentration thereof are respectively 60g/L glucose, 8g/L yeast extract and 3g/L, Na yeast powder2SO4 5g/L、KCl 0.5g/L、MgSO4 1.0g/L、K2SO4 0.5g/L、KH2PO41.0g/L、(NH4)2SO4 2.0g/L、CaCl2 0.5g/L、CuSO4 0.001g/L、ZnSO40.001g/L, and adjusting the initial pH value to 6.0 by using alkali (sodium hydroxide solution or ammonia water) after the preparation is finished; the seed culture medium 2 consists of solute and solvent, the solvent is water, the solute and the concentration thereof are respectively 150g/L glucose, 25g/L yeast extract and 8g/L, Na yeast powder2SO4 20g/L、KCl 1.5g/L、MgSO4 3.0g/L、K2SO4 2.5g/L、KH2PO4 2.0g/L、(NH4)2SO4 5.0g/L、CaCl2 2.5g/L、CuSO40.02g/L、ZnSO40.02g/L, and adjusting the initial pH value to 6.0 by using alkali (sodium hydroxide solution or ammonia water) after the preparation is finished.
The fermentation medium 1 comprises solute and solvent, wherein the solvent is water, the solute and its concentration are glucose 60g/L, respectively8g/L of mother cream and 3g/L, Na of yeast powder2SO4 5g/L、KCl 0.5g/L、MgSO4 1.0g/L、K2SO4 0.5g/L、KH2PO41.0g/L、(NH4)2SO4 2.0g/L、CaCl2 0.5g/L、CuSO4 0.001g/L、ZnSO40.001g/L, biotin 0.001g/L and corn starch 0.1g/L, and adjusting the initial pH value to 6.0 by using alkali (sodium hydroxide solution or ammonia water) after the preparation is finished; the fermentation medium 2 consists of solute and solvent, the solvent is water, the solute and the concentration thereof are respectively 150g/L glucose, 25g/L yeast extract and 8g/L, Na yeast powder2SO4 20g/L、KCl 1.5g/L、MgSO4 3.0g/L、K2SO4 2.5g/L、KH2PO4 2.0g/L、(NH4)2SO4 5.0g/L、CaCl2 2.5g/L、CuSO4 0.02g/L、ZnSO40.02g/L, 0.06g/L biotin, 10g/L corn starch and 20g/L pea protein powder, and adjusting the initial pH value to 6.0 by using alkali (sodium hydroxide solution or ammonia water) after the preparation is finished; the fermentation culture medium 3 consists of solute and solvent, the solvent is water, the solute and the concentration thereof are respectively 60g/L glucose, 8g/L yeast extract and 3g/L, Na yeast powder2SO4 5g/L、KCl 0.5g/L、MgSO4 1.0g/L、K2SO4 0.5g/L、KH2PO4 1.0g/L、(NH4)2SO4 2.0g/L、CaCl2 0.5g/L、CuSO4 0.001g/L、ZnSO40.001g/L, biotin 0.001g/L and starch sodium octenyl succinate 0.1g/L, and adjusting the initial pH value to 6.0 by using alkali (sodium hydroxide solution or ammonia water) after the preparation is finished; the fermentation culture medium 4 consists of solute and solvent, the solvent is water, the solute and the concentration thereof are respectively 150g/L glucose, 25g/L yeast extract and 8g/L, Na yeast powder2SO4 20g/L、KCl 1.5g/L、MgSO4 3.0g/L、K2SO4 2.5g/L、KH2PO4 2.0g/L、(NH4)2SO4 5.0g/L、CaCl2 2.5g/L、CuSO4 0.02g/L、ZnSO40.02g/L, biotin 0.06g/L, sodium starch octenyl succinate 10g/L, whey protein powder 20g/L, and the preparation is finishedThe initial pH is adjusted to 6.0 with a base (sodium hydroxide solution or ammonia).
Preparation and index detection of schizochytrium limacinum powder
1. Preparation of
Inoculating schizochytrium limacinum HS01 into a shake flask culture medium 1, and culturing for 24h under the conditions of the rotation speed of 200rpm and the temperature of 20 ℃ to obtain a shake flask culture solution 1; inoculating the shake flask culture solution 1 into a seed culture medium 1, culturing for 48h under the conditions that the dissolved oxygen is 10-80% (the dissolved oxygen is a dynamic process in the growth process) and the temperature is 20 ℃, keeping the pH of the culture solution between 4.5-6.5 in the culture process, reducing the pH in the fermentation process, and adjusting by ammonia water or sodium hydroxide solution to obtain a seed culture solution 1; inoculating the seed culture solution 1 into a fermentation culture medium 1 according to the inoculation amount of 10%, culturing for 120h under the conditions of dissolved oxygen of 10-80% (dissolved oxygen is a dynamic process in the growth process) and the temperature of 20 ℃ to obtain a fermentation solution, marking the fermentation solution as the fermentation solution 1, keeping the pH of the culture solution between 4.5 and 6.5 in the culture process, reducing the pH in the fermentation process, and adjusting by ammonia water or a sodium hydroxide solution.
Inoculating schizochytrium limacinum HS01 into a shake flask culture medium 2, and culturing for 48h under the conditions of the rotation speed of 400rpm and the temperature of 30 ℃ to obtain a shake flask culture solution 2; inoculating the shake flask culture solution 2 into a seed culture medium 2, culturing for 24h under the conditions that the dissolved oxygen is 10-80% and the temperature is 30 ℃, keeping the pH of the culture solution between 4.5 and 6.5 in the culture process, reducing the pH in the fermentation process, and adjusting by ammonia water or sodium hydroxide solution to obtain a seed culture solution 2; inoculating the seed culture solution 2 into a fermentation culture medium 2 according to the inoculation amount of 20%, culturing for 72h under the conditions of dissolved oxygen of 10-80% and the temperature of 30 ℃ to obtain a fermentation solution, recording the fermentation solution as the fermentation solution 2, keeping the pH of the culture solution between 4.5 and 6.5 in the culture process, reducing the pH in the fermentation process, and adjusting by ammonia water or a sodium hydroxide solution.
Inoculating schizochytrium limacinum HS01 into a shake flask culture medium 1, and culturing for 24h under the conditions of the rotation speed of 200rpm and the temperature of 20 ℃ to obtain a shake flask culture solution 1; inoculating the shake flask culture solution 1 into a seed culture medium 1, culturing for 48h under the conditions that the dissolved oxygen is 10-80% and the temperature is 20 ℃, keeping the pH of the culture solution between 4.5 and 6.5 in the culture process, reducing the pH in the fermentation process, and adjusting by ammonia water or sodium hydroxide solution to obtain a seed culture solution 1; inoculating the seed culture solution 1 into a fermentation culture medium 3 according to the inoculation amount of 10%, culturing for 120h under the conditions of dissolved oxygen of 0-80% and the temperature of 20 ℃ to obtain a fermentation solution, recording the fermentation solution as the fermentation solution 3, keeping the pH value of the culture solution between 4.5 and 6.5 in the culture process, reducing the pH value in the fermentation process, and adjusting by ammonia water or a sodium hydroxide solution.
Inoculating schizochytrium limacinum HS01 into a shake flask culture medium 2, and culturing for 24h under the conditions of the rotation speed of 400rpm and the temperature of 30 ℃ to obtain a shake flask culture solution 2; inoculating the shake flask culture solution 2 into a seed culture medium 2, culturing for 24h under the conditions that the dissolved oxygen is 10-80% and the temperature is 30 ℃, keeping the pH value of the culture solution between 4.5-6.5 in the culture process, reducing the pH value in the fermentation process, and adjusting by ammonia water or sodium hydroxide solution to obtain a seed culture solution 2; inoculating the seed culture solution 2 into a fermentation culture medium 4 according to the inoculation amount of 20%, culturing for 72h under the conditions of dissolved oxygen of 10-80% and the temperature of 30 ℃ to obtain a fermentation solution, recording the fermentation solution as the fermentation solution 4, keeping the pH value of the culture solution between 4.5 and 6.5 in the culture process, reducing the pH value in the fermentation process, and adjusting by ammonia water or a sodium hydroxide solution.
After fermentation is finished, adding an antioxidant 1 (the antioxidant 1 is composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, wherein the mass ratio of the natural mixed tocopherol to the rosemary to the tea polyphenol to the isoascorbic acid to the phytic acid is 20:2:10:10:2) into the fermentation liquor 1 to obtain a mixed liquor, wherein the mass percentage content of the natural mixed tocopherol to the rosemary to the tea polyphenol to the isoascorbic acid to the phytic acid is respectively 0.2%, 0.02%, 0.1% and 0.02%, and emulsifying and uniformly mixing the mixed liquor to obtain a stable fermentation liquor 1; and (3) carrying out pasteurization on the stable fermentation liquor 1, and then carrying out spraying, rolling or freeze drying to prepare the schizochytrium limacinum powder 1.
Adding an antioxidant 2 (the antioxidant 2 is composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, wherein the mass ratio of the natural mixed tocopherol to the rosemary to the tea polyphenol to the isoascorbic acid to the phytic acid is 40:3:20:20:4) into the fermentation liquid 2 to obtain a mixed liquid, wherein the mass percentage contents of the natural mixed tocopherol, the rosemary to the tea polyphenol to the isoascorbic acid and the phytic acid in the mixed liquid are respectively 0.4%, 0.03%, 0.2% and 0.04%, and emulsifying and uniformly mixing the mixed liquid to obtain a stable fermentation liquid 2; and (3) carrying out pasteurization on the stable fermentation liquor 2, and then carrying out spraying, rolling or freeze drying to prepare the schizochytrium limacinum powder 2.
Centrifuging the fermentation liquor 3 to collect schizochytrium limacinum cell mud, adding sterile deionized water with the same volume according to the volume of the schizochytrium limacinum cell mud, uniformly mixing, centrifuging, and repeatedly cleaning for 2-3 times to obtain the schizochytrium limacinum cell mud; adding antioxidant 3 (the antioxidant 3 is composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, wherein the mass ratio of the natural mixed tocopherol to the rosemary to the tea polyphenol to the isoascorbic acid to the phytic acid is 60:2:40:30:6) into the schizochytrium limacinum cell mud to obtain a mixture, wherein the mass percentage content of the natural mixed tocopherol, the rosemary to the tea polyphenol, the isoascorbic acid to the phytic acid in the mixture is respectively 0.6%, 0.02%, 0.4%, 0.3% and 0.06%, and emulsifying and uniformly mixing the mixture to obtain stable cell mud; pasteurizing the stable cell paste, and then drying (spray drying, roller drying or freeze drying, or any one of the three) to obtain schizochytrium limacinum powder, and marking the schizochytrium limacinum powder as schizochytrium limacinum powder 3.
Centrifuging the fermentation liquor 4 to collect schizochytrium limacinum cell mud, adding sterile deionized water with the same volume according to the volume of the schizochytrium limacinum cell mud, uniformly mixing, centrifuging, and repeatedly cleaning for 2-3 times to obtain the schizochytrium limacinum cell mud; adding antioxidant 4 (the antioxidant 4 is composed of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid, wherein the mass ratio of the natural mixed tocopherol to the rosemary to the tea polyphenol to the isoascorbic acid to the phytic acid is 80:2:40:40:8) into the schizochytrium limacinum cell mud to obtain a mixture, wherein the mass percentage content of the natural mixed tocopherol, the rosemary to the tea polyphenol, the isoascorbic acid to the phytic acid in the mixture is respectively 0.8%, 0.02%, 0.4% and 0.08%, and emulsifying and uniformly mixing the mixture to obtain stable cell mud; pasteurizing the stable cell paste, and then drying (spray drying, roller drying or freeze drying, or any one of the three) to obtain schizochytrium limacinum powder, and marking the schizochytrium limacinum powder as schizochytrium limacinum powder 4.
2. Index detection
And (3) respectively detecting the contents of protein, moisture, fatty acid, ash and DHA in the schizochytrium limacinum powder 1-4 obtained in the step (1), wherein the specific detection method comprises the following steps:
detection of proteins: the method is carried out according to GB 5009.9 determination of protein in food safety national standard.
And (3) detection of moisture: the method is carried out according to GB 5009.3 determination of moisture in national food standard of food safety.
And (3) detection of ash: the method is carried out according to GB 5009.4 determination of ash content in food according to national food safety standard.
Detection of fatty acids: according to GB 5009.168 determination of fatty acids in food safety national standard.
And (3) detection of DHA: according to GB 26400 national standard food additive docosahexaenoic acid oil (fermentation method) for food safety.
The result shows that the schizochytrium limacinum powder obtained in the step has the mass content of 10-60% of protein, 0.5-3.0% of moisture, 3-12% of ash and 25-50% of fatty acid. In the fatty acid, the mass content of unsaturated fatty acid DHA is 10-24%, the mass content of DPA is 2.0-6.0%, and the mass content of EPA is 0.1-0.5%.
TABLE 1 index test results of schizochytrium limacinum powder
Note: the content of each index in table 1 means the mass percentage of each substance in the dry powder; the other fatty acids are fatty acids except C8:0 octanoic acid, C10:0 decanoic acid, C16:0 palmitic acid, C18:0 stearic acid, C22:5DPA, C20:5EPA and C22:6 DHA.
Fistula experiment test of schizochytrium limacinum powder
The rumen fistula experiment of the dairy cows by using the schizochytrium powder 1 and the schizochytrium powder 2 is carried out by adopting a nylon bag method. The operation steps are as follows:
1. test animals and daily rations
Cows with permanent gastric fistulas (holstein cows). The pre-feeding period is 7 days, during which anthelmintic action is carried out, 1% trichlorfon solution is used to expel ectoparasites, and 0.8mg/kg body weight levamisole hydrochloride is orally taken to expel endoparasites. Feeding under the condition that the nutrient level is 1.3 times of the maintenance requirement, feeding twice a day with equal amount, and feeding the feed at the ratio of 07: 00 and 16: 00 feeding once and drinking water freely after feeding.
2. Preparation of the samples
The schizochytrium limacinum powder is randomly sampled by adopting a quartering method, dried to constant weight at 65 ℃ and then filled into a ground bottle for later use.
3. Production of nylon bag
Cutting into rectangle of 170mm × 130mm with 300 mesh nylon cloth, folding, sewing with terylene thread, making into nylon bag of 120mm × 80mm, and ironing with iron. Before the test, the nylon bag is put into the rumen for balancing for 72h, taken out, cleaned, dried and used when no damage is detected.
4. Test design and determination method
The nylon bag method rumen degradation is carried out according to the proposal of the dairy cow feeding standard scientific research cooperative group and the like, the test adopts the design of random unit groups, and each cow is provided with two repetitions at each time point.
About 10g of schizochytrium limacinum powder is filled in each nylon bag, and the variance analysis difference of the samples is not obvious (P is more than 0.05). Every 2 bags are tied on a half-polyethylene tube with the length of 30cm, the nylon bag is placed at the abdominal sac part of the rumen 2h after feeding in the morning, and the other end of the tube is hung on the fistula cover. 6 tubes of 12 bags are put in each cattle tumor stomach at the same time. Respectively taking out a tube from the rumen of each cow at 0h, 2h, 4h, 6h, 12h, 24h, 36h and 48h 8 time points after bagging, cleaning with clear water, rinsing in a washing machine for 7 minutes until the water is clear, drying at 65 ℃ to constant weight, and weighing.
Determination of the Dry Matter (DM) was carried out according to the method of GB 6435-86, and determination of DHA was carried out by Xiamen Congestion Bio Inc. The degradation rate of a certain nutrient/% (1-mass of a certain residual nutrient/total mass of a certain nutrient put in the bag) x 100% at a certain time point (t) of the sample.
The results are shown in table 2 and show that the maximum degradation rate of schizochytrium limacinum powder through the rumen of cows is 54.7%.
TABLE 2 fistula test results of schizochytrium limacinum powder
The schizochytrium limacinum powder in the embodiment 3 and the embodiment 2 can improve the DHA content in milk
This embodiment is through feeding the schizochytrium limacinum powder of embodiment 2 to the milk cow, detects the DHA content in the milk, confirms the influence of schizochytrium limacinum powder to DHA content in the milk, and the experiment is repeated three times.
Firstly, a breeding method comprises the following steps:
selecting 60 healthy Holstein cows with no obvious difference in weight and age of one month, randomly dividing the cows into six groups, and dividing 10 cows in each group, namely a free-ranging experimental group 1, a free-ranging experimental group 2, a free-ranging blank control group, a captive experimental group 1, a captive experimental group 2 and a captive blank control group.
Firstly, carrying out pre-feeding period cultivation on the dairy cows of each experimental group (the pre-feeding period is 15 days, and the feeding amount of schizochytrium limacinum powder is gradually increased according to the adaptive condition of the dairy cows); in the later formal feeding period, the schizochytrium limacinum powder is added into the feed according to a certain adding proportion, stirred and mixed, and fed once every 8 hours.
The schizochytrium limacinum powder added to the feed of the free-ranging experimental group 1 and the free-ranging experimental group 2 is schizochytrium limacinum powder 1 and 2 in the embodiment 1 respectively, the schizochytrium limacinum powder added to the feed of the captive experimental group 1 and the captive experimental group 2 is schizochytrium limacinum powder 1 and 2 in the embodiment 1 respectively, and the specific feeding method is as follows:
a pre-feeding period: the amount of schizochytrium limacinum powder fed to the experimental group is gradually increased, and the method specifically comprises the following steps:
the feeding amount of schizochytrium limacinum powder on the 1 st and 2 nd days in the pre-feeding period is 50 mg/day/head;
the feeding amount of schizochytrium limacinum powder on the 3 rd and 4 th days in the pre-feeding period is 75 mg/day/head;
the feeding amount of schizochytrium limacinum powder on 5 th and 6 th days in the pre-feeding period is 100 mg/day/head;
the feeding amount of schizochytrium limacinum powder on 7 th and 8 th days in the pre-feeding period is 125 mg/day/head;
the feeding amount of schizochytrium limacinum powder on 9 th and 10 th days in the pre-feeding period is 150 mg/day/head;
the feeding amount of schizochytrium limacinum powder on 11 th and 12 th days in the pre-feeding period is 200 mg/day/head;
the feeding amount of schizochytrium limacinum powder on days 13, 14 and 15 in the pre-feeding period is 250 mg/day/head.
The formal feeding period is 250 mg/day/head of schizochytrium limacinum powder.
The feed for the blank control group dairy cows is not added with schizochytrium limacinum powder, the rest components are the same as those of the experimental group, and the feeding time and the feeding amount are the same as those of the experimental group.
The dairy cows of the free-ranging experimental group 1, the free-ranging experimental group 2 and the free-ranging blank control group are subjected to free-ranging, and no other edible food is left in a free-ranging field; the cows in the captive experimental group 1, the captive experimental group 2 and the captive blank control group are captive and have no other edible food in the pens.
Second, milk data detection
And (3) DHA milk sample collection: collecting milk samples of all the fed cows in the morning, the noon and the evening, and performing mixed sampling and inspection; according to the national standard GB5413.27-2010 determination of fatty acid in infant food and dairy products; and detecting the content indexes of milk fat, milk protein and DHA in the milk.
The results (table 3) show that the milk fat and milk protein content in the experimental group milk were not significantly different from the blank control group, whether in free-range or captive. After the schizochytrium limacinum powder in example 1 is added into the feed, the DHA content in the milk is gradually increased along with the increase of the feeding time, the DHA content in the milk of each experimental group is obviously higher than that in the milk of the control group, and the DHA content in the milk of the two control groups has no obvious difference.
TABLE 3 milk DHA content test results (mg/100g milk)
Note: the end of the prefeed period is day 15 of the prefeed period, days 7, 15, 30, 45, 60 and 75 of the full scale period are days 7, 15, 30, 45, 60 and 75 of the full scale period, respectively, and after 75 days of the full scale period is day 100.
Example 4 and example 2 of schizochytrium limacinum powder can improve the quality of eggs and the egg laying performance of laying hens
This embodiment is through feeding the schizochytrium limacinum powder of embodiment 2 to the laying hen, detects the influence of schizochytrium limacinum powder to laying hen egg laying performance and egg quality.
1. Test animal
360 healthy laying hens (Hailan white chickens) with no significant difference in body weight in the egg producing period are randomly selected, the month ages of the laying hens are not significantly different, the laying hens are randomly divided into 4 groups (a control group, a 0.5% experimental group, a 1.0% experimental group and a 1.5% experimental group), each group is 6 in parallel, and each group is 15 in parallel.
2. Feeding management
Adopting 3 layers of cage culture and continuously illuminating. The test diets were fed as dry powder, 3 times daily, with free access to food and water, and daily food intake was recorded in cages. The basic ration of the laying hens of the experimental groups is corn-soybean meal type ration, each experimental group is fed with the basic ration added with the schizochytrium limacinum powder of the example 2, the mass content of 0.5% of the schizochytrium limacinum powder 1 in the food of the experimental group is 0.5%, the mass content of 1.0% of the food of the experimental group is 1.0%, and the mass content of 1.5% of the food of the experimental group is 1.5%; the control group was fed basal diet. The day that the experimental group was fed with schizochytrium limacinum containing meal was recorded as day 1. The laying rate was counted daily, the cholesterol content of the eggs was measured on day 15 and day 25, respectively (the results on day 25 are shown in table 4), and the changes in laying rate, egg cholesterol and DHA content were calculated and shown in table 5.
Table 4, results of determination of laying rate, cholesterol and DHA content on day 25 of feeding schizochytrium limacinum powder 1
TABLE 5 change in laying rate and egg Nutrition composition on day 15 and day 25 of rearing
Note: in table 5, "increase/decrease in laying rate" means the amount of change in laying rate of the control group of the laying hens at the same time period, "increase/decrease in cholesterol" means the amount of change in cholesterol content of the control group of the laying hens at the same time period, "+" indicates an increase, and "-" indicates a decrease.
The laying rate, egg weight, feed intake and egg-feed ratio on day 25 were counted, and the lipid content and cholesterol content of the eggs were measured, and the results are shown in table 6.
TABLE 6 influence of feeding different amount of schizochytrium limacinum powder on layer production performance and lipid at day 25
And (3) carrying out significance analysis on each item (index) among the groups, wherein the significance analysis of the laying rate is as follows:
0.5% group statistics
| Group of | N | Mean value | Standard deviation of | Standard deviation of mean |
| Control group | 6 | 91.600 | 3.4774 | 1.4196 |
| 0.5% of the group | 6 | 87.617 | 4.4441 | 1.8143 |
0.5% group independent sample assay
1.0% group statistics
| Group of | N | Mean value | Standard deviation of | Standard deviation of mean |
| Control | 6 | 91.600 | 3.4774 | 1.4196 |
| 1.0% of the group | 6 | 95.600 | 1.8580 | .7585 |
1.0% group independent sample assay
1.5% group statistics
| Group of | N | Mean value | Standard deviation of | Standard deviation of mean |
| Control | 6 | 91.600 | 3.4774 | 1.4196 |
| 1.5% of the groups | 6 | 93.500 | 2.1392 | .8733 |
1.5% group independent sample assay
From the significance analysis, the egg laying rates of the 0.5% group and the 1.5% group on the 25 th day were not significantly different from those of the control group (P > 0.05), but the 1.0% group was able to significantly increase the egg laying rate of the laying hens (P < 0.05), indicating that the egg laying rate of the laying hens was able to be increased by feeding a specific amount of schizochytrium limacinum powder 1.
The DHA content of the layer chicken fed with schizochytrium limacinum powder 1 at 15 days can be increased by 317.71% to 150mg/100g in 0.5% group compared with the control group; the 1.0% group can improve 579.48% and reach 244mg/100 g; while the 1.5% group can improve 885.79% and reach 354mg/100 g. After 25 days of feeding, the 0.5% and 1.0% groups remained essentially unchanged, while the 1.5% group was slightly decreased, but still significantly increased compared to the control group. The DHA content in the eggs of the laying hens can be improved by feeding the schizochytrium limacinum powder 1, and the DHA content in the eggs is higher when the addition amount of the schizochytrium limacinum powder 1 in the food is more.
When the layer chicken is fed with schizochytrium limacinum powder 1, the cholesterol content in eggs is obviously reduced; the decrease amount of the cholesterol content of the eggs has no obvious relation with the content of the schizochytrium limacinum powder 1 in the food, but the cholesterol content of the eggs tends to further decrease along with the increase of the feeding time. The result shows that the content of cholesterol in eggs of laying hens can be reduced by feeding schizochytrium limacinum powder 1.
According to the method, the schizochytrium limacinum powder 1 in the steps is replaced by the schizochytrium limacinum powder 2, and other steps are not changed, so that the result with the same change trend is obtained, and the schizochytrium limacinum powder 1 and the schizochytrium limacinum powder 2 in the example 1 have the same functions.
<110> Xiamen Huishi Biometrics Ltd
<120> application of schizochytrium limacinum and schizochytrium limacinum preparation in improving egg quality
<160> 2
<170> PatentIn version 3.5
<210> 1
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<213> Artificial sequence
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Claims (2)
1. Use of Schizochytrium limacinum (Schizochytrium limacinum) preparation for increasing DHA content and reducing cholesterol content in egg;
the preparation is prepared according to a method comprising the following steps: culturing Schizochytrium limacinum HS01 to obtain fermentation liquid; adding an antioxidant into the fermentation liquor and then drying to obtain the preparation;
the preservation number of the Schizochytrium limacinum HS01 in the common microorganism center of China Committee for culture Collection of microorganisms is CGMCC No. 1374;
the antioxidant is a mixed antioxidant consisting of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid; the mixture ratio of the substances in the mixed antioxidant is p1) or p 2):
p1) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 20:2:10:10: 2;
p2) the mass ratio of natural mixed tocopherol, rosemary, tea polyphenol, isoascorbic acid and phytic acid is 40:3:20:20: 4.
2. Use according to claim 1, characterized in that: culturing the Schizochytrium limacinum HS01 according to claim 1 by using a fermentation medium, wherein the fermentation medium is composed of a solvent and a solute, the solvent is water, and the solute and the concentration thereof are respectively 60-150 g/L of glucose, 8-25 g/L of yeast extract and 3-8 g/L, Na of yeast powder2SO4 5～20g/L、KCl 0.5～1.5g/L、MgSO4 1.0～3.0g/L、K2SO4 0.5～2.5g/L、KH2PO4 1.0～2.0g/L、(NH4)2SO4 2.0～5.0g/L、CaCl2 0.5～2.5g/L、CuSO4 0.001～0.02g/L、ZnSO40.001-0.02 g/L, biotin 0.001-0.06 g/L, starch 0.1-10 g/L and albumen powder 0-20 g/L, and the pH value is 4.5-6.5.
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| EP18913727.6A EP3756472A4 (en) | 2018-04-04 | 2018-11-14 | Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product |
| PCT/CN2018/115327 WO2019192182A1 (en) | 2018-04-04 | 2018-11-14 | Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product |
| AU2018417303A AU2018417303A1 (en) | 2018-04-04 | 2018-11-14 | Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product |
| AU2018102207A AU2018102207A4 (en) | 2018-04-04 | 2018-11-14 | Application of schizochytrium limacinum and preparation thereof in improvement of quality and yield of animal product |
| JP2020529495A JP7191102B2 (en) | 2018-04-04 | 2018-11-14 | Use of Schizochytrium rimascinum and preparations thereof in improving the quality and yield of animal products |
| US17/043,468 US11583566B2 (en) | 2018-04-04 | 2018-11-14 | Use of Schizochytrium limacinum and its preparation in improving the quality and yield of animal product |
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