A method for preparing a dha algal flour, and products and uses thereof

Abstract

The application provides a preparation method, product and application of DHA algal powder, and the method comprises the following steps: inoculating a schizochytrium seed liquid into a fermentation medium for fermentation, inoculating a yeast mixed fermentation into the fermentation medium in the late fermentation stage, and obtaining a fermentation liquid containing DHA until the fermentation is completed, and then performing spray drying to obtain the DHA algal powder. The preparation method provided by the application utilizes the mixed fermentation of schizochytrium and yeast, reduces the content of extracellular polysaccharide in the fermentation liquid, solves the problems of product wall sticking and equipment blockage in the spray drying process, has higher substrate utilization rate, higher product yield, lower equipment maintenance cost and a DHA algal powder product with better performance, conforms to the energy-saving and green production concept, and is beneficial to the protection of the ecological environment.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a preparation method of DHA algal powder, and products and applications thereof. BACKGROUND

[0002] Marine algae resources are rich, and are mainly divided into red algae, green algae, brown algae and microalgae. Due to the large number of microalgae species, large quantity and wide distribution, microalgae play a positive role in the entire marine ecosystem. Due to the extreme characteristics of high salt, high pressure, low temperature, low nutrition, low light and other characteristics of the ocean and the unique water environment of being in a flowing state all the time, some marine organisms must have corresponding adhesion structures or sticky substances to ensure a relatively stable ecological environment.

[0003] Docosahexaenoic acid (DHA), abbreviated as DHA, has a molecular formula of C 22 H 32 O2, is a long-chain omega-3 polyunsaturated fatty acid. Schizochytrium limacinum, also known as Schizochytrium, belongs to marine fungi of water mold order and thraustochytrid family. Thraustochytrid is a saprophytic microorganism, accounting for 30% of the total number of microorganisms in decaying brown algae. The bacteria widely exist in environments rich in organic matter and high in salinity, with a content of 2.1×10 3 -4.1×10 4 L in seawater, especially in mangrove forests rich in organic matter, with a larger biomass. The DHA contained in Schizochytrium is a polyunsaturated fatty acid essential for the human body. Because the growth of Schizochytrium is not affected by seasons, it has many advantages such as no pollution to the environment and stable nutritional composition, and has become an ideal strain for fermentation production of DHA. Schizochytrium is an ideal additive for aquatic and poultry feed, and its safety has been recognized by the Food and Drug Administration (FDA). Studies have shown that feeding fish, shrimp and shellfish with Schizochytrium dry powder can improve the immunity of their larvae and increase the survival rate, and has a significant promoting effect on the growth of their larvae. Adding Schizochytrium powder to the feed of poultry can increase the content of DHA in poultry products such as meat, eggs and milk.

[0004] The microalgae of this kind will secrete a large amount of polysaccharides while fermenting to produce DHA. The polysaccharides or polysaccharide complexes exist outside the cell wall and are easy to separate from the bacteria, and are called exopolysaccharides (EPS). EPS is composed of continuous oligosaccharide units, and the oligosaccharide units contain 73-85% galactose, 5.5-8.5% glucose, 2.5-8% mannose, 2-6% rhamnose, 1.5-3.8% fucose and 1.2-2.50% xylose. The HPGPC method can detect four polysaccharide components SP1, SP2, SP3 and SP4, and the molecular weights are 43 kDa, 1494 kDa, 473 kDa and 1625 kDa, respectively. SP1 mainly contains D-glucose, D-mannose and galactose; SP2 contains two of D-mannose, D-idose, galactose and D-glucose; SP3 and SP4 are similar and contain D-mannose, idose and galactose.

[0005] These exopolysaccharides will increase the viscosity of the fermentation broth and sharply reduce the maximum dissolved oxygen, thereby affecting the progress of fermentation. Therefore, these exopolysaccharides are often discarded as by-products. In practical applications, especially in the preparation process of algal powder, the schizochytrium fermentation broth contains a large amount of exopolysaccharides. The sticky properties of the polysaccharides will cause wall sticking and clogging in the spray drying process, which not only causes a large amount of product loss, but also affects the quality of the product.

[0006] CN103204951B introduces a method for separating and purifying schizochytrium exopolysaccharides. The method includes the following steps: 1) using schizochytrium fermentation broth as raw material, precipitating polysaccharides in the fermentation broth with ethanol, washing the insoluble matter with an organic solvent, dissolving the insoluble matter with deionized water, removing proteins by trichloroacetic acid method, precipitating the polysaccharide crude product with ethanol again, and preparing a fractionation solution with deionized water as solvent; 2) gradient elution of the fractionation solution prepared in step 1) through fast-flow cellulose agarose gel anion exchange resin, collecting each tube, determining the polysaccharide concentration of each tube by the sulfuric acid phenol method, drawing an elution curve, combining each part collected, concentrating, removing small molecular impurities through a dialysis membrane, and freeze-drying to obtain the separated and purified schizochytrium exopolysaccharides. The operation is simple, efficient, suitable for large-scale industrial production, and the obtained polysaccharides have high physiological activity.

[0007] CN105685766B utilizes schizochytrium strain fermentation to prepare an exopolysaccharide composition. The exopolysaccharide contains, in terms of molar percentage, 73-85% galactose, 5.5-8.5% glucose, 2.5-8% mannose, 2-6% rhamnose, 1.5-3.8% fucose and 1.2-2.5% xylose. The invention also relates to a preparation method of the composition, and the composition prepared by the method. The composition of the invention has significant antioxidant activity and can be applied to food antioxidant and fruit and vegetable preservation.

[0008] Due to the positive interaction between different microorganisms, it is found that the mixed fermentation of two or more microorganisms can better solve many problems in practice. In the past few years, the application of mixed fermentation of microorganisms and the mechanism of interaction between microorganisms have made obvious progress, mainly including the following aspects (1) degradation and utilization of biomass (2) degradation of environmental pollutants (3) production of specific metabolites (4) mixed fermentation process. The applicant has used mixed fermentation of fermentation broth, and a method for producing DHA and astaxanthin by mixed culture of schizochytrium and haematococcus pluvialis is disclosed in patent document CN110408671B. Schizochytrium is inoculated in the fermentation broth after the growth period of haematococcus pluvialis to carry out mixed fermentation culture, which has the characteristics of simplifying the culture process, reducing the production cost, improving the stability during oil extraction, etc.

[0009] The utilization and separation of extracellular polysaccharide of schizochytrium in the prior art are obviously not suitable for the feed field which pursues efficiency and cost. Therefore, it is very meaningful to study the interaction of microorganisms and develop the potential of mixed culture of microorganisms to solve the problems encountered in fermentation production. SUMMARY

[0010] In view of the deficiencies of the prior art, the purpose of the present application is to provide a preparation method and product and application of algal powder, and in particular to provide a preparation method and product and application of DHA algal powder. The preparation method provided by the present application utilizes mixed fermentation of schizochytrium and yeast, reduces the content of extracellular polysaccharide in the fermentation broth, solves the problem of product sticking to the wall and equipment blocking during spray drying, has higher substrate utilization rate, higher product yield, lower equipment maintenance cost and DHA algal powder product with better performance, conforms to the production concept of energy saving and green, and is beneficial to the protection of ecological environment.

[0011] In order to achieve the purpose of the present application, the following technical solutions are adopted:

[0012] In a first aspect, the present application provides a preparation method of DHA algal powder, which comprises the following steps: inoculating schizochytrium seed liquid into fermentation medium for fermentation, inoculating yeast for mixed fermentation in the late stage of fermentation culture, and obtaining fermentation broth containing DHA after fermentation is completed, and then carrying out spray drying to obtain the DHA algal powder.

[0013] The preparation method described in this invention involves inoculating a certain amount of activated yeast solution into the fermentation broth during the later stages of Schizochytrium fermentation for mixed-culture fermentation. The fermentation broth during the later stages of Schizochytrium fermentation often contains a large amount of extracellular polysaccharides, a byproduct. These extracellular polysaccharides cause a decrease in the maximum dissolved oxygen content and an increase in viscosity, which is detrimental to the subsequent growth and fermentation of Schizochytrium. The viscous nature of the fermentation broth also causes wall adhesion and equipment blockage during spray drying, further affecting the quality of the DHA algal powder product. Adding yeast during the later stages of fermentation allows the yeast to utilize the extracellular polysaccharides as a substrate. The yeast first consumes the monosaccharides in the fermentation broth, then secretes extracellular enzymes to degrade the polysaccharides, breaking them down into monosaccharides for absorption. Since there are very few monosaccharides remaining in the system during the later stages of fermentation, the yeast can quickly utilize the polysaccharides, thereby reducing the extracellular polysaccharide content in the fermentation broth and effectively reducing its viscosity. This solves the problem of wall adhesion and blockage caused by excessive fermentation broth viscosity during spray drying from the source of the fermentation process. Furthermore, the resulting product contains less extracellular polysaccharides, resulting in a high product yield, high DHA content, and excellent performance. Furthermore, this preparation method is simple to operate and suitable for industrial scale-up.

[0014] Preferably, the amount of yeast inoculated is 5-15% of the volume of the Schizochytrium fermentation broth.

[0015] The specific values ​​in the 5-15% range can be selected from 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15%, etc. Other specific point values ​​within the above ranges can also be selected, which will not be elaborated here.

[0016] When the yeast inoculation amount involved in this invention is less than 5% of the volume of the Schizochytrium fermentation broth, the extracellular polysaccharides are not completely consumed by the yeast, and the problem of extracellular polysaccharides in the fermentation broth is not well solved. When the yeast inoculation amount is higher than 15%, the excessive yeast will over-consume the nutrients in the fermentation broth, interfering with the later metabolism of Schizochytrium and causing a decrease in DHA, an important component in algal powder. Therefore, when the yeast inoculation amount is between 5% and 15%, the extracellular polysaccharides can be fully consumed by the yeast, while the yeast content in the fermentation broth is moderate and has almost no impact on the metabolism of Schizochytrium. Furthermore, the extracellular polysaccharide content in the fermentation broth becomes lower, the viscosity of the fermentation broth is lower, and the lower extracellular polysaccharide content provides a more favorable environment for mixed fermentation, resulting in a higher DHA product yield and higher DHA content in the product.

[0017] Preferably, during the mixed fermentation process, the pH of the mixed fermentation broth is maintained at no less than 6. For example, the pH can be 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10, etc. More preferably, the pH is between 6 and 9. For example, the pH can be 6, 6.5, 7, 7.5, 8, 8.5 or 9, etc. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0018] The pH of the fermentation broth in the mixed fermentation system according to the present application is not less than 6, especially between 6 and 9, so that the two bacteria in the mixed bacteria system have better cell state, lower content of extracellular polysaccharide, lower viscosity of the fermentation broth, and the DHA algal powder product is less likely to stick to the wall or block the pipeline, the product yield is higher, and the DHA content in the product is higher.

[0019] Preferably, the pH of the mixed fermentation broth is maintained by using an acid-base regulator.

[0020] Preferably, the acid-base regulator comprises any one or a combination of at least two of citric acid, sodium bicarbonate, sodium carbonate, hydrochloric acid or sodium hydroxide, and the combination of the at least two can be a combination of citric acid and sodium bicarbonate, a combination of sodium bicarbonate and sodium carbonate, a combination of sodium carbonate and hydrochloric acid, etc., and any other combination can be selected, which will not be described one by one here.

[0021] Preferably, the acid-base regulator is citric acid and sodium hydroxide.

[0022] In the mixed fermentation process of the present application, the yeast is added at the late stage of the Schizochytrium sp. fermentation, and in the process of consuming monosaccharides and polysaccharides in the fermentation broth, the pH of the fermentation broth decreases, which is not conducive to the growth of the mixed bacteria. The addition of the acid-base regulator in the mixed fermentation process is more suitable for the growth and metabolism of the two bacteria and the production fermentation of Schizochytrium sp. When the acid-base regulator is specifically selected as a combination of citric acid and sodium hydroxide, the pH of the fermentation broth in the mixed fermentation process is more stable, and compared with sodium carbonate and sodium bicarbonate, citric acid and sodium hydroxide will not produce carbon dioxide gas in the pH adjustment process, which will not affect the gas system in the fermentation process.

[0023] Preferably, the late stage of the fermentation culture is 20-40h before the end of the Schizochytrium sp. fermentation.

[0024] The specific values in the 20-40h can be 20h, 24h, 28h, 32h, 36h or 40h, and other specific point values within the above numerical range can be selected, which will not be described one by one here.

[0025] Preferably, the total fermentation culture time is 110-130h.

[0026] The values in the 110-130h can be 110h, 114h, 118h, 122h, 126h, 130h, and other specific point values within the above numerical range can be selected, which will not be described one by one here.

[0027] It can be understood that the time of adding the yeast in the present application can be determined according to the total fermentation time, for example, when the total fermentation time is 120 h, the yeast is added at 80-100 h. If the yeast is added too early, the growth of the Schizochytrium sp. and the fermentation production will be affected, and the DHA content in the DHA algal powder product will be reduced. If the yeast is added less than 20 h before the end of the fermentation, the fermentation time of the yeast is short, and the yeast cannot fully consume the exocellular polysaccharide in the fermentation broth. Further, the viscosity of the fermentation broth is large, and the wall sticking and blocking phenomenon occurs in the spray drying process, and the yield of the DHA algal powder is low. Therefore, when the yeast is added 20-40 h before the end of the fermentation of the Schizochytrium sp., the wall sticking and blocking phenomenon in the spray drying process is less, and the yield of the DHA algal powder and the DHA content in the product are higher.

[0028] Preferably, the yeast includes Saccharomyces cerevisiae.

[0029] Since the polysaccharide decomposition ability of Saccharomyces cerevisiae is universal, and the main purpose of the present application is not the metabolic product of Saccharomyces cerevisiae, the type of Saccharomyces cerevisiae is not limited herein.

[0030] When the yeast in the present application includes Saccharomyces cerevisiae, the exocellular polysaccharide content of the fermentation broth in the mixed fermentation process is lower, and the DHA algal powder product has higher nutritional value.

[0031] Preferably, the inlet air temperature of the spray drying is 160-220℃.

[0032] The values in the range of 160-220℃ can be 160℃, 170℃, 180℃, 190℃, 200℃, 210℃ or 220℃, and other specific point values in the above range can be selected, which will not be described one by one herein.

[0033] Preferably, the outlet air temperature of the spray drying is 70-100℃.

[0034] The values in the range of 70-100℃ can be 70℃, 75℃, 80℃, 85℃, 90℃, 95℃ or 100℃, and other specific point values in the above range can be selected, which will not be described one by one herein.

[0035] When the inlet air temperature of the spray drying is selected to be 160-220℃ and the outlet air temperature is 70-100℃, the efficiency of the spray drying is higher.

[0036] Preferably, the spray drying further includes a centrifugal dehydration step of the mixed product containing DHA, so that the solid content is more than 20% of the mixed product content.

[0037] The specific values of the 20% or more include 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90%, and other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0038] Preferably, the preparation method of the Schizochytrium seed liquid comprises activation and expansion culture of Schizochytrium.

[0039] Preferably, the inoculation amount of the Schizochytrium seed liquid is 3-10% of the volume of the fermentation medium.

[0040] The specific values of the 3-10% include 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, and other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0041] Preferably, the activation culture time of the Schizochytrium is 30-50h.

[0042] The numerical values in the 30-50h can be 30h, 34h, 38h, 42h, 46h, or 50h, and other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0043] Preferably, the culture time of the expansion culture is 20-40h.

[0044] The numerical values in the 20-40h can be 20h, 24h, 28h, 32h, 36h, or 40h, and other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0045] Preferably, the temperature of the fermentation of the Schizochytrium seed liquid inoculated into the fermentation medium and the mixing fermentation is independently 25-30℃, and the aeration amount of the fermentation is independently 1-2vvm.

[0046] The numerical values in the 25-30℃ can be 25℃, 26℃, 27℃, 28℃, 29℃, or 30℃, and other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0047] The numerical values in the 1-2vvm can be 1vvm, 1.2vvm, 1.4vvm, 1.6vvm, 1.8vvm, or 2vvm, and other specific point values within the above-mentioned numerical ranges can be selected, which will not be repeated here.

[0048] Preferably, the yeast is activated and expanded before inoculation.

[0049] Preferably, the culture time of the activation is 30-50h.

[0050] The median value in the range of 30-50h can be 30h, 34h, 38h, 42h, 46h or 50h, and other specific point values in the above-mentioned numerical range can be selected, which will not be repeated here.

[0051] Preferably, the culture time of the scale-up culture is 20-40h.

[0052] The median value in the range of 20-40h can be 20h, 24h, 28h, 32h, 36h or 40h, and other specific point values in the above-mentioned numerical range can be selected, which will not be repeated here.

[0053] Preferably, the dissolved oxygen amount in the first 30h of the fermentation of the Aureobasidium pullulans seed liquid inoculated into the fermentation medium is 20-80%, and the dissolved oxygen amount after 30h is 5-80%.

[0054] The specific value of the range of 20-80% can be 20%, 30%, 40%, 50%, 60%, 70% or 80%, and other specific point values in the above-mentioned numerical range can be selected, which will not be repeated here.

[0055] The specific value of the range of 5-80% can be 5%, 20%, 35%, 50%, 65%, 70%, 75% or 80%, and other specific point values in the above-mentioned numerical range can be selected, which will not be repeated here.

[0056] In the second aspect, the present application provides a DHA algal powder, which is prepared according to the preparation method of the first aspect.

[0057] Preferably, the DHA algal powder contains yeast, and the content of DHA in the DHA algal powder is greater than 20% and the moisture content is less than 5%.

[0058] The specific value of greater than 20% can be 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% or 90%, and other specific point values in the above-mentioned numerical range can be selected, which will not be repeated here.

[0059] The specific value of less than 5% can be 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4% or 4.5%, and other specific point values in the above-mentioned numerical range can be selected, which will not be repeated here.

[0060] The DHA algal powder product of the present application contains algal powder and yeast powder, is rich in DHA, and also contains yeast powder compared with single Schizochytrium algal powder product, the yeast powder is low in fat, low in sugar, low in cholesterol, rich in protein and vitamins, so that the DHA algal powder has more diversified nutritional elements, and the mixed fermentation process also produces some organic acids and ketone substances, improving the nutritional value of the final product. In addition, the DHA algal powder has low sugar content, the distribution of various nutritional elements of the product is more uniform, and the quality of each batch of product is more stable.

[0061] In a third aspect, the present application provides the use of the preparation method of the first aspect or the DHA algal powder of the second aspect in the preparation of an animal nutritional agent.

[0062] Compared with the prior art, the present application has the following beneficial effects:

[0063] The preparation method of the present application is to inoculate a certain amount of yeast activation liquid into the fermentation broth in the late fermentation of Schizochytrium for mixed fermentation culture, the fermentation broth in the late fermentation of Schizochytrium usually contains a large amount of by-product extracellular polysaccharide, which will cause the maximum dissolved oxygen capacity of the fermentation broth to decrease and the viscosity to increase, which is not conducive to the subsequent growth and fermentation of Schizochytrium, and the sticky properties of the fermentation broth will also cause the wall sticking and equipment blockage phenomenon to occur in the spray drying process, which will further affect the quality of the DHA algal powder product. The yeast is added in the late fermentation culture, the yeast can use extracellular polysaccharide as a substrate, the yeast first consumes monosaccharide in the fermentation broth, and then secretes extracellular enzymes to degrade polysaccharide, and after the polysaccharide is decomposed into monosaccharide, the yeast absorbs it, and the monosaccharide in the system is almost exhausted in the late fermentation, so the yeast will quickly utilize the polysaccharide, thereby reducing the content of extracellular polysaccharide in the fermentation broth, effectively reducing the viscosity of the fermentation broth, solving the problem of wall sticking and blockage in the spray drying process due to the excessive viscosity of the fermentation broth from the source of the fermentation process, and the product prepared has less extracellular polysaccharide, high product yield, uniform product particles, high DHA content, and excellent performance. In addition, the preparation method is simple to operate and suitable for industrial scale-up. DETAILED DESCRIPTION

[0064] The technical solutions of the present application will be further described through specific embodiments. Those skilled in the art should understand that the embodiments are only used to help understand the present application, and should not be regarded as specific limitations on the present application.

[0065] If the specific techniques or conditions are not specified in the embodiments, the techniques or conditions described in the literature in the art or according to the product instructions are used. If the reagents or instruments are not specified by the manufacturer, they are all conventional products that can be commercially available through regular channels.

[0066] The preparation method of the culture medium involved in the following examples, comparative examples and test examples is as follows:

[0067] Primary activation liquid medium, secondary expansion liquid medium formula: 4% glucose, 3% sodium glutamate, 0.5% yeast extract, 2% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.5% magnesium sulfate, 0.02% calcium chloride, supplemented with water to 100%, sterilized after preparation for standby.

[0068] Fermentation medium ingredients: 5% glucose, 3% sodium glutamate, 0.9% yeast extract, 0.5% sodium chloride, 0.2% potassium dihydrogen phosphate, 0.2% magnesium sulfate, 0.02% calcium chloride, 1% sodium sulfate, 0.1% potassium chloride, 0.2% ammonium sulfate, 0.013% sodium bicarbonate, 0.1% vitamin mixture (9.5 g / L thiamine, 3.2 g / L calcium pantothenate, 0.06 g / L biotin), the rest is supplemented with water to 100%, sterilized and ready for use.

[0069] Preparation Example 1

[0070] Schizochytrium seed liquid preparation

[0071] Schizochytrium sp. (Biological preservation number: CCTCC M 2019990) was inoculated into the primary activation liquid medium at an inoculation amount of 3%, and cultured at a temperature of 28°C and a shaking speed of 220 r / min for 40 h to obtain a primary activation liquid. The primary activation liquid was inoculated into the secondary expansion liquid medium at an inoculation amount of 2.5%, and cultured at a temperature of 28°C and a shaking speed of 220 r / min for 30 h to obtain a Schizochytrium seed liquid.

[0072] It is worth noting that the secretion of the Schizochytrium extracellular polysaccharide in the present application is a universal phenomenon in Schizochytrium. Although the present preparation example uses Schizochytrium sp. CABIO-A-2-Ⅱ strain (Biological preservation number: CCTCC M 2019990) to embody the content of the application, other Schizochytrium strains, including but not limited to Schizochytrium sp. CABIO-A-2-III strain (Biological preservation number: CCTCC NO: M 2021564), Schizochytrium sp. CABIO-A-2-IV (Biological preservation number: CCTCC NO: M 2021566), etc. can also be used to achieve the effect of the application.

[0073] Preparation Example 2

[0074] Yeast liquid preparation

[0075] Saccharomyces cerevisiae is a commercially available product of Angel Yeast. It was inoculated into YPD medium at an inoculation amount of 3%, and cultured at a temperature of 28°C and a shaking speed of 220 r / min for 40 h to obtain a primary activation liquid. The primary activation liquid was inoculated into YPD medium at an inoculation amount of 3%, and cultured at a temperature of 28°C and a shaking speed of 220 r / min until the OD 600 = 20, to obtain a Saccharomyces cerevisiae liquid.

[0076] Example 1

[0077] The present example provides a DHA algal powder, which is prepared as follows: Schizochytrium sp. seed liquid is inoculated into fermentation medium at an inoculation amount of 5%, and a 50L fermenter is used for cultivation, the cultivation temperature is 28°C, the rotation speed is 300r / min, the aeration amount is 1.5vvm, the dissolved oxygen amount is 40% in the first 30h of fermentation culture, and the dissolved oxygen amount is 20% after 30h, citric acid is used to control the pH to 7.8 during the fermentation culture, and the residual sugar is supplemented according to the residual sugar, and 5% is supplemented at one time when the residual sugar is less than 5g / L;

[0078] Schizochytrium sp. is mixed with Saccharomyces cerevisiae liquid at a volume ratio of 10% when the fermentation culture reaches 90h, sodium hydroxide is used to adjust the pH to maintain the pH of the fermentation liquid to 7, the mixed fermentation is terminated after 30h, the fermentation liquid is collected, and a mixed product containing DHA is obtained;

[0079] The mixed product is centrifuged to a solid content of 40% of the total mass, and then spray dried, the inlet air temperature is 200°C, the outlet air temperature is 80°C, the feed concentration is 100g / L, the outlet air temperature is controlled to reach 90°C by controlling the feed amount, and the dried product is collected, which is the DHA algal powder.

[0080] Example 2

[0081] The present example provides a DHA algal powder, which is prepared as follows: Schizochytrium sp. seed liquid is inoculated into fermentation medium at an inoculation amount of 3%, and a 50L fermenter is used for cultivation, the cultivation temperature is 28°C, the rotation speed is 200r / min, the aeration amount is 1vvm, the dissolved oxygen amount is 20% in the first 30h of fermentation culture, and the dissolved oxygen amount is 5% after 30h, citric acid is used to control the pH to maintain the pH to 7.8 during the fermentation culture, and the residual sugar is supplemented according to the residual sugar, and 5% is supplemented at one time when the residual sugar is less than 8g / L;

[0082] Schizochytrium sp. is mixed with Saccharomyces cerevisiae liquid at a volume ratio of 5% when the fermentation culture reaches 100h, sodium hydroxide is used to adjust the pH to maintain the pH of the fermentation liquid to 7.5, the mixed fermentation is terminated after 20h, the fermentation liquid is collected, and a mixed product containing DHA is obtained;

[0083] The mixed product is centrifuged to a solid content of 50% of the total mass, and then spray dried, the inlet air temperature is 220°C, the outlet air temperature is 100°C, the feed concentration is 100g / L, the outlet air temperature is controlled to reach 90°C by controlling the feed amount, and the dried product is collected, which is the DHA algal powder.

[0084] Example 3

[0085] The embodiment provides a DHA algal powder, and a preparation method thereof is as follows: Schizochytrium sp. seed liquid is inoculated into a fermentation medium at an inoculation amount of 10%, a 50L fermentation tank is used for culture, the culture temperature is 28 DEG C, the rotating speed is 400r / min, the ventilation amount is 2vvm, the dissolved oxygen amount is 50% in the first 30h of fermentation culture, the dissolved oxygen amount is 30% after 30h, citric acid is used for controlling the pH to be 7.8 in the fermentation culture process, the residual sugar is supplemented, and 5% is supplemented at a time when the residual sugar is less than 5g / L;

[0086] Schizochytrium sp. is fermented and cultured to 80h, and then 15% of the fermentation liquid of Schizochytrium sp. is mixed with Saccharomyces cerevisiae liquid for mixed fermentation, sodium carbonate is used for adjusting the pH so that the pH of the fermentation liquid is maintained to 6.5, the mixed fermentation is stopped after 40h, the fermentation liquid is collected, and a mixed product containing DHA is obtained;

[0087] The mixed product is centrifuged and dewatered to 30% of solid matter in total mass, and then spray drying is carried out, the inlet air temperature is 160 DEG C, the outlet air temperature is 70 DEG C, the feeding concentration is 100g / L, the outlet air temperature reaches 90 DEG C by controlling the feeding amount, and the dried product is collected, which is the DHA algal powder.

[0088] Embodiment 4

[0089] The embodiment provides a DHA algal powder, and a preparation method thereof is as follows: Schizochytrium sp. seed liquid is inoculated into a fermentation medium at an inoculation amount of 10%, a 50L fermentation tank is used for culture, the culture temperature is 28 DEG C, the rotating speed is 400r / min, the ventilation amount is 2vvm, the dissolved oxygen amount is 50% in the first 30h of fermentation culture, the dissolved oxygen amount is 30% after 30h, citric acid is used for controlling the pH to be 7.8 in the fermentation culture process, the residual sugar is supplemented, and 5% is supplemented at a time when the residual sugar is less than 5g / L;

[0090] Embodiment 5

[0091] The embodiment provides a DHA algal powder, and a preparation method thereof is as follows: Schizochytrium sp. seed liquid is inoculated into a fermentation medium at an inoculation amount of 10%, a 50L fermentation tank is used for culture, the culture temperature is 28 DEG C, the rotating speed is 400r / min, the ventilation amount is 2vvm, the dissolved oxygen amount is 50% in the first 30h of fermentation culture, the dissolved oxygen amount is 30% after 30h, citric acid is used for controlling the pH to be 7.8 in the fermentation culture process, the residual sugar is supplemented, and 5% is supplemented at a time when the residual sugar is less than 5g / L;

[0092] Embodiment 6

[0093] The embodiment provides a DHA algal powder, and a preparation method thereof is as follows: Schizochytrium sp. seed liquid is inoculated into a fermentation medium at an inoculation amount of 10%, a 50L fermentation tank is used for culture, the culture temperature is 28 DEG C, the rotating speed is 400r / min, the ventilation amount is 2vvm, the dissolved oxygen amount is 50% in the first 30h of fermentation culture, the dissolved oxygen amount is 30% after 30h, citric acid is used for controlling the pH to be 7.8 in the fermentation culture process, the residual sugar is supplemented, and 5% is supplemented at a time when the residual sugar is less than 5g / L;

[0094] Embodiment 7

[0095] The embodiment provides a DHA algal powder, and a preparation method thereof is as follows: Schizochytrium sp. seed liquid is inoculated into a fermentation medium at an inoculation amount of 10%, a 50L fermentation tank is used for culture, the culture temperature is 28 DEG C, the rotating speed is 400r / min, the ventilation amount is 2vvm, the dissolved oxygen amount is 50% in the first 30h of fermentation culture, the dissolved oxygen amount is 30% after 30h, citric acid is used for controlling the pH to be 7.8 in the fermentation culture process, the residual sugar is supplemented, and 5% is supplemented at a time when the residual sugar is less than 5g / L;

[0096] Embodiment 8

[0097] The present example provides a DHA algal powder, the preparation method of which is different from that of Example 1 only in that the inoculation time of the yeast is 110 h of the cultivation of the Schizochytrium sp., and the rest is the same as that of Example 1.

[0098] Comparative Example 1

[0099] The present comparative example provides a DHA algal powder, the preparation method of which is as follows: the Schizochytrium sp. seed liquid is inoculated into the fermentation medium at a 5% inoculation amount, and a 50L fermenter is used for cultivation, the cultivation temperature is 28°C, the rotation speed is 300r / min, the aeration amount is 1.5vvm, the dissolved oxygen amount is 40% in the first 30h of the fermentation cultivation, and the dissolved oxygen amount is 20% after 30h, citric acid is used to control the pH at 7.8 during the fermentation cultivation, the residual sugar is supplemented according to the residual sugar, and when the residual sugar is less than 10g / L, 5% is supplemented at one time; the Schizochytrium sp. fermentation cultivation is terminated at 120h.

[0100] The Schizochytrium sp. fermentation liquid is centrifuged to 40% of the solid matter in the total mass, and then spray drying is performed, the inlet air temperature is 200°C, the outlet air temperature is 80°C, the feeding concentration is 100g / L, the outlet air temperature reaches 90°C by controlling the feeding amount, and the dried product is collected, which is the DHA algal powder.

[0101] Test Example 1

[0102] The extracellular polysaccharide content of the fermentation liquid after the fermentation of Examples 1-8 and Comparative Example 1 is evaluated.

[0103] The specific evaluation method of the exopolysaccharide index is as follows: (1) Take the fermentation broth, add 4 times the volume of 60% ethanol and stir at 60°C for 1 h, centrifuge at 8000 rpm for 10 min to obtain supernatant and precipitate; (2) Add 3 times the amount of 60% ethanol to the precipitate and stir at 60°C for 1 h to obtain supernatant, and mix the supernatant with the supernatant of (1); (3) Remove the ethanol in the mixed supernatant by vacuum concentration, then add petroleum ether, repeat 2 times to remove lipids, add an organic solution of chloroform: n-butanol at a ratio of 4:1, mix well, stand, take the lower liquid, repeat 5 times to remove proteins, and concentrate the lower liquid by vacuum concentration; (4) Take the concentrated liquid, add ethanol, stand at 4°C for 12 h, centrifuge at 8000 rpm for 10 min, and collect the precipitate; (5) Wash the precipitate with 95% ethanol twice, resuspend with water, and freeze-dry to obtain crude polysaccharide; (6) Use the phenol-sulfuric acid method to analyze the polysaccharide content. First, prepare a standard curve: accurately weigh 20 mg of standard dextran (or glucose) into a 500 mL volumetric flask, add water to the mark, respectively take 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6 and 1.8 mL, each to 2.0 mL with distilled water, then add 1 mL of 6% phenol and 5 mL of concentrated sulfuric acid, shake well and stand for 30 min, then detect the absorbance at 490 nm. Take 2 mL of water as a blank group, with the same color developing operation. The horizontal coordinate is the polysaccharide, and the vertical coordinate is the absorbance. The standard curve is obtained. Then take 1 mL of crude polysaccharide sample, add 1 mL of distilled water, then add 1 mL of 6% phenol, quickly add 5 mL of concentrated sulfuric acid, shake on a vortex instrument, mix well, stand for 30 min, and detect the absorbance at 490 nm. According to the standard curve, the polysaccharide concentration is calculated.

[0104] The exopolysaccharide difference rate = the exopolysaccharide content at the end of fermentation of each example or comparative example / the exopolysaccharide content at the end of fermentation of Example 1 x 100%.

[0105] Table 1

[0106] Exopolysaccharide difference rate (%) Example 1 100 Example 2 113 Example 3 98 Example 4 235 Example 5 148 Example 6 106 Example 7 129 Example 8 266 Comparative Example 1 Comparative Example 2 435

[0107] From the data in Table 1, the inoculation amount of yeast in Example 4 is 2% of the volume of the Schizochytrium fermentation broth. Too little yeast cannot completely consume the exopolysaccharide, resulting in a large difference in exopolysaccharide. Compared with Example 1, more exopolysaccharide remains, indicating that when the inoculation amount of yeast is 5-15%, the efficiency of mixed fermentation in reducing exopolysaccharide is higher. In Example 6, the pH of the fermentation system is adjusted to 5.5. Compared with Example 1, the difference in intracellular polysaccharide in Example 6 is greater than that in Example 1 after fermentation, indicating that the pH of the mixed fermentation affects the utilization of exopolysaccharide by yeast in the fermentation system. When the pH is maintained between 6 and 9, the utilization efficiency of exopolysaccharide by yeast is stronger. In Examples 7 and 8, yeast is inoculated into the Schizochytrium fermentation broth at 72 h and 110 h, respectively. Compared with Example 1, the difference in exopolysaccharide in Examples 7 and 8 is greater than that in Example 1, indicating that too early or too late inoculation of yeast affects the utilization of polysaccharide by yeast, resulting in too much polysaccharide remaining at the end of fermentation. Comparative Example 1 does not inoculate yeast, and the difference in exopolysaccharide is higher, indicating that there is no utilization of exopolysaccharide by yeast, and more exopolysaccharide accumulates in the fermentation broth.

[0108] Test Example 2

[0109] The DHA content in the DHA algal powder prepared in Examples 1-8 was evaluated. The DHA content determination method is as follows: gas chromatography analysis (Agilent 7890, USA). Gas chromatography parameters: capillary column (CP Sil-88: 50.0 m x 250 μm x 0.20 μm), FID detector, injection temperature 250°C, injection volume 1 μL, column temperature conditions: 80°C for 2 min, then increased to 120°C at a rate of 10°C / min, then increased to 180°C at a rate of 5°C / min, maintained for 2 min, then increased to 230°C at a rate of 2°C / min, and maintained at 230°C for 5 min. Helium was used as the carrier gas. C19:0 was used as the internal standard to calculate the percentage content of DHA in the mixed dry bacterial powder of Schizochytrium and yeast.

[0110] Table 2

[0111]

[0112]

[0113] Test Example 3

[0114] The spray drying product yield and product wall sticking, clogging phenomena of the DHA algal powder prepared in Examples 1-8 and Comparative Example 1 were evaluated. The yield of the product prepared in Examples 1-8 and Comparative Example 1 was calculated, product yield = product mass / feed mass x 100%, and the average of 3 times was taken.

[0115] Table 3

[0116]

[0117]

[0118] All examples and comparative moisture contents are less than 5%.

[0119] From the data in Tables 2 and 3, the inoculation amount of yeast in Example 4 and 5 is 2% and 20% of the volume of the Schizochytrium sp. fermentation broth, respectively. Although the percentage of DHA in Example 4 is higher than that in Example 1, the product yield is lower. The product yield in Example 5 is low, and the percentage of DHA is also low, indicating that when the inoculation amount of yeast is 5-15%, the comprehensive performance of the product is better. In Example 6, the pH of the fermentation system is adjusted to 5.5. Compared with Example 1, the percentage of DHA in Example 6 is lower under the condition of lower pH, indicating that the pH of the mixed fermentation will affect the content of DHA in the fermentation system. When the pH is maintained between 6-9, the utilization efficiency of yeast on extracellular polysaccharide is strong, the product yield is high, and the content of DHA is appropriate. In Example 7 and 8, yeast is inoculated into the Schizochytrium sp. fermentation broth at 72h and 110h, respectively. Compared with Example 1, the product yield of the algal powder in Example 7 is similar, but the content of DHA is low. In Example 8, the percentage of DHA in the algal powder is high, but the product yield is low, and there is a wall sticking phenomenon in the spray drying process, indicating that when yeast is inoculated between 80-100h of Schizochytrium sp. fermentation, the product does not stick to the wall, the yield is high, and the content of DHA is high. In Comparative Example 1, yeast is not inoculated, the product yield is lower than that in Example 1, and the blocking phenomenon occurs more frequently, indicating that the mixed fermentation of yeast and Schizochytrium sp. can significantly reduce the extracellular polysaccharide of the fermentation broth, improve the product yield and product quality of DHA algal powder, and has higher production cost performance.

[0120] The above describes the preferred embodiments of the present application in detail, but the present application is not limited to the specific details in the above embodiments. Within the technical concept of the present application, various simple modifications can be made to the technical solutions of the present application, and these simple modifications all belong to the protection scope of the present application.

[0121] In addition, it should be noted that each specific technical feature described in the above specific embodiments can be combined in any appropriate manner without contradiction. In order to avoid unnecessary repetition, the present application will not describe various possible combinations again.

Claims

1. A method for preparing DHA algal powder, characterized in that, The method includes the following steps: The seed culture of Schizochytrium was inoculated into a fermentation medium for fermentation. In the later stage of fermentation, yeast was added for mixed fermentation. After the fermentation was completed, a fermentation broth containing DHA was obtained, which was then spray-dried to obtain the DHA algal powder. The later stage of fermentation is 20-40 hours before the end of fermentation; the total fermentation time is 110-130 hours. The inoculation amount of the yeast is 5-15% of the volume of the Schizochytrium fermentation broth; the yeast includes Saccharomyces cerevisiae. The temperatures for inoculating the Schizochytrium seed culture into the fermentation medium and for the mixed fermentation are independently 25-30°C. During the mixed fermentation process, the pH of the mixed fermentation broth is maintained at no less than 6.

2. The method as described in claim 1, characterized in that, During the mixed fermentation process, the pH of the mixed fermentation broth is maintained between 6 and 9.

3. The method as described in claim 1, characterized in that, An acid-base regulator is used to maintain the pH of the mixed fermentation broth.

4. The method as described in claim 3, characterized in that, The acid-base regulator includes any one or a combination of at least two of citric acid, sodium bicarbonate, sodium carbonate, hydrochloric acid, or sodium hydroxide.

5. The method as described in claim 1, characterized in that, The inlet air temperature for the spray dryer is 160-220℃.

6. The method as described in claim 1, characterized in that, The outlet air temperature of the spray dryer is 70-100℃.

7. A DHA algae powder, characterized in that, The DHA algae powder is prepared by the method according to any one of claims 1-6.

8. The DHA algae powder as described in claim 7, characterized in that, The DHA algae powder contains yeast powder, and the DHA content in the DHA algae powder is greater than 20%, while the moisture content is less than 5%.

9. The preparation method according to any one of claims 1-6 or the application of DHA algae powder according to claim 7 or 8 in the preparation of animal nutrients.

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