Preparation method of DHA (docosahexaenoic acid) algae powder and application of DHA algae powder in increasing DHA content of milk

Abstract

The invention discloses a preparation method of DHA (docosahexaenoic acid) algae powder and application of the DHA algae powder in increasing DHA content of milk. The preparation method of the DHA algae powder comprises the following steps: (1) fermenting schizochytrium limacinum to obtain schizochytrium limacinum fermentation liquor; (2) the schizochytrium limacinum fermentation liquor is not treated or is subjected to biological enzyme wall breaking pretreatment; and (3) adding protein, a wall material, an emulsifier, an antioxidant, a dispersing agent, a pH regulator and water into the untreated or treated schizochytrium limacinum fermentation liquor sample, and performing spray drying to obtain the DHA algae powder. The DHA algae powder with the DHA content being 20% or above is prepared and further added into basic ration to serve as a dairy cow feed additive. The dairy cow feed additive provided by the invention can improve the content of DHA in milk, and the high-DHA milk with the natural source can be used as a safer and more effective way for people to take in DHA.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for preparing DHA algae powder and its application in increasing the DHA content of milk. Background Technology

[0002] DHA (docosahexaenoic acid) is an omega-3 polyunsaturated fatty acid, an important component of brain cell membranes, involved in brain cell formation and development, maintaining normal physiological activity of nerve cells, and participating in the brain's thinking and memory formation processes. Breast milk contains long-chain polyunsaturated fatty acids. Studies have found that breastfed infants have significantly higher cognitive development scores than formula-fed infants, and supplementing formula-fed infants with DHA significantly improves their developmental rate. DHA supplementation promotes brain volume development after birth and improves infants' tolerance to formula.

[0003] DHA is closely related to brain health. Increasing the DHA content in milk helps raise DHA levels in the brain, thereby enhancing learning and memory functions and promoting healthy brain and nerve development. In addition, it may help prevent vision loss and Alzheimer's disease. Studies have shown that with age, the DHA content in platelet and erythrocyte membrane lipids decreases, and superoxide dismutase (SOD) activity declines. Supplementing with adequate DHA enhances SOD activity in erythrocyte membrane lipids, indicating that DHA has antioxidant and anti-aging effects.

[0004] Milk contains almost no DHA. Currently, most DHA fortification is achieved through exogenous addition of DHA algal oil or DHA microcapsule powder. However, because DHA contains multiple unsaturated double bonds, it is easily oxidized by air and metals, often resulting in a fishy smell and deteriorating taste, flavor, and quality. Furthermore, in recent years, people have increasingly favored nutrients from natural sources. Compared to exogenous DHA milk, endogenous DHA milk has a higher absorption rate and lower excretion rate, indicating a higher effective utilization rate of endogenous DHA in the human body. Studies have found that high-DHA-content endogenous DHA milk can significantly improve learning and memory abilities in mice. In summary, endogenous DHA milk not only has advantages in improving DHA absorption and bioavailability but may also improve learning and memory abilities in children and adolescents. These findings are significant for developing functional dairy products rich in DHA, contributing to human health and cognitive development. Therefore, developing milk containing native DHA is of great importance.

[0005] Endogenous DHA-rich milk has significant scientific and economic value, but it currently faces multiple technical challenges, such as the rumen degradation of DHA. Rumen microorganisms in dairy cows hydrogenate most unsaturated fatty acids (including DHA) into saturated fatty acids, resulting in a DHA absorption rate of less than 10% in the small intestine. Even with the addition of unprotected DHA algae powder, the DHA enrichment rate in milk is typically only 1%–3%. Summary of the Invention

[0006] Purpose of the invention: To address the problems existing in the prior art, this invention provides a DHA algae powder and its preparation method. The DHA algae powder prepared by this invention can not only increase the DHA content in milk, but also significantly alleviate the problem of DHA degradation in the rumen of dairy cows by using a specific combination of wall materials such as sodium octenyl succinate starch, thereby significantly increasing the DHA content in milk.

[0007] The present invention also provides applications of the DHA algae powder.

[0008] Technical Solution: To achieve the above objectives, the present invention provides a DHA algal powder comprising Schizochytrium fermentation broth (with or without pretreatment with cell-wall-breaking enzymes), protein, wall material, emulsifier, antioxidant, dispersant, pH adjuster, and water; wherein the protein accounts for 0.5-5.0% of the weight of the Schizochytrium fermentation broth; the wall material accounts for 0.2-2.5% of the weight of the Schizochytrium fermentation broth and is one or more of sodium octenyl succinate starch, maltodextrin, corn cob powder, monoglycerides, citrate fatty acid glycerides, sucrose esters, β-cyclodextrin, and sodium caseinate; the emulsifier accounts for 0.1-0.3% of the weight of the Schizochytrium fermentation broth; the antioxidant accounts for 0-0.3% of the weight of the Schizochytrium fermentation broth; the dispersant accounts for 0.1-1.0% of the weight of the Schizochytrium fermentation broth; and the pH adjuster accounts for 0.05-0.4% of the weight of the Schizochytrium fermentation broth.

[0009] The Schizochytrium sp. HX-308 is a known Schizochytrium sp. ATCC 20888.

[0010] The protein is any one or more of corn protein, soy protein, sunflower seed protein, and soy protein isolate, wherein the weight of corn protein accounts for 1.0-3.0% of the weight of the fermentation broth, the weight of soy protein accounts for 1.0-5.0% of the weight of the fermentation broth, the weight of sunflower seed protein accounts for 1.0-4.0% of the weight of the fermentation broth, and the weight of soy protein isolate accounts for 0.5-2.5% of the weight of the fermentation broth.

[0011] The wall material is one or more of the following: sodium octenyl succinate starch, maltodextrin with a DE value of 16-20, corn cob powder, monoglyceride, citric acid fatty acid glyceride, sucrose ester, β-cyclodextrin, and sodium caseinate. The weight of sodium octenyl succinate starch accounts for 0.2-1.5% of the weight of the fermentation broth, the weight of maltodextrin accounts for 0.1-2.0% of the weight of the fermentation broth, and the weight of corn cob powder accounts for 0.5-2.5% of the weight of the fermentation broth.

[0012] Preferably, the wall material is a combination of sodium octenyl succinate starch and maltodextrin with a DE value of 16-20, wherein the weight of sodium octenyl succinate starch accounts for 0.2-1.5% of the weight of the fermentation broth, and the weight of maltodextrin with a DE value of 16-20 accounts for 0.1-2.0% of the weight of the fermentation broth.

[0013] The emulsifier is polyglycerol fatty acid ester, accounting for 0.1-0.3% of the fermentation broth weight; the antioxidant is one of L-ascorbic acid palmitate, bamboo leaf antioxidant, tea polyphenols, ascorbic acid, rosemary extract, mixed tocopherols, and grape seed extract, accounting for 0-0.3% of the fermentation broth weight; the dispersant is fumed silica, accounting for 0.1-1.0% of the fermentation broth weight; the pH adjuster is one or more of citric acid and vitamin C, wherein citric acid accounts for 0.05-0.2% of the fermentation broth weight; and vitamin C accounts for 0.1-0.2% of the fermentation broth weight.

[0014] The method for preparing DHA algal powder according to the present invention includes the following steps:

[0015] (1) Fermenting Schizochytrium to obtain Schizochytrium fermentation broth;

[0016] (2) The above-mentioned Schizochytrium fermentation broth is either left untreated or pretreated with bio-enzymes to break down cell walls.

[0017] (3) Add protein, wall material, emulsifier, antioxidant, dispersant, pH adjuster and water to the above untreated or treated Schizochytrium fermentation broth sample, and then shear and spray dry to obtain the DHA algal powder.

[0018] In step (1), the fermentation medium solvent is water, including 10-60 g / L glucose, 1-3 g / L KH2PO4, 0.2-3 g / L K2SO4, 1-3 g / L MgSO4, 1-4 g / L (NH4)2SO4, 0.1-0.4 g / L CaCl2·2H2O, 5-15 g / L Na2SO4, 0.2-2 g / L KCl, 5-20 g / L sodium glutamate, 6-10 g / L malic acid, 8-25 g / L yeast powder, and 0.5-3 g / L corn steep liquor powder, with a pH of 6.0-6.5; the fermentation conditions are 28-30℃ and fermentation time is 100-120 h.

[0019] The feature is that the pretreatment of cell wall breaking by the bio-enzyme in step (2) is carried out at a temperature of 40-60℃ for 1-3 h, and the mass-volume ratio of the amount of bio-enzyme to the fermentation liquid after fermentation of Schizochytrium is (0.1-2.5) kg: 1000 L. The bio-enzyme is at least one of alkaline protease, neutral protease, papain, cellulase, β-glucanase and xylanase.

[0020] Preferably, the temperature for biological enzyme treatment of Schizochytrium is 50°C and the time is 2 h, and the mass ratio of the added cell wall breaking enzyme to the volume ratio of the fermentation broth is 1 kg: 1000 L.

[0021] In step (3), the crude fat content of the DHA algae powder is more than 25% of the total weight of the DHA algae powder, and the DHA content is more than 20% of the total weight of the DHA algae powder.

[0022] The total weight of the substances added to the fermentation broth shall not exceed 30% of the dry matter weight of the fermentation broth.

[0023] In this invention, the density of the fermentation broth is 1 g / mL, and the volume and mass are interchangeable at a 1:1 ratio.

[0024] The present invention discloses a dairy cow feed comprising a basal diet and the aforementioned DHA algae powder. The DHA algae powder accounts for 2%-10% of the total dry matter weight of the basal diet. The basal diet, on a dry matter basis, includes roughage and concentrate. Roughage accounts for 40%-75% of the dry matter weight of the basal diet, and concentrate accounts for 25%-60% of the dry matter weight of the basal diet. The roughage includes alfalfa hay and corn silage, and the concentrate includes two or more of the following: extruded soybeans, corn, wheat bran, vitamins, soybean meal, carrots, salt, beets, bone meal, distiller's grains, and tofu residue.

[0025] This invention provides a method for producing DHA algae powder feed, and adds it in an appropriate proportion to the basal diet of dairy cows to increase the DHA content in raw milk.

[0026] The DHA algae powder of the present invention comprises: Schizochytrium fermentation broth or fermentation broth after biological enzyme treatment, water, protein, wall material, emulsifier, antioxidant, dispersant, pH adjuster, etc., which are mixed, stirred, sheared, homogenized and dried to obtain a suspension.

[0027] The application of the DHA algae powder or the dairy cow feed described in this invention in increasing the DHA content of milk.

[0028] The application involves feeding DHA algae powder 2-3 times daily, at a rate of 50-250 g / day per head, with an interval of 5-10 hours between feedings, for 30-45 days or more.

[0029] The DHA algal powder of this invention comprises: Schizochytrium fermentation broth, water, wall material, protein, emulsifier, antioxidant, dispersant, and pH adjuster. After mixing, stirring, shearing, and homogenizing, a suspension is obtained and then dried. The dairy cow feed of this invention mainly consists of a basal diet and DHA algal powder produced from Schizochytrium. By adding protein, wall material, emulsifier, antioxidant, dispersant, pH adjuster, and water to the Schizochytrium fermentation broth, mixing, and then spray drying, DHA algal powder with a DHA content of over 20% is obtained and added to the basal diet. The dairy cow feed additive provided by this invention can increase the DHA content in milk. This naturally sourced high-DHA milk can serve as a safer and more effective way for people to obtain DHA.

[0030] In this invention, a specific combination of sodium octenyl succinate starch and maltodextrin with a DE value of 16-20 is used as the wall material. After the sodium octenyl succinate starch is dissolved in water, high-speed stirring or homogenization disperses DHA oil droplets in the Schizochytrium fermentation broth into tiny particles. The molecules are oriented on the surface of the oil droplets, forming an emulsion layer, reducing interfacial tension and preventing droplet aggregation. The amphiphilic structure of sodium octenyl succinate starch results in high emulsification efficiency, capable of encapsulating over 90% of DHA within microcapsules, reducing the oxidative loss of free DHA. Maltodextrin with a DE value of 16-20 readily forms a continuous, dense film structure during spray drying, serving as an "outer shell" to encapsulate the DHA microdroplets emulsified by the sodium octenyl succinate starch.

[0031] This invention, through experiments, reveals that among numerous wall materials or combinations thereof, using sodium octenyl succinate starch and maltodextrin with a DE value of 16-20 as a wall material to encapsulate DHA algae powder improves the encapsulation rate. This not only increases the DHA content in milk but also significantly alleviates the problem of DHA degradation in the rumen of dairy cows, thus significantly increasing the DHA content in milk. Conversely, using sodium octenyl succinate starch or maltodextrin with a DE value of 16-20 as a single wall material, or using either of these alone in combination with other wall materials such as corn cob powder, or in combination with other wall materials, all result in significantly reduced effectiveness.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0033] 1. This invention proposes a novel method for preparing DHA algae powder and its application in increasing the DHA content of milk. The endogenous DHA milk developed by this invention not only has a high DHA content, but also effectively avoids the problem of DHA degradation in the rumen.

[0034] 2. This invention uses sodium octenyl succinate starch and maltodextrin with a DE value of 16-20 as composite wall materials to prepare DHA algae powder. The encapsulation rate and stability are significantly improved, the antioxidant properties are strong, the degradation in the rumen of dairy cows is effectively inhibited, the shelf life of the product is extended, and it is acid and salt resistant, making it suitable for addition to a variety of food systems.

[0035] 3. By adding the DHA algae powder prepared in this invention to the basic diet, the DHA content in milk is significantly increased, and the endogenous DHA milk has no fishy smell or other off-flavors, thus enhancing palatability. Attached Figure Description

[0036] Figure 1 The PAV values ​​of different DHA algal powders after accelerated oxidation. Detailed Implementation

[0037] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0038] The various experimental operations involved in the specific embodiments are all conventional techniques in the art. For parts not specifically annotated herein, those skilled in the art can refer to various commonly used reference books, scientific and technological literature, or related instructions and manuals prior to the filing date of this invention. Unless otherwise specified, the materials, reagents, instruments, etc., used in the following embodiments are all commercially available. All quantitative experiments in the following embodiments are performed in triplicate, and the results are averaged.

[0039] The soy protein is a commercially available food-grade soy protein powder, purchased from Guangdong Osman Biotechnology Co., Ltd.

[0040] The corn gluten was commercially available feed-grade corn gluten powder, purchased from Jinan Baishun Chemical Co., Ltd.

[0041] The sunflower seed protein was commercially available food-grade sunflower seed protein, purchased from Shanxi Chengzhi Biotechnology Co., Ltd.

[0042] The soy protein isolate was purchased from Jiangsu Pepta Biotechnology Co., Ltd., under the brand name Shansong, model SD100.

[0043] Alkaline protease, sodium octenyl succinate starch, and grape seed extract were purchased from Henan Anrui Biotechnology Co., Ltd. The alkaline protease was branded as Longket, 200,000 IU; the sodium octenyl succinate starch was type D, branded as Ruilin; and the grape seed extract was a commercially available food-grade grape seed extract.

[0044] Neutral protease was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and its product name was 1398 neutral protease; papain was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and its product name was Papain; cellulase was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and its product name was cellulase; xylanase was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and its product name was xylanase; β-glucanase was purchased from Nanning Dongheng Huadao Biotechnology Co., Ltd., and its product name was dextranase.

[0045] Maltodextrin (MD20) with a DE value of 16-20 was purchased from Guangzhou Julan Chemical Technology Co., Ltd., under the brand name Xiwang, with the product number PO18092803.

[0046] L-Ascorbate palmitate, CAS 137-66-6, is a commercially available food-grade L-ascorbate palmitate, purchased from Shanghai JueTu Biotechnology Co., Ltd.

[0047] The polyglycerol fatty acid ester was a commercially available food-grade emulsifier, PGFE, purchased from Jiangsu Yanke Bioengineering Co., Ltd., product number 101.

[0048] Rosemary extract was purchased from Guangzhou Hecheng Sansheng Biotechnology Co., Ltd.; tea polyphenols were purchased from Henan Wanbang Chemical Technology Co., Ltd.; bamboo leaf antioxidants (bamboo leaf antioxidants AOB natural antioxidants), a conventional commercially available food-grade ingredient, were purchased from Shanghai Qianbaiwei Biotechnology Co., Ltd.; and mixed tocopherols (natural antioxidant mixed tocopherols_FORTIUM) were purchased. TM MT was purchased from Kemin (China) Technology Co., Ltd., with batch number 2404103375.

[0049] Silica, a commercially available food anti-caking agent, was purchased from Shandong Jiquan Biotechnology Co., Ltd., product number 21.

[0050] All raw materials used in the embodiments of this invention are commercially available conventional materials.

[0051] The schizochytrium is Schizochytrium sp. HX-308. Specifically, the schizochytrium sp. ATCC 20888 was provided by Nanjing Normal University.

[0052] In the example, fermentation broth 1 was obtained using fermentation medium 1: deionized water as solvent, glucose concentration 12 g / L, KH2PO4 2 g / L, K2SO4 3 g / L, MgSO4 3 g / L, (NH4)2SO4 2 g / L, CaCl2·2H2O 0.4 g / L, Na2SO4 15 g / L, KCl 2 g / L, monosodium glutamate 20 g / L, malic acid 10 g / L, yeast powder 25 g / L, corn steep liquor powder 3 g / L, pH 6.0, fermentation conditions 28℃ for 120 h.

[0053] In the example, fermentation broth 2 was obtained using fermentation medium 2: deionized water as solvent, glucose concentration 12 g / L, KH2PO4 1 g / L, K2SO4 0.65 g / L, MgSO4 2 g / L, (NH4)2SO4 2 g / L, CaCl2·2H2O 0.17 g / L, Na2SO4 12 g / L, KCl 0.5 g / L, monosodium glutamate 10 g / L, malic acid 6 g / L, yeast powder 10 g / L, corn steep liquor powder 5 g / L, pH 6.0, fermented at 28℃ for 120 h.

[0054] In the example, the density of the fermentation broth was 1 g / mL.

[0055] Example 1 Schizochytrium sp. HX-308 cultured to the logarithmic phase was added to fermentation medium 1 or 2 at a volume ratio of 5%, and fermented at 28°C for 120 h at a rotation speed of 500 rpm.

[0056] After fermentation, citric acid solution was added to the fermentation broth to adjust the pH to 6.0. Then, different types and concentrations of cell-wall-breaking enzymes were added to the fermentation broth. β-glucanase (1.0 g / L) was added to either fermentation broth 1 or 2. β-glucanase and papain were added to either fermentation broth 1 or 2, with β-glucanase concentrations of 0.5 g / L and 0.4 g / L, and papain concentrations of 0.5 g / L and 0.6 g / L, respectively. Papain (1.0 g / L) was added to either fermentation broth 1 or 2. The mixture was then enzymatically hydrolyzed at 50℃ and 200 r / min for 2 h. The DHA content was then determined according to Method I of GB 5009.168-2016.

[0057] Table 1. DHA content after enzymatic hydrolysis with different amounts of enzyme added.

[0058]

[0059] A comparison of DHA production effects between Schizochytrium malaccensis treated with cell-wall-breaking enzymes and those without shows that enzyme treatment has significant advantages in improving DHA release efficiency and product quality. In untreated Schizochytrium malaccensis, the DHA is encapsulated within the intact cell wall, releasing only slowly through mechanical extrusion or natural osmosis, resulting in generally low DHA extraction rates. Furthermore, the extraction process is susceptible to oxidation and residual impurities, leading to low product purity and poor stability. However, after treatment with a combination of β-glucanase and papain, the cell wall of Schizochytrium malaccensis is directionally degraded, significantly increasing the DHA release rate. Simultaneously, the gentle enzymatic hydrolysis conditions avoid the damage to DHA caused by high temperature and pressure. In addition, the hydrolyzed DHA is more easily encapsulated by subsequent cell wall materials (such as sodium octenyl succinate starch), significantly enhancing its application performance in food and health products.

[0060] Furthermore, experiments conducted in this invention revealed that using equal amounts of alkaline protease, neutral protease, cellulase, xylanase (1.0 g / L), or combinations of other enzymes, was significantly less effective than using a specific ratio of β-glucanase and papain in combination.

[0061] Example 2

[0062] This embodiment provides a method for producing DHA algae powder, which is used as a supplement to the basic daily diet of dairy cows.

[0063] The fermentation broth 1 or 2 used was treated with 0.4 g / L β-glucanase and 0.6 g / L papain, and then enzymatically hydrolyzed according to the method of Example 1. After adding one or more of protein, wall material, emulsifier, antioxidant, dispersant, pH adjuster, and water, the mixture was sheared for 10 min using a shearing machine and then spray-dried using conventional methods to prepare DHA algal powder.

[0064] The specific implementation formula is as follows:

[0065] 1. DHA algae powder 1: fermentation broth 1 900 L, water 500 L.

[0066] 2. DHA algae powder 2: fermentation liquid 2 900 L, water 500 L.

[0067] 3. DHA Algae Powder 3: Fermentation liquid 2900 L, water 500 L, 1.5 kg of white carbon black, 11.5 kg of soy protein isolate, 4 kg of sodium octyl succinate starch, 1 kg of L-ascorbic acid palmitate, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0068] 4. DHA Algae Powder 4: Fermentation liquid 2900 L, water 500 L, 1.5 kg of precipitated silica, 11.5 kg of soy protein isolate, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of L-ascorbic acid palmitate, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0069] 5. DHA Algae Powder 5: Fermentation broth 2900 L, water 500 L, 1.5 kg of precipitated silica, 11.5 kg of corn protein, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of L-ascorbic acid palmitate, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0070] 6. DHA Algae Powder 6: Fermentation liquid 2900 L, water 500 L, 1.5 kg of white carbon black, 11.5 kg of soybean protein, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of L-ascorbic acid palmitate, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0071] 7. DHA Algae Powder 7: Fermentation broth 2900 L, water 500 L, 1.5 kg of precipitated silica, 11.5 kg of sunflower seed protein, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of L-ascorbic acid palmitate, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0072] 8. DHA Algae Powder 8: Fermentation liquid 2900 L, water 500 L, 1.5 kg of white carbon black, 11.5 kg of soy protein isolate, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of mixed tocopherols, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0073] 9. DHA Algae Powder 9: Fermentation liquid 2900 L, water 500 L, 1.5 kg of white carbon black, 11.5 kg of soy protein isolate, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of bamboo leaf antioxidants, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0074] 10. DHA Algae Powder 10: Fermentation liquid 2900 L, water 500 L, 1.5 kg of white carbon black, 11.5 kg of soy protein isolate, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of tea polyphenols, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0075] 11. DHA Algae Powder 11: Fermentation broth 2900 L, water 500 L, 1.5 kg of white carbon black, 11.5 kg of soy protein isolate, 2.5 kg of sodium octyl succinate starch, 1.5 kg of maltodextrin with a DE value of 16-20, 1 kg of rosemary extract, 1 kg of polyglycerol fatty acid ester, and 0.5 kg of citric acid.

[0076] 12. DHA Algae Powder 12: Fermentation broth 2900 L, water 500 L, fumed silica 1.5 kg, soy protein isolate 11.5 kg, sodium octyl succinate starch 2.5 kg, maltodextrin with DE value of 16-20 1.5 kg, grape seed extract 1 kg, polyglycerol fatty acid ester 1 kg, citric acid 0.5 kg.

[0077] 13. DHA Algae Powder 13: Fermentation liquid 2900 L, water 500 L, white carbon black 1.5 kg, soy protein isolate 11.5 kg, maltodextrin with DE value of 16-20 4 kg, L-ascorbic acid palmitate 1 kg, polyglycerol fatty acid ester 1 kg, citric acid 0.5 kg.

[0078] Example 3

[0079] The crude protein, crude fat, DHA, polysaccharides, ash, and pH of the Schizochytrium powder obtained in each group of Example 2 were measured separately, and the specific detection methods are as follows:

[0080] Crude protein detection: Performed according to GB / T 6432-2018 "Determination of crude protein in feed - Kjeldahl method".

[0081] Crude fat content was determined in accordance with GB 5009.6-2016 "Determination of fat in food".

[0082] DHA testing: conducted according to Method I of GB 5009.168-2016.

[0083] Polysaccharide detection: conducted in accordance with GB / T 15672-2009 "Determination of total sugar content in edible fungi".

[0084] Ash content determination: conducted in accordance with GB 5009.4-2016 "Determination of Ash Content in Food".

[0085] The results are shown in Table 2. The DHA algal powder obtained in the above steps has a crude protein content of 15-25%, a crude fat content of over 38%, a DHA content of around 20%, a polysaccharide content of over 0.5%, an ash content of around 12%, and a pH of 4.8-6.

[0086] Based on the test results of algae powders 4, 5, 6, and 7, when the added protein is soy protein isolate, the crude protein content of the algae powder is ensured to be above 20%, and the DHA content is kept stable at around 20%. For other added proteins, the crude protein content of the algae powder is guaranteed to be above 15%. Soy protein isolate has a protein content of over 90%, a reasonable amino acid composition, and complete essential amino acids, providing high-quality protein for dairy cows. It is easily digested and absorbed, helping to maintain the health and milk production performance of dairy cows.

[0087] Based on the test results of algae powders 3, 4, and 13, the added packaging material is either sodium octenyl succinate starch or maltodextrin with a DE value of 16-20, or a mixture of both, with the total amount of both accounting for 0.4-0.6% of the fermentation broth weight. Sodium octenyl succinate starch molecules contain both hydrophilic starch chains and hydrophobic octenyl succinic acid groups, exhibiting excellent emulsifying properties. Maltodextrin with a DE value of 16-20 has moderate viscosity and hygroscopicity; a DE value of 16-20 indicates a moderate degree of polymerization, providing sufficient viscosity for encapsulation without affecting the spray drying process due to excessive viscosity. The oligosaccharide chains of maltodextrin (DP≈10-15) encapsulate the outer layer through hydrogen bonds, forming a "bilayer protective shell," significantly improving the encapsulation rate compared to a single wall material. When used in combination, the glass transition temperature of the wall material can be adjusted, enhancing the density and mechanical strength of the microcapsule wall, further improving the stability of DHA, and also reducing production costs.

[0088] Table 2. Component test results of different DHA algae powder formulations

[0089]

[0090] Example 4

[0091] The accelerated oxidation of DHA algal powders 4 and 8-12 in Example 2 was tested in a constant temperature and humidity oven at 65℃. The changes in their anisidine values ​​were tested according to GB / T24304-2009 "Determination of Anisidine Value in Animal and Vegetable Oils". The results are as follows. Figure 1 As shown.

[0092] Based on the test results, the antioxidant was identified as either a mixture of biophenols or L-ascorbate palmitate. By comparing the effects of different antioxidants on the oxidative stability of DHA algal powder, the advantages of L-ascorbate palmitate were determined. It possesses both hydrophilic and lipophilic properties, allowing it to distribute evenly in the DHA oil phase, directly scavenging free radicals and inhibiting lipid oxidation. Furthermore, it synergistically forms a composite protective barrier with wall materials and emulsifiers, reducing oxygen contact. In the experiment, algal powder 16 with this antioxidant showed a significantly lower rate of increase in anisidine value during accelerated oxidation at 65℃ compared to the group containing mixed tocopherols, tea polyphenols, and other antioxidants, demonstrating the best antioxidant performance. In addition, its lipophilicity makes its antioxidant efficiency in oil systems higher than that of water-soluble antioxidants, effectively delaying DHA oxidation and maintaining the stability of algal powder quality.

[0093] Example 5

[0094] The experiment was conducted at the dairy farm of Inner Mongolia Mengniu Dairy (Group) Co., Ltd.

[0095] Two hundred healthy Holstein cows were selected based on age and parity (1st to 3rd parity). The test herd was managed according to the dairy farm's current feeding system, including tethering, regular brushing of the cows and cleaning of the pen, and free access to water.

[0096] Dairy cow feed includes a basal diet and DHA algae powder. The basal diet, on a dry matter basis, includes roughage and concentrate. Roughage includes alfalfa hay and corn silage; concentrate includes two or more of the following: extruded soybeans, corn, wheat bran, vitamins, soybean meal, carrots, salt, beets, bone meal, distillers' grains, and tofu residue. Roughage should account for 40% to 75% of the dry matter of the basal diet, and concentrate should account for 25% to 60% of the dry matter of the basal diet. The ratio should meet the nutritional needs of lactating dairy cows.

[0097] The embodiments of this invention employ the following specific basic diet formulation:

[0098] Roughage: Alfalfa hay accounts for 25% of the dry matter of the diet, and corn silage accounts for 30% of the dry matter of the diet.

[0099] Concentrates: Corn accounts for 20% of the dry matter of the diet, soybean meal accounts for 12%, puffed soybeans account for 5%, wheat bran accounts for 4%, carrots account for 2%, salt accounts for 0.5%, bone meal accounts for 1%, and premix accounts for 0.5%. The premix is ​​a mixture of vitamins A, D, and E.

[0100] The alfalfa hay was purchased from Shandong Huixin Grassland Ecological Technology Co., Ltd., and the corn silage was purchased from Beijing Hejiayuan Agricultural Technology Co., Ltd., with the variety name being: Silage Corn Tieyan 53.

[0101] The bone meal was multivitamin bone meal purchased from Tianjin Sanshang Feed Co., Ltd.

[0102] The premixed feed brand is Xingwang Veterinary Medicine, and it is a conventional commercially available animal vitamin AD3E. The feed approval number is: Su Si Yu Zi (2014) 826201.

[0103] All other feed ingredients used in this invention are conventional commercially available feeds.

[0104] Example 6

[0105] The DHA algal powder 3 prepared in Example 2 and the basal diet in Example 5 were used.

[0106] Specifically, the following method was used in this embodiment: The milk was from Holstein cows (lactation days: 90±28 days, weight: 628±103 kg). Each experimental group consisted of 25 cows, and the experimental period was 45 days. The feeding method for the DHA algae powder prepared in Example 2 was as follows: Single cows were precisely fed, with a gradual increase in the amount added. The intake per cow was 50 g / day on days 1-3, 100 g / day on days 4-6, 150 g / day on days 7-8, 200 g / day on day 9, and 250 g / day on days 10-45. The feed mixing method was as follows: 1 / 10 of the daily feed intake per cow (the basal diet in Example 5) was mixed with the algae powder. The algae powder was fed twice daily, with an interval of 5-10 hours between feedings.

[0107] Sampling began in the second week, with samples taken every Tuesday. On the day of sampling, the amount of milk collected three times a day (morning, noon, and evening) was recorded to evaluate whether algae powder affected milk production. The test included milk production, milk fat percentage, and DHA content in the milk.

[0108] The DHA algae powder 3 prepared in Example 2 was used in an experiment on a farm according to the above method. The control group dairy cows were fed feed without algae powder, but the other components were the same as the experimental group. Feeding time and amount were also the same as the experimental group. The results are shown in Table 3. After feeding DHA algae powder 3, compared with the control group, the DHA content in milk gradually increased with the number of feeding days, began to decrease after 29 days, and reached 17.4 mg / 100 g at 45 days, with a milk fat percentage of 3.3%. The results indicate that after adding DHA algae powder 3 to the diet, the native DHA content in milk increases after digestion and absorption by the cows, while milk production decreases slightly.

[0109] Table 3. Milk yield, DHA content, and milk fat content of algae powder 3 after being fed to dairy cows.

[0110]

[0111] Example 7

[0112] The DHA algae powder 4 prepared in Example 2 was used in an experiment on a dairy farm. The control group dairy cows were fed feed without any added *Schizochytrium* powder, but the other components were the same as the experimental group. Feeding time and amount were also the same as the experimental group. Other feeding conditions were the same as in Example 6. The results are shown in Table 4. After feeding DHA algae powder 4, compared with the control group, the DHA content in milk gradually increased with the number of feeding days, reaching 24.5 mg / 100 g after 45 days, with a milk fat percentage of 4.1%. The results indicate that after adding DHA algae powder 4 to the diet, the native DHA content in milk significantly increased after digestion and absorption by the cows, while milk production remained almost unchanged.

[0113] Table 4. Milk yield, DHA content, and milk fat content of milk after DHA algae powder was used to feed dairy cows.

[0114]

[0115] Example 8

[0116] The DHA algae powder 13 prepared in Example 2 was used in a farm experiment. The control group dairy cows were fed feed without any added *Schizochytrium* powder, but the other components were the same as the experimental group. Feeding time and amount were also the same as the experimental group. Other feeding conditions were the same as in Example 6. The results are shown in Table 5. After feeding DHA algae powder 13, compared with the control group, the DHA content in milk gradually increased with the number of feeding days, reaching only 14.3 mg / 100 g after 45 days, with a milk fat percentage of 3.5%. The results indicate that after adding DHA algae powder 13 to the diet, the native DHA content in milk increases after digestion and absorption by the cows, while milk production decreases slightly.

[0117] Table 5. Milk yield, DHA content in milk, and milk fat content after DHA algae powder 13 was used to feed dairy cows.

[0118]

[0119] Furthermore, DHA algae powder was prepared by replacing sodium octenyl succinate starch or maltodextrin with a DE value of 16-20 in group 4 of Example 2 with an equal amount of corn cob powder, gum arabic, acetylated starch, hydroxypropyl starch, β-cyclodextrin, or sodium caseinate and then testing it on a ranch under the same conditions as in Example 6. As the number of feeding days increased, the DHA content in the milk gradually increased, but it remained below 20 mg / 100 g, and the milk fat percentage was below 3.8%. Especially at 45 days, the effect was significantly less than that of DHA algae powder 4 in Example 2 of this invention. When the above wall material was used alone, the DHA content was even lower, and the milk fat percentage was further reduced. This further demonstrates the importance and specificity of the specific wallpaper combination in this invention.

[0120] In summary, Examples 6, 7, and 8 demonstrate that the composite wall material of sodium octenyl succinate starch and maltodextrin with a DE value of 16-20 achieves efficient encapsulation and protection of DHA algae powder through a triple mechanism of "emulsification-film formation-barrier". The DHA algae powder exhibits good stability, significantly increasing the content of native DHA in milk, while maintaining a high and stable milk fat percentage, effectively alleviating the problem of DHA degradation in the rumen of dairy cows. Its advantages stem from the complementary physicochemical properties of the materials themselves, as well as their processing adaptability and cost control, making it an ideal solution for functional oil encapsulation in the food industry.

Claims

1. A DHA algal powder, characterized in that, The Schizochytrium fermenting liquid, protein, wall material, emulsifier, antioxidant, dispersant, pH regulator and water are included. The protein accounts for 0.5-5.0% of the weight of the Schizochytrium fermenting liquid; the wall material accounts for 0.2-2.5% of the weight of the Schizochytrium fermenting liquid and is one or more of sodium octenyl succinate starch, malt dextrin, corn cob powder, monoglyceride, citric acid fatty acid glyceride, sucrose ester, β-cyclodextrin and sodium caseinate; the emulsifier accounts for 0.1-0.3% of the weight of the Schizochytrium fermenting liquid; the antioxidant accounts for 0-0.3% of the weight of the Schizochytrium fermenting liquid; the dispersant accounts for 0.1-1.0% of the weight of the Schizochytrium fermenting liquid; and the pH regulator accounts for 0.05-0.4% of the weight of the Schizochytrium fermenting liquid.

2. The DHA algal powder according to claim 1, characterized in that, The Schizochytrium is preferably Schizochytrium sp. HX-308; and the Schizochytrium fermenting liquid is a product formed after fermentation of the Schizochytrium and includes Schizochytrium cells and residual culture medium components after fermentation.

3. The DHA algal powder of claim 1, wherein, The protein is any one or more of corn protein, soybean protein, sunflower seed protein and soybean protein isolate, wherein the corn protein accounts for 1.0-3.0% of the weight of the fermenting liquid, the soybean protein accounts for 1.0-5.0% of the weight of the fermenting liquid, the sunflower seed protein accounts for 1.0-4.0% of the weight of the fermenting liquid, and the soybean protein isolate accounts for 0.5-2.5% of the weight of the fermenting liquid.

4. The DHA algal powder of claim 1, wherein, The wall material is one or more of sodium octenyl succinate starch, malt dextrin with a DE value of 16-20, corn cob powder, monoglyceride, citric acid fatty acid glyceride, sucrose ester, β-cyclodextrin and sodium caseinate, wherein the sodium octenyl succinate starch accounts for 0.2-1.5% of the weight of the fermenting liquid, the malt dextrin accounts for 0.1-2.0% of the weight of the fermenting liquid, and the corn cob powder accounts for 0.5-2.5% of the weight of the fermenting liquid.

5. The DHA algal powder of claim 1, wherein, The wall material is a combination of sodium octenyl succinate starch and malt dextrin with a DE value of 16-20, wherein the sodium octenyl succinate starch accounts for 0.2-1.5% of the weight of the fermenting liquid and the malt dextrin with a DE value of 16-20 accounts for 0.1-2.0% of the weight of the fermenting liquid.

6. The DHA algal powder of claim 1, wherein, The emulsifier is polyglycerol fatty acid ester and accounts for 0.1-0.3% of the weight of the fermenting liquid; the antioxidant is one of L-ascorbyl palmitate, bamboo leaf antioxidant, tea polyphenol, ascorbic acid, rosemary extract, mixed tocopherol and grape seed extract and accounts for 0-0.3% of the weight of the fermenting liquid; the dispersant is white carbon black and accounts for 0.1-1.0% of the weight of the fermenting liquid; and the pH regulator is one or more of citric acid and vitamin C, wherein the citric acid accounts for 0.05-0.2% of the weight of the fermenting liquid and the vitamin C accounts for 0.1-0.2% of the weight of the fermenting liquid.

7. A method of preparing the DHA algal flour of claim 1, characterized by, The method comprises the following steps: (1) fermenting Schizochytrium to obtain a Schizochytrium fermenting liquid; (2) using the Schizochytrium fermenting liquid without treatment or after biological enzyme cell wall pretreatment. (3) adding protein, wall material, emulsifier, antioxidant, dispersing agent, pH regulator, water to the untreated or treated Schizochytrium fermenting liquid sample, and shearing and spray drying to obtain the DHA algal powder.

8. The method of producing DHA algal powder according to claim 7, characterized in that, The solvent of the fermentation medium in step (1) is water, including 10-60 g / L of glucose, 1-3 g / L of KH2PO4, 0.2-3 g / L of K2SO4, 1-3 g / L of MgSO4, 1-4 g / L of (NH4)2SO4, 0.1-0.4 g / L of CaCl2·2H2O, 5-15 g / L of Na2SO4, 0.2-2 g / L of KCl, 5-20 g / L of sodium glutamate, 6-10 g / L of malic acid, 8-25 g / L of yeast powder, 0.5-3 g / L of corn syrup powder, and pH of 6.0-6.5; the fermentation condition is 28-30°C, and the fermentation time is 100-120 h.

9. The method of claim 7, wherein the DHA algal powder is prepared by, The biological enzyme wall-breaking pretreatment in step (2) is at a temperature of 40-60°C for 1-3 h, and the mass / volume ratio of the biological enzyme to the fermentation liquid after Schizochytrium fermentation is (0.1-2.5) kg:1000 L, and the biological enzyme is at least one of alkaline protease, neutral protease, papain, cellulase, β-glucanase and xylanase.

10. A feed for dairy cattle, characterized in that, The DHA algal powder of claim 1 is included in the basic daily ration, and the DHA algal powder accounts for 2%-10% of the total weight of the dry matter of the basic daily ration, the basic daily ration contains coarse feed and concentrate feed in terms of dry matter, the coarse feed accounts for 40%-75% of the mass of the dry matter of the basic daily ration, and the concentrate feed accounts for 25%-60% of the mass of the dry matter of the basic daily ration, wherein the coarse feed includes alfalfa hay and corn silage, and the concentrate feed includes a combination of two or more of puffed soybeans, corn, bran, vitamins, soybean meal, carrots, salt, sugar beets, bone meal, distiller's grains, and bean curd residue.

11. Use of the DHA algal powder of claim 1 or the dairy cow feed of claim 10 in increasing the DHA content of milk.

12. Use according to claim 11, characterized in that, The use is feeding the DHA algal powder 2-3 times a day, with a feeding amount of 50-250 g / day / head, and a feeding interval of 5-10 hours, and feeding the dairy cow for more than 30-45 days.

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