Use of <i>Schizochytrium limacinum </i>and its preparation in improving the quality and yield of animal product

Abstract

The invention discloses the use of the Schizochytrium limacinum and its preparation in improving the quality and yield of animal product. The deposit number of Schizochytrium limacinum in the present invention is CGMCC No. 13746 in the China General Microbiological Culture Collection Center. The Schizochytrium limacinum powder produced by the Schizochytrium limacinum may increase the DHA content in an animal product, reduce the cholesterol content in an animal product, and also improve the egg production performance of poultry. This animal product with high DHA content from natural sources is organic, safe, stable, and easy to be absorbed. It may be used as a safer and effective way for people to ingest natural DHA, and it may also cater to and meet consumer needs. Thus, Schizochytrium limacinum and Schizochytrium limacinum powder of the present application have a wide range of application in the field of general food and livestock breeding.

Claims

1. A method for improving quality and/or increasing a yield of an animal product, including feeding an animal with Schizochytrium limacinum or its preparation to improve the quality and/or increase the yield of the animal product; and wherein the Schizochytrium limacinum is Schizochytrium limacinum HS01 and has a deposit number of CGMCC No. 13746 in the China General Microbiological Culture Collection Center.

2. The method according to claim 1, wherein the improvement of the quality of animal product is c1) and/or c2) and/or c3): c1) increase a DHA content in the animal product; c2) increase a Sn-2 DHA content in the animal product; c3) reduce a cholesterol content in the animal product.

3. The method according to claim 2, wherein the animal is fed with the Schizochytrium limacinum preparation having been produced using a method comprising the steps of: cultivating the Schizochytrium limacinum HS01 to obtain a fermentation broth; and using the fermentation broth to produce the Schizochytrium limacinum preparation.

4. The method according to claim 3, wherein the culture of the Schizochytrium limacinum is carried out using a fermentation medium, which consists of a solvent and solutes, the solvent is water, the solutes and their concentrations are 60-150 g/L of glucose, 8-25 g/L of yeast extract, 3-8 g/L of yeast powder, 5-20 g/L of Na.sub.2SO.sub.4, 0.5-1.5 g/L of KCl, 0-3.0 g/L of MgSO.sub.4, 0.5-2.5 g/L of K.sub.2SO.sub.4, 1.0-2.0 g/L of KH.sub.2PO.sub.4, 2.0-5.0 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5-2.5 g/L of CaCl.sub.2), 0.001-0.02 g/L of CuSO.sub.4, 0.001-0.02 g/L of ZnSO.sub.4, 0.001-0.06 g/L of biotin, 0.1-10 g/L of starch and 0-20 g/L of protein powder, respectively, and the pH of the fermentation medium is 4.5-6.5.

5. The method according to claim 3, wherein the preparation is a Schizochytrium limacinum powder.

6. The method according to claim 3, wherein producing the preparation by using the fermentation broth includes drying the fermentation broth to obtain the preparation.

7. The method according to claim 3, wherein producing the preparation by using the fermentation broth includes adding an antioxidant to the fermentation broth after obtaining the fermentation broth and then drying to obtain the preparation.

8. The method according to claim 7, wherein the antioxidant is an oil-soluble antioxidant and/or a water-soluble antioxidant; wherein the oil-soluble antioxidant is rosemary, mixed natural tocopherols, polyphenols and/or ascorbyl palmitate; and wherein the water-soluble antioxidant is phytic acid, ascorbic acid and/or erythorbic acid.

9. The method according to claim 1, wherein the animal is a1) or a2) or a3) or b1) or b2): a1) poultry; a2) chicken; a3) Beijing white chicken, Hy-Line white chicken, Hy-Line brown chicken or Hy-Line pink chicken; b1) ruminant; b2) cow; and wherein the animal product is an egg or milk.

Description

BEST MODE OF IMPLEMENTING THE INVENTION

(1) The present invention will be further described in detail below referring to specific embodiments, and the examples provided are only to illustrate the present invention, not to limit the scope of the present invention. Unless otherwise specified, the experimental methods in the following examples are conventional methods. Unless otherwise specified, the materials, reagents, and instruments used in the following examples are commercially available. In the following quantitative experiments, three repeated experiments are carried out, and the results are averaged.

(2) Screening liquid medium: dissolve 50 g of glucose and 15 g of yeast powder in 1 L of mixed solution (obtained by mixing 1 part by volume of natural seawater and 1 part by volume of distilled water), and the pH is natural.

(3) Screening plate: pour the screening solid medium at about 55° C. into a petri dish, and cool to obtain a solid plate.

(4) Fermentation medium: 60 g of glucose, 10 g of glutamic acid or sodium glutamate, 10 g of corn syrup dry powder, 14 g of NaSO.sub.4, 0.5 g of KCl, 2.0 g of MgSO.sub.4, 1.0 g of K.sub.2SO.sub.4, 1.0 g of KH.sub.2PO.sub.4, 1.0 g of (NH.sub.4).sub.2SO.sub.4 and 0.5 g of CaCl.sub.2) is dissolved in 1 L of distilled water, and the pH is adjusted to 6.0.

(5) Malt juice agar medium: dissolve 150 g of malt infusion powder in 1 L of mixed solution (composed of 1 part by volume of natural seawater and 1 part of volume of distilled water), and the pH value is natural; then add agar powder to a concentration of 15 g/100 mL, to obtain a medium.

(6) Natural mixed tocopherol is a product of ADM company, with the product number of MTS-90. Rosemary is a product of Guangzhou Branch of Kenaiou Trading (Shanghai) Co., Ltd., with the product number of ROSEMARY 41-19-58. Polyphenol is a product of Fujian Likangyuan Biological Engineering Co., Ltd., with the product number of TP-98. Erythorbic acid is a product of Zhengzhou Tuoyang Experimental Co., Ltd., with the content is ≥98%. Phytic acid is a product of Laiyang Wanjiwei Biological Engineering Co., Ltd.

Example 1. Separation and Identification of Schizochytrium limacinum HS01

(7) I. Separation of HS01

(8) 1. Schizochytrium limacinums from a number of mangroves in Yunxiao County, Zhangzhou City, Fujian Province is collected by the inventor of the present application, and mixed to obtain a mixed solution. 0.5 mL of the mixed solution are inoculated into 5 mL of the screening liquid medium, and then cultured at 25° C., 200 rpm/min for 2 days, to obtain a cultured bacterial solution.

(9) 2. The cultured bacterial solution obtained in step 1 is spread evenly on the screening plate and incubated at 25° C. for 2 days to produce a single colony.

(10) 3. After step 2 is completed, single colonies are picked and inoculated into 5 mL of fermentation medium, and then cultured at 25° C., 200 rpm/min for 2 days to obtain a cultured bacterial solution.

(11) 4. Take the cultured bacterial solution obtained in step 3, centrifuge at 4° C., 2000 rpm for 5 min, and collect bacteria.

(12) 5. Take 1.0-2.0 g of bacteria into a measuring cylinder with stopper (the specification is 100 mL), add 15 mL of HCl aqueous solution (with the concentration of 8.3 mol/L) first, close the lid, and place it in a water bath at 70-80° C. and hydrolyze for 50-60 minutes (during this period, measuring cylinder is placed on a vortex mixer and shaken once every 10 minutes); after cooling to room temperature, first add 10 mL of 95% (v/v) ethanol aqueous solution, shake fully and then add 20 mL of anhydrous ether to fully shake and extract for 1-2 minutes, and finally add 20 mL of petroleum ether, shake fully and extract for 1-2 minutes, let stand for layering, place the upper organic phase in a glass weighing dish, which has been dried and the empty weight thereof has been weighed, and place the glass weighing dish on a boiling water bath in a fume hood to fully evaporate the organic phase (be sure to fully evaporate), and the liquid phase is the grease.

(13) 6. Take the grease extracted in step 5 and detect the DHA content according to GB 26400-2011 National Food Safety Standard, and the composition and content of fatty acid according to the method of AOAC996.06.

(14) Strains with higher DHA content are selected and purified repeatedly 24 times. A strain of Schizochytrium limacinum strain screened is named Schizochytrium limacinum HS01.

(15) The Schizochytrium limacinum HS01 is inoculated into the fermentation medium for monoclonal culturing 12 consecutive passages and the DHA content is detected according to the above steps. The results show that the stability of the Schizochytrium limacinum HS01 for producing DHA is good.

(16) II. Identification of the Schizochytrium limacinum HS01

(17) 1. Morphological Identification

(18) The Schizochytrium limacinum HS01 is inoculated on the wort agar medium and cultured in the dark at 25° C. After 5 days, the morphology of the colonies is observed and the morphological characteristics of the cells are observed by high-resolution transmission electron microscopy.

(19) The results show that the colony diameter of the Schizochytrium limacinum HS01 is 2-4.3 mm, white (light orange in the later period), and the edges are uneven; the bacteria proliferated in a fission manner, the cell wall is thin, spherical, colorless or light orange, transparent, with a size of 4.5-15.5 μm. Zoospores and exoplasmic reticulum are not seen.

(20) 2. 18s rDNA sequence homology analysis

(21) The partial sequence of the 18s rDNA of the Schizochytrium limacinum HS01 is shown as SEQ ID No. 1 in the sequence listing.

(22) The partial sequence of the 18s rDNA of the Schizochytrium limacinum HS01 is shown as SEQ ID No. 2 in the sequence listing.

(23) Combining the above identification results, the Schizochytrium limacinum HS01 is a Schizochytrium limacinum.

(24) III. Deposition of Schizochytrium limacinum HS01

(25) Schizochytrium limacinum HS01 is deposited on Mar. 10, 2017 at China General Microbiological Culture Collection Center (CGMCC for short, address: Building 3, No. 1 West Beichen Road, Chaoyang District, Beijing, China). The deposition number is CGMCC No. 13746.

Example 2. Production of Schizochytrium limacinum Powder

(26) The procedure for producing Schizochytrium limacinum powder by using Schizochytrium limacinum HS01 in Example 1 is as follows. The experiment is repeated three times, and the results are averaged:

(27) I. Production of a Culture Medium

(28) Shake flask medium 1 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 60 g/L of glucose, 5 g/L of yeast extract, respectively. Shake flask medium 2 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 150 g/L of glucose and 25 g/L of yeast extract, respectively.

(29) Seed medium 1 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 60 g/L of glucose, 8 g/L of yeast extract, 3 g/L of yeast powder, 5 g/L of Na.sub.2SO.sub.4, 0.5 g/L of KCl, 1.0 g/L of MgSO.sub.4, 0.5 g/L of K.sub.2SO.sub.4, 1.0 g/L of KH.sub.2PO.sub.4, 2.0 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5 g/L of CaCl.sub.2), 0.001 g/L of CuSO.sub.4, 0.001 g/L of ZnSO.sub.4. After the production is completed, the initial pH is adjusted to 6.0 with alkali (sodium hydroxide solution or ammonia water). The seed medium 2 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 150 g/L of glucose, 25 g/L of yeast extract, 8 g/L of yeast powder, 20 g/L of Na.sub.2SO.sub.4, 1.5 g/L of KCl, 3.0 g/L of MgSO.sub.4, 2.5 g/L of K.sub.2SO.sub.4, 2.0 g/L of KH.sub.2PO.sub.4, 5.0 g/L of (NH.sub.4).sub.2SO.sub.4, 2.5 g/L of CaCl.sub.2), 0.02 g/L of CuSO.sub.4, 0.02 g/L of ZnSO.sub.4, After the production is completed, the initial pH is adjusted to 6.0 with alkali (sodium hydroxide solution or ammonia water).

(30) Fermentation medium 1 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 60 g/L of glucose, 8 g/L of yeast extract, 3 g/L of yeast powder, 5 g/L of Na.sub.2SO.sub.4, 0.5 g/L of KCl, 1.0 g/L of MgSO.sub.4, 0.5 g/L of K.sub.2SO.sub.4, 1.0 g/L of KH.sub.2PO.sub.4, 2.0 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5 g/L of CaCl.sub.2), 0.001 g/L of CuSO.sub.4, 0.001 g/L of ZnSO.sub.4, 0.001 g/L of biotin, 0.1 g/L of corn starch. After the production is completed, the initial pH is adjusted to 6.0 with alkali (sodium hydroxide solution or ammonia water). Fermentation medium 2 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 150 g/L of glucose, 25 g/L of yeast extract, 8 g/L of yeast powder, 20 g/L of Na.sub.2SO.sub.4, 1.5 g/L of KCl, 3.0 g/L of MgSO.sub.4, 2.5 g/L of K.sub.2SO.sub.4, 2.0 g/L of KH.sub.2PO.sub.4, 5.0 g/L of (NH.sub.4).sub.2SO.sub.4, 2.5 g/L of CaCl.sub.2), 0.02 g/L of CuSO.sub.4, 0.02 g/L of ZnSO.sub.4, 0.06 g/L of biotin, 10 g/L of corn starch, 20 g/L of pea protein powder. After the production is completed, the initial pH is adjusted to 6.0 with alkali (sodium hydroxide solution or ammonia water). Fermentation medium 3 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 60 g/L of glucose, 8 g/L of yeast extract, 3 g/L of yeast powder, 5 g/L of Na.sub.2SO.sub.4, 0.5 g/L of KCl, 1.0 g/L of MgSO.sub.4, 0.5 g/L of K.sub.2SO.sub.4, 1.0 g/L of KH.sub.2PO.sub.4, 2.0 g/L of (NH.sub.4).sub.2SO.sub.4, 0.5 g/L of CaCl.sub.2), 0.001 g/L of CuSO.sub.4, 0.001 g/L of ZnSO.sub.4, 0.001 g/L of biotin, 0.1 g/L of sodium starch octenyl succinate. After the production is completed, the initial pH is adjusted to 6.0 with alkali (sodium hydroxide solution or ammonia water). Fermentation medium 4 is composed of a solute and a solvent. The solvent is water. The solute and their concentrations are 150 g/L of glucose, 25 g/L of yeast extract, 8 g/L of yeast powder, 20 g/L of Na.sub.2SO.sub.4, 1.5 g/L of KCl, 3.0 g/L of MgSO.sub.4, 2.5 g/L of K.sub.2SO.sub.4, 2.0 g/L of KH.sub.2PO.sub.4, 5.0 g/L of (NH.sub.4).sub.2SO.sub.4, 2.5 g/L of CaCl.sub.2), 0.02 g/L of CuSO.sub.4, 0.02 g/L of ZnSO.sub.4, 0.06 g/L of biotin, 10 g/L of sodium starch octenyl succinate, 20 g/L of whey protein powder. After the production is completed, the initial pH is adjusted to 6.0 with alkali (sodium hydroxide solution or ammonia water).

(31) II. Production and Indicator Detection of Schizochytrium limacinum Powder

(32) 1. Production

(33) Inoculate Schizochytrium limacinum HS01 into the shake flask medium 1 and incubate at 200 rpm and a temperature of 20° C. for 24 h to obtain a shake flask culture solution 1; inoculate the shake flask culture solution 1 into the seed medium 1, and culture for 48 h under a condition of 10-80% of dissolved oxygen (dissolved oxygen is a dynamic process during the growth process) and a temperature of 20° C. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution to obtain a seed culture solution 1. Inoculate the seed culture solution 1 into the fermentation medium 1 at an inoculation amount of 10%, and culture under a condition of 10-80% dissolved oxygen (dissolved oxygen is a dynamic process during the growth process) and a temperature of 20° C. for 120 h to obtain a fermentation broth, recorded as fermentation broth 1. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution.

(34) Inoculate Schizochytrium limacinum HS01 into the shake flask medium 2 and culture at 400 rpm and 30° C. for 48 h to obtain a shake flask culture solution 2; inoculate the shake flask culture solution 2 into the seed medium 2, culture for 24 h under a condition of 10-80% of dissolved oxygen and a temperature of 30° C. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution, to obtain a seed culture solution 2; inoculate the seed culture solution 2 into the fermentation medium 2 at an inoculation amount of 20%, and culture for 72 h under a condition of 10-80% of dissolved oxygen and a temperature of 30° C. to obtain a fermentation broth, referred to as a fermentation broth 2. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution.

(35) Inoculate the Schizochytrium limacinum HS01 into the shake flask medium 1 and culture at 200 rpm and a temperature of 20° C. for 24 h to obtain a shake flask culture solution 1. Inoculate the shake flask culture solution 1 into a seed medium 1, and culture for 48 h under a condition of 10-80% of dissolved oxygen and a temperature of 20° C. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution to obtain a seed culture solution 1. Inoculate the seed culture solution 1 into the fermentation medium 3 at an inoculation amount of 10%, and culture for 120 h under a condition of 0 to 80% of dissolved oxygen and a temperature of 20° C. to obtain a fermentation broth, referred to as a fermentation broth 3. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution.

(36) Inoculate the Schizochytrium limacinum HS01 into a shake flask medium 2 and culture at 400 rpm and 30° C. for 24 h to obtain a shake flask culture solution 2. Inoculate the shake flask culture solution 2 into a seed medium 2, and culture for 24 h under a condition of 10-80% of dissolved oxygen and a temperature of 30° C. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH will decrease during the fermentation, and it may be adjusted by ammonia or sodium hydroxide solution to obtain a seed culture solution 2. Inoculate the seed culture solution 2 into a fermentation medium 4 at an inoculation amount of 20%, and culture for 72 h under a condition of 10-80% of dissolved oxygen and a temperature of 30° C. to obtain a fermentation broth, referred to as a fermentation broth 4. During the culture, the pH of the culture solution is maintained between 4.5 and 6.5. The pH of the fermentation process will decrease, and the pH is adjusted by ammonia or sodium hydroxide solution.

(37) After the fermentation is completed, an antioxidant 1 (the antioxidant 1 is composed of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid, wherein the mass ratio of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid is 20:2:10:10:2) is added to the fermentation broth 1 to obtain a mixed solution, in which the content of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid by mass percentage are 0.2%, 0.02%, 0.1%, 0.1% and 0.02%, respectively. The mixed solution is emulsified and mixed to obtain a stable fermentation broth 1. The stable fermentation broth 1 is pasteurized and then sprayed, roller or freeze-dried to obtain a Schizochytrium limacinum powder 1.

(38) Add an antioxidant 2 (the antioxidant 2 is composed of mixed natural tocopherols, rosemary, polyphenols, erythorbic acid and phytic acid, wherein the mass ratio of mixed natural tocopherols, rosemary, polyphenols, erythorbic acid and phytic acid is 40:3:20:20:4) to the fermentation broth 2, to obtain a mixed solution, in which the content of natural mixed tocopherol, rosemary, polyphenol, erythorbic acid and phytic acid by mass percentage are 0.4%, 0.03%, 0.2%, 0.2% and 0.04%, respectively. The mixed solution is emulsified and mixed to obtain a stable fermentation broth 2. The stable fermentation broth 2 is pasteurized and then sprayed, roller or freeze-dried to obtain a Schizochytrium limacinum powder 2.

(39) Centrifuge the fermentation broth 3 to collect Schizochytrium limacinum cell slurry, add the same volume of sterile deionized water according to the volume of Schizochytrium limacinum cell slurry, and then centrifuge, repeat washing 2 to 3 times to obtain a Schizochytrium limacinum cell slurry. Add an antioxidant 3 (the antioxidant 3 is composed of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid, wherein the mass ratio of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid is 60:2:40:30:6) into the Schizochytrium limacinum cell slurry to obtain a mixture, in which the content of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid by mass percentage is 0.6%, 0.02%, 0.4%, 0.3% and 0.06%, respectively. The mixture is emulsified and mixed to obtain a stable cell slurry. The stable cell slurry is pasteurized and then dried (one of the following three is selected: spray, roller or freeze-drying), to obtain a Schizochytrium limacinum powder, referred to as a Schizochytrium limacinum powder 3.

(40) The fermentation broth 4 is centrifuged to collect a Schizochytrium limacinum cell slurry, add the same volume of sterile deionized water according to the volume of Schizochytrium limacinum cell slurry, and then centrifuge, repeat washing 2 to 3 times to obtain the Schizochytrium limacinum cell slurry. Add an antioxidant 4 (the antioxidant 4 is composed of mixed natural tocopherols, rosemary, polyphenols, erythorbic acid and phytic acid, in which the mass ratio of mixed natural tocopherols, rosemary, polyphenols, erythorbic acid and phytic acid is 80:2:40:40:8) into the Schizochytrium limacinum cell slurry to obtain a mixture. The content of natural mixed tocopherol, rosemary, polyphenols, erythorbic acid and phytic acid in the mixture by mass percentage is 0.8%, 0.02%, 0.4%, 0.4% and 0.08%, respectively. The mixture is emulsified and mixed to obtain a stable cell slurry. The stable cell slurry is pasteurized and then dried (one of the following three is selected: spray, roller or freeze-drying) to obtain a Schizochytrium limacinum powder, referred to as Schizochytrium limacinum powder 4.

(41) 2. Indicator Detection

(42) The content of protein, moisture, fatty acid, ash and DHA of Schizochytrium limacinum powders 1-4 obtained in step 1 is detected respectively. The specific detection methods are as follows:

(43) Protein detection is carried out according to GB 5009.9 “National Food Safety Standards Determination of Protein in Food”.

(44) Moisture detection is carried out according to GB 5009.3 “National Food Safety Standards Determination of Moisture in Food”.

(45) Ash detection is carried out according to GB 5009.4 “National Food Safety Standards Determination of Ash in Food”.

(46) Fatty acid detection is carried out according to GB 5009.168 “National Food Safety Standards Determination of Fatty Acids in Foods”.

(47) DHA detection is carried out according to GB 26400 “National Food Safety Standards Food Additives Docosahexaenoic Acid Grease (Fermentation Method)”.

(48) Detection of the proportion of Sn-2 DHA to total DHA in milk and the fatty acid composition are carried out according to GB 5009.168-2016 “National Food Safety Standards Determination of Fatty Acids in Food” and GB/T 24894-2010/ISO 6800:1997.

(49) The results show that the protein content of Schizochytrium limacinum powder obtained in the above step is 10-60%, the mass content of moisture 0.5-3.0%, the content of ash is 3-12%, and the mass content of the fatty acid is 25-50%. Among fatty acids, 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%.

(50) TABLE-US-00001 TABLE 1 Indicator test results of the Schizochytrium limacinum powder Schizochytrium Schizochytrium Schizochytrium Schizochytrium limacinum limacinum limacinum limacinum Indicator powder 1 powder 2 powder 3 powder 4 Protein 20 25 30 45 Moisture ≤3.0 ≤3.0 ≤3.0 ≤3.0 Ash 9.5 11.0 4.0 4.0 Fatty C8:0 2.0 2.5 2.5 3.0 acids Caprylic acid C10:0 1.0 1.5 2.0 2.5 Capric acid C16:0 3.0 6.0 3.0 6.0 Palmitic acid C18:0 0.5 0.5 0.5 0.5 Stearic acid C22:5 4.5 4.0 4.0 3.0 DPA C20:5 0.2 0.25 0.25 0.3 EPA C22:6 20.0 17.0 19.5 20.0 DHA Other 5.0 3.0 4.0 0.7 fatty acids Note: The content of each indicator in Table 1 refers to the mass percentage of each substance in the dry powder. Other fatty acids refer to C8:0 Caprylic acid, C10:0 Capric acid, C16:0 Palmitic acid, C18:0 Stearic acid, C22:5 DPA, C20:5 EPA and C22:6 Fatty acids other than DHA.

(51) III. The Fistula Experimental Test of Schizochytrium limacinum Powders

(52) Nylon bag method is used in rumen fistula experiment of Schizochytrium limacinum powders 1 and 2. The operation steps are as follows:

(53) 1. Test Animals and Diets

(54) Dairy cows (Holstein dairy cows) possess a permanent gastric fistula. The pre-feeding period is 7 days. During this period, deworming is performed, and ectoparasites are expelled with 1% trichlorfon. 0.8 mg/kg body weight of levamisole hydrochloride is orally administrated to expel internal parasites. Dairy cows are bred under a condition that the nutritional level is 1.3 times to maintain the need, twice a day in equal amounts, once at 07:00 and 16:00, respectively, and water is drunk freely after feeding.

(55) 2. Production of Samples

(56) Schizochytrium limacinum powders are randomly sampled by the “quartile method”, dried at 65° C. to a constant weight, and put it into a milled bottle for use.

(57) 3. Production of Nylon Bag

(58) A 300 mesh nylon cloth is cut into a 170 mm×130 mm rectangle. After folding in half, double stitch with polyester is threaded to make a nylon bag with a size of 120 mm×80 mm. Flatten the loose edges with a soldering iron. Before the test, place the nylon bag in the rumen for balancing 72 h, take it out, wash and dry it, and check it for use without any damage.

(59) 4. Test Design and Measurement Method

(60) The rumen degradation of the nylon bag method is carried out according to the scheme proposed by the Dairy Cow Breeding Standards Research Collaboration Group, and the like. The test is designed with a random unit group, and two repeats for each cow at each time point are set.

(61) Each nylon bag is filled with about 10 g of Schizochytrium limacinum powders, and the variance analysis of the samples is not significant (P>0.05). Every 2 bags are fastened to a 30 cm long semi-polyethylene tube. 2 hours later, after feeding in the morning, the nylon bag is placed in the abdominal sac of the rumen. The other end of the tube is hung on the fistula cover. 6 tubes are put into each rumen of a cow at the same time, totally 12 bags. Take a tube from the rumen of each cow at 8 time points of 0 h, 2 h, 4 h, 6 h, 12 h, 24 h, 36 h and 48 h, respectively after putting the bag, wash it with clean water and rinse it in the washing machine for 7 minutes, until the water is clear, then bake at 65° C. to a constant weight and weigh.

(62) The determination of dry matter (DM) is carried out according to the method of GB6435-86, and the determination of DHA is undertaken by Xiamen Huisheng Biological Co., Ltd. The degradation rate of a certain nutrient at a certain time point (t) of the sample/%=(1−the mass of a certain nutrient remaining/the total mass of a nutrient put in the bag)×100%

(63) The results are shown in Table 2. The results show that the highest degradation rate of Schizochytrium limacinum powder over the rumen of dairy cows is 54.7%.

(64) TABLE-US-00002 TABLE 2 Fistula test results of the Schizochytrium limacinum powder Items Schizochytrium limacinum Schizochytrium limacinum powder 1 powder 2 degradation rate degradation rate of of Schizochytrium degradation Schizochytrium degradation Time in limacinum rate of DHA limacinum rate of DHA rumen (h) powder (%) (%) powder (%) (%) 0 18.5 1.0 14.6 0 2 37.0 5.2 33.8 2.0 4 47.5 5.2 40.7 3.3 6 50.2 6.0 44.7 5.0 12 50.6 7.0 45.7 6.2 24 53.8 7.0 47.3 6.5 36 54.7 10.0 50.9 8.1 48 54.7 10.0 50.3 8.5

Example 3. The Schizochytrium limacinum Powder of Example 2 May Increase the DHA Content in Milk

(65) In this example, after feeding cows with the Schizochytrium limacinum powder of Example 2, the DHA content in the milk is detected to determine the effect of Schizochytrium limacinum powders on the DHA content in the milk, and the experiment is repeated three times.

(66) I. Feeding Methods:

(67) 60 healthy Holstein dairy cows with no significant difference in body weight and month age are selected and randomly divided into six groups with 10 heads in each group, that is, a free-range experimental group 1, a free-range experimental group 2, a free-range blank control group, a captive experimental group 1, a captive experiment group 2 and a captive blank control group.

(68) Firstly, cows in each experimental group are bred in the pre-feeding period (the pre-feeding period is 15 days, and a large feeding amount of Schizochytrium limacinum powder is gradually added according to the adaptation of the cows); in the later formal feeding period, Schizochytrium limacinum powder is added according to a certain proportion into the feed for stirring and mixing, feeding once every 8 hours.

(69) The Schizochytrium limacinum powder added to the feeds of the free-range experimental group 1 and the free-range experimental group 2 are the Schizochytrium limacinum powder 1 and 2 in example 1, respectively, and the Schizochytrium limacinum powder added to the feed of the captive experiment group 1 and the captive experiment group 2 is Schizochytrium limacinum powder 1 and 2 in example 1, respectively. The specific feeding method is as follows:

(70) In the pre-feeding period, the feeding amount of the Schizochytrium limacinum powder in the experimental group increases gradually as follows:

(71) The feeding amount of the Schizochytrium limacinum powder on days 1 and 2 of the pre-feeding period is 50 mg/day/head;

(72) The feeding amount of the Schizochytrium limacinum powder on days 3 and 4 of the pre-feeding period is 75 mg/day/head;

(73) The feeding amount of the Schizochytrium limacinum powder on days 5 and 6 of the pre-feeding period is 100 mg/day/head;

(74) The feeding amount of the Schizochytrium limacinum powder on days 7 and 8 of the pre-feeding period is 125 mg/day/head;

(75) The feeding amount of the Schizochytrium limacinum powder on days 9 and 10 of the pre-feeding period is 150 mg/day/head;

(76) The feeding amount of the Schizochytrium limacinum powder on days 11 and 12 of the pre-feeding period is 200 mg/day/head;

(77) The feeding amount of the Schizochytrium limacinum powder on days 13, 14 and 15 of the pre-feeding period is 250 mg/day/head.

(78) In the formal feeding period, the feeding amount of the Schizochytrium limacinum powder is 250 mg/day/head.

(79) In the blank control group, no Schizochytrium limacinum powder is added into the feed of cows, and the rest of the ingredients are the same as those in the experimental group. The feeding time and feeding amount are the same as that in the experimental group.

(80) Cows in the free-range experimental group 1, the free-range experimental group 2 and the free-range blank control group are free-ranged, and there are no other edible foods for the cows in the free-range facility. The cows in the captive experimental group 1, the captive experimental group 2 and the captive blank control group are captive, without any other edible food.

(81) II. Milk Data Detection

(82) DHA milk sample collection: collect milk samples from all cows fed three times in the morning, noon and evening and mix and send them for inspection. According to the national standard GB5413.27-2010 Determination of fatty acids in infant food and dairy products, indicators of milk fat, milk protein, DHA content, the proportion of Sn-2 DHA in milk to the total DHA are detected.

(83) The results (Table 3) show that no matter whether it is free-range or captive, there is no significant difference between the experimental group and the blank control group in the milk fat and milk protein content. However, after adding the Schizochytrium limacinum powder of Example 1, the DHA content in milk gradually increases with the increase of feeding time, and the DHA content in milk of each experimental group is significantly higher than that of the control group. There is no significant difference in DHA content between two control groups.

(84) TABLE-US-00003 TABLE 3 Test results of milk DHA content (mg/100 g milk) Time After Day Day Day Day Day Day Day 7 15 in 30 in 45 in 60 in 75 in 75 in End of the in the the the the the the the pre-feeding formal formal formal formal formal formal formal Group period period period period period period period period free-range 4.0 5.7 7.9 8.5 9.0 12.5 13.0 20.0 experimental group 1 free-range 6.0 8.0 9.4 11.0 14.2 15.5 20.0 25.5 experimental group 2 free-range 0 0.2 0.2 0.1 0.5 0 0.3 0.2 blank control group captive 12 14.5 15.5 18.0 21.5 25.4 26.5 30 experimental group 1 captive 16 18.3 19.5 21.0 22.9 23.8 24.5 28.5 experimental group 2 captive 0 0.2 0.2 0.1 0.5 0 0.3 0.2 blank control group Note: The end of the pre-feeding period is the day 15 of the pre-feeding period, and the days 7, 15, 30, 45, 60 and 75 of the formal period are the day 7, 15, 30, 45, 60 and 75 of the formal feeding period, respectively. After the day 75 of the formal period means day 100.

(85) TABLE-US-00004 TABLE 4 The proportion of Sn-2 DHA to total DHA in milk (%) Time After Day Day Day Day Day Day Day 7 15 in 30 in 45 in 60 in 75 in 75 in End of the in the the the the the the the pre-feeding formal formal formal formal formal formal formal Group period period period period period period period period free-range 30 40 50 55 60 65 70 80 experimental group 1 free-range 30 50 55 55 60 70 80 95 experimental group 2 free-range 0 0 0 0 0 0 0 0 blank control group captive 30 40 50 55 60 65 70 80 experimental group 1 captive 30 50 55 55 60 70 80 95 experimental group 2 captive 0 0 0 0 0 0 0 0 blank control group Note: There is no obvious difference between free-range groups and captive groups in the proportion of Sn-2 DHA to the total DHA in milk; and the proportion generally fluctuates in a range of 30-95% without any special rule.

Example 4. The Schizochytrium limacinum Powder of Example 2 May Improve Egg Quality and Laying Performance of Laying Hens

(86) In this example, after feeding the Schizochytrium limacinum powder of Example 2 to laying hens, the effect of Schizochytrium limacinum powder on the performance of laying hens and egg quality is detected.

(87) 1. Test Animals

(88) 360 healthy laying hens (Hy-Line white chicken) in laying period with no significant difference in body weight are randomly selected. There is no significant difference in the age of month among each laying hens. The laying hens are randomly divided into 4 groups (control group, 0.5% experiment group, 1.0% experimental group and 1.5% experimental group). For each group, there are 6 parallel groups, and for each parallel group, there are 15 laying hens.

(89) 2. Feeding Management

(90) 3 layers of cages and keep light are used. The experimental diet is fed in the form of dry powder, 3 times a day, free to eat and drink, and the daily intake is recorded for each cage. The basic diet for laying hens in the experimental group is a corn-soybean diet. Each experimental group is fed with the basic diet supplemented with the Schizochytrium limacinum powder of Example 2. The mass content of the Schizochytrium limacinum powder in the 0.5% experimental group is 0.5%. The mass content of the Schizochytrium limacinum powder in the 1.0% experimental group is 1.0%. The mass content of the Schizochytrium limacinum powder in the 1.5% experimental group is 1.5%. The control group is fed a basic diet. The day when the experimental group is fed with the Schizochytrium limacinum powder is recorded as day 1. Calculate the laying rate every day, detect the cholesterol content in eggs on day 15 and day 25 (the results on day 25 are shown in Table 5), and calculate the changes in laying rate, egg cholesterol and DHA content, the results are shown in Table 6.

(91) TABLE-US-00005 TABLE 5 Determination results of laying rate, cholesterol and DHA content on day 25 laying rate cholesterol content DHA content Groups (%) (mg/100 g) (mg/100 g) control Parallel 88.9 307.43 33.37 group 1 parallel 90.6 304.54 32.63 2 parallel 94.2 314.35 35.50 3 parallel 93.7 318.72 37.67 4 parallel 86.6 320.31 39.22 5 parallel 95.6 301.43 37.07 6 mean 91.6 311.1 35.9 0.5% Parallel 85.3 250.07 142.00 Group 1 parallel 84.4 243.58 163.00 2 parallel 89.8 239.43 145.00 3 parallel 91.1 233.75 147.00 4 parallel 93.3 249.37 146.00 5 parallel 81.8 248.61 157.00 6 mean 87.6 243.1 150.0 1.0% Parallel 96.8 213.85 259.00 Group 1 parallel 98.2 217.24 244.00 2 parallel 92.8 234.06 241.00 3 parallel 96.0 235.23 263.00 4 parallel 94.7 228.09 242.00 5 parallel 95.1 233.84 245.00 6 mean 95.6 227.1 249.0 1.5% Parallel 90.2 250.57 320.00 Group 1 parallel 92.8 243.07 328.00 2 parallel 95.1 227.51 309.00 3 parallel 93.7 250.67 333.00 4 parallel 96.4 224.99 311.00 5 parallel 92.8 230.67 319.00 6 mean 93.5 237.9 320.0

(92) TABLE-US-00006 TABLE 6 Changes in laying rate and egg nutrient composition on days 15 and 25 Group Day 15 in experimental Day 25 in experimental Group Group Day 25 in 0.5% 1.0% 1.5% 0.5% 1.0% 1.5% Item control group Group Group Group Group Group Group Change in laying +0.05 +4.40 +2.00 +0.10 +4.37 +2.07 rate (%) Change in −18.54 −22.11 −19.46 −21.85 −27.02 −23.53 cholesterol (%) DHA (mg/100 g) 35.91 150 244 354 150 249 320 Note: In Table 6, “Change in laying rate” refers to the change amount compared with the laying rate of the same period of laying hens in the control group. “Change in cholesterol” refers to the change amount compared with the cholesterol content in eggs of the same period in the control group. “+” means increase, “−” means decrease.

(93) The laying rate, egg weight, feed intake, egg-to-feed ratio on day 25 are counted, and the lipid content and cholesterol content in eggs are measured. The results are shown in Table 7.

(94) TABLE-US-00007 TABLE 7 Effect of feeding different amounts of Schizochytrium limacinum powder on the performance of laying hens and lipid on day 25 Groups control 0.5% 1.0% 1.5% group experimental experimental experimental (Day Group Group Group item 25) (Day 25) (Day 25) (Day 25) laying rate (%) 91.6 87.6 95.6 93.5 egg weight (g) 56.08 57.11 56.87 56.61 feed intake 113.19 119.36 125.87 109.79 (g/hen/day) egg-to-feed ratio 2.20 2.38 2.36 2.15 lipid content in eggs 9.16 10.77 9.28 11.11 (g/100 g) cholesterol content 311.13 243.14 227.05 237.91 in eggs (mg/100 g)

(95) Significance analysis is performed on each item (indicator) among groups, and the significance analysis of laying rate is as follows:

(96) TABLE-US-00008 Statistics of 0.5% Group Standard Standard Deviation of Groups N Mean Deviation Mean Control group 6 91.600 3.4774 1.4196 0.5% Group 6 87.617 4.4441 1.8143

(97) TABLE-US-00009 Independent sample test in 0.5% Group T-test of Mean Variance Levene test The 95% of Variance standard confidence interval Equation Mean error for the difference F Sig. t df P difference difference lower limit upper limit Assume 1.024 0.336 1.729 10 0.114 3.9833 2.3037 −1.1496 9.1163 that the variances are equal Assume 1.729 9.453 0.116 3.9833 2.3037 −1.1901 9.1568 that the variances are not equal

(98) TABLE-US-00010 Statistics of 1.0% Group Standard Standard Deviation of Groups N Mean Deviation Mean Control 6 91.600 3.4774 1.4196 1.0% Group 6 95.600 1.8580 .7585

(99) TABLE-US-00011 Independent sample test in 1.0% Group T-test of Mean Variance Levene test The 95% of Variance standard confidence interval Equation Mean error for the difference F Sig. t df P difference difference lower limit upper limit Assume 4.355 0.063 −2.485 10 0.032 −4.0000 1.6096 −7.5863 −.4137 that the variances are equal Assume −2.485 7.640 0.039 −4.0000 1.6096 −7.7423 −.2577 that the variances are not equal

(100) TABLE-US-00012 Statistics of 1.5% Group Standard Standard Deviation of Groups N Mean Deviation Mean Control 6 91.600 3.4774 1.4196 1.5% Group 6 93.500 2.1392 .8733

(101) TABLE-US-00013 Independent sample test in 1.5% Group T-test of Mean Variance Levene test The 95% of Variance standard confidence interval Equation Mean error for the difference F Sig. t df P difference difference lower limit upper limit Assume 2.938 0.117 −1.140 10 0.281 −1.9000 1.6667 −5.6137 1.8137 that the variances are equal Assume −1.140 8.310 0.286 −1.9000 1.6667 −5.7187 1.9187 that the variances are not equal

(102) From the significance analysis, it can be seen that the laying rate of the 0.5% group and the 1.5% group on day 25 is not significantly different from the control group (P>0.05), but the 1.0% group may significantly increase the egg laying rate (P<0.05), indicating that feeding a specific amount of the Schizochytrium limacinum powder 1 may increase the laying rate of laying hens.

(103) When the laying hens are fed the Schizochytrium limacinum powder 1, as for the DHA content on the day 15, compared with the control group, in the 0.5% group, it could increase 317.71% to 150 mg/100 g. In the 1.0% group, it could increase 579.48% to 244 mg/100 g. In the 1.5% group, it may increase 885.79% to 354 mg/100 g. After 25 days of feeding, the 0.5% group and 1.0% group remained basically unchanged, while the 1.5% group slightly decreased, but compared with the control group, it still increased significantly. It shows that feeding the Schizochytrium limacinum powder 1 may increase the DHA content in laying hen eggs, and the more the addition of the Schizochytrium limacinum powder 1 in food, the higher the DHA content in eggs.

(104) When the laying hens are fed the Schizochytrium limacinum powder 1, the cholesterol content in eggs decreased significantly. The decrease in the cholesterol content in eggs had no obvious relationship with the content of the Schizochytrium limacinum powder 1 in the food, but with the feeding time increases, cholesterol content has a further downward trend. It shows that feeding the Schizochytrium limacinum powder 1 may reduce the cholesterol content of laying hen eggs.

(105) According to the above method, when the Schizochytrium limacinum powder 1 in the above steps is replaced with the Schizochytrium limacinum powder 2, and other steps remain unchanged, the same change trend results are obtained, indicating that both Schizochytrium limacinum powder 1 and 2 of Example 1 have the same function.

INDUSTRIAL APPLICATION

(106) The Schizochytrium limacinum powder produced by Schizochytrium limacinum in the present invention may increase the DHA content in animal products, reduce the cholesterol content in animal products, and also improve the egg production performance of poultry. This animal product with high DHA content from natural sources is organic, safe, stable, and easy to be absorbed. It may be used as a safer and effective way for people to ingest natural DHA, and it may also cater to and meet consumer needs. Thus, Schizochytrium limacinum and Schizochytrium limacinum powder of the present application have a wide range of application in the field of general food and livestock breeding.