JPWO2019130535A1 - Labyrinthula culture method - Google Patents

Abstract

An object of the present invention is to provide a method for cultivating Labyrinthula, which is capable of cultivating Labyrinthula, which is a heterotrophic organism, at a low cost, and which hardly impairs the availability of a culture composition obtained by culturing. The method for cultivating Labyrinthuras is characterized by culturing Labyrinthulas using a medium containing whey. It is preferable that the culture medium for culturing Labyrinthuras has a composition not containing a medium component that is a protein degradation product, for example, a casein degradation product such as tryptone, peptone, and casamino acid.

Description

  The present invention relates to a method for cultivating a heterotrophic labyrinthulas.

  In recent years, techniques for producing useful substances using microalgae have been actively developed. Certain types of microalgae accumulate a large amount of lipids in cells. Therefore, practical use of production of functional components, physiologically active substances, biofuels, and the like utilizing this ability has been promoted. Further, microalgae such as chlorella, spirulina, and euglena are rich in nutrients, and their use is expanding for edible uses such as health foods and feeds.

  In recent years, Labyrinthula, which is a protist closely related to microalgae, has attracted attention. Labyrinthuras are heterotrophic marine eukaryotic microorganisms that do not perform photosynthesis, and are widely distributed mainly in the subtropics and the tropics. Labyrinthuras have been reported to accumulate large amounts of omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), and higher hydrocarbons such as squalene in cells. Are fast and have a high amino acid score.

  Conventionally, marine fish such as blue fish have been mainly used as a supply source of DHA and EPA. Fatty acids in fish oil collected from marine fish are transesterified and distilled for the purpose of removing oxidized substances generated during transportation, chemical substances accumulated in the sea, and increasing the purity of DHA and EPA in the fatty acid group. And a purification treatment such as hydrophobic chromatography. On the other hand, the use of Labyrinthuras eliminates competition with fisheries resources, enables stable supply, and is expected to reduce deterioration due to oxidation and contamination of impurities, thus reducing the cost of DHA and EPA. Is expected to advance.

  As described in Patent Literature 1 and Patent Literature 2, a GTY medium is generally used as a medium for growing heterotrophs Labyrinthula. The GTY medium has a basic composition containing glucose at a concentration of 20 g / L, tryptone at a concentration of 10 g / L, and yeast extract at a concentration of 5 g / L, and is prepared using natural seawater or artificial seawater.

JP-A-2006-304686 International Publication No. 2012/077799

  Heterotrophic organisms such as Labyrinthuras can be grown on media containing nutrients necessary for growth, such as energy sources and essential nutrients. Conventionally, in a general medium, a casein hydrolyzate such as tryptone or peptone has been used as a part of essential nutrients. However, protein degradation products such as casein degradation products are relatively expensive, although they are excellent in nutrient utilization efficiency. Therefore, there is a problem that it is difficult to make a profit in a medium containing expensive proteolytic products, such as a commonly used GTY medium, when mass-cultivating heterotrophic organisms such as labyrinturas. .

  Until now, the present inventors have attempted to use a fish-derived extract as a nutrient alternative to tryptone. The fish-derived extract is a proteolytic product obtained by treating fish meat with an enzyme, but can also be prepared using waste as a raw material, and can be obtained at a lower cost compared to tryptone and the like. In addition, it has been confirmed that the fish-derived extract has a good content ratio for each type of amino acid and is excellent as a nutrient source.

  However, the fish-derived extract is low-cost, but has a strong odor. Therefore, there is a problem that odor is transferred to the cultured heterotrophic organism and useful substances extracted from the cultured heterotrophic organism. Fish odor is unsuitable for use when the cultured heterotrophic organism is converted into food or when extracted useful substances are used. Usage is restricted. In addition, the production amount of fish-derived extract is easily affected by fishing restrictions and edible demand, so that it may become difficult to procure in the future and may not be worth the cost.

  Therefore, an object of the present invention is to provide a method for cultivating labulinturula, which is capable of cultivating a heterotrophic labulinturula at a low cost and hardly impairing the availability of a culture composition obtained by culturing. And

  In order to solve the above-mentioned problems, the present inventors have intensively searched for and evaluated inexpensive medium components that can be used for culturing heterotrophic organisms among unused resources. As a result, it has been found that whey (whey) obtained by separating a main solid content from milk is effective as a component of a medium for culturing heterotrophic organisms such as Labyrinthuras.

  Animal-derived whey is known, for example, as a by-product during cheese production.The solid content in whey is used as a food material or feed, and a specific protein called whey protein is functional. There is an example used as a component. Also, soy whey called soy whey, soy milk whey, or the like (hereinafter sometimes simply referred to as “whey”) is also known. However, in Japan and the like, there are few examples in which the whole whey is effectively used for both animal-derived whey and soy whey, and most of them are disposed of. Therefore, whey can be stably obtained as a by-product, and is an inexpensive material as compared with a protein decomposition product such as tryptone.

  In addition, cheese whey is a protein such as β-lactoglobulin, α-lactalbumin, immunoglobulin, lactoferrin, free amino acids, non-protein nitrogen compounds such as urea, vitamins such as riboflavin, Ca, Na , K and minerals, lactose, etc., and are excellent as nutrient sources. The nutrients originally contained in soy milk remain in soy milk whey, which is a squeezed liquid during tofu production, and its utility value is high. In addition, whey has a weak odor peculiar to dairy products, but has no strong odor and has a characteristic that odor transfer hardly occurs.

  The method for cultivating Labyrinthulas according to the present invention invented on the basis of such findings is characterized by culturing Labyrinthulas using a medium containing whey.

  According to the method for cultivating Labyrinthuras according to the present invention, Labyrinthulas, which are heterotrophic organisms, can be cultured at low cost, and the usability of the culture composition obtained by the culture is not easily impaired. Even in the case where the food itself is converted into a food, the use of the cultured labulinturula itself is hardly restricted. The cultivation composition containing rabirintula as a main component obtained by cultivation has a small odor transfer from the medium and has a weak odor, so that it is subjected to a deodorizing treatment for removing odor, or a useful substance produced by labirintula is extracted. Even if it does not, the Labyrinthula itself or the produced useful substance can be used for any purpose.

It is a figure which shows the evaluation result of the assimilation property of the disaccharide by auranthiochitrium. FIG. 2 is a graph showing the influence of whey concentration on the culture of Aurantiochytrium. It is a growth curve of Aurantiochitrium cultured by changing the conditions of preculture. It is a growth curve of Aurantiochitrium cultured by adding a feed solution containing whey. It is a growth curve of Aurantiochytrium cultured by adding glucose.

  Hereinafter, the method for culturing Labyrinthulas according to one embodiment of the present invention will be described in detail.

  The method for cultivating Labyrinthuras according to the present embodiment relates to a method for culturing Labyrinthulas, which are heterotrophs, using a medium containing whey. In the present specification, whey refers to an aqueous solution obtained by separating a main solid content from a protein source such as milk. Whey is an aqueous solution mainly containing a water-soluble component, but may contain a trace amount of a solid component or a fat-soluble component.

  In the present embodiment, whey is used as an essential nutrient that promotes the growth of the labulinturula when preparing a medium for culturing the labulinturula. As the medium, an appropriate medium such as a liquid medium, a solid medium, and a semi-solid medium can be prepared. However, it is preferable to prepare a liquid medium because a large amount of Labyrinthula can be cultured.

  Whey may be used in a liquid state or a solid state. After the liquid whey is separated from a protein source such as milk, it may be diluted to an appropriate solid content or may be concentrated. The solid whey can be used in an appropriate state such as a powder or a granule by spray drying or the like. Whey is particularly preferably used in a liquid state. Liquid whey eliminates the need for the operation of dissolving the solid in the medium, so that a large amount of medium can be efficiently prepared. In addition, since the initial state of whey obtained as a by-product is liquid, it is efficient from the viewpoint of using by-products.

  As whey, at least one of animal-derived whey separated from milk and soy whey separated from beans, which is a vegetable protein source, can be used. As whey, one kind may be used alone for the preparation of the culture medium, or a plurality of kinds having different origins may be mixed and used.

  Animal-derived whey can be obtained by separating the water-soluble fraction from milk. For example, during the production of cheese, milk is fermented by lactic acid bacteria fermentation as a starter, by the addition of milk-clotting enzymes such as chymosin, pepsin, and rennet, or by the addition of an acid, or during the production of yogurt, milk is fermented by lactic acid bacteria. By doing so, a curd containing protein or fat as a main component is obtained. By squeezing or draining such curd, whey can be obtained as a water-soluble fraction.

  Milk used as a raw material of animal-derived whey may have defatted milk fat or may not have defatted milk fat. The animal-derived whey may be an acidic whey obtained by coagulating milk at a pH of about 4.6, or a sweet whey obtained by coagulating milk with rennet or the like. You may. In addition, animal-derived whey may or may not be desalted with sodium, potassium and the like.

  Specifically, milk, buffalo milk, goat milk, sheep milk, yak milk, horse milk, camel milk, and the like can be used as milk. Among these, milk, buffalo milk, goat milk, or sheep milk is preferred among these, and milk is particularly preferred, because it is easily available and inexpensive.

  Soy whey can be obtained by separating the water-soluble fraction from vegetable protein sources such as beans. For example, when beans are ground and immersed in water, treated under acidic conditions with a pH of about 4.5 to about 5.0 or under heating conditions of about 80 ° C. or more to separate and remove solids such as fibers, An extract containing mainly water-soluble components is obtained. In addition, when producing processed bean products such as tofu and fried oil, if the solids are strained after boiling down the ground beans, soy milk can be obtained. When the insoluble protein contained in the thus obtained extract or soy milk is salted out with a coagulant such as magnesium chloride, soy whey can be obtained as a water-soluble fraction.

  The vegetable protein source used as a raw material for soy whey may be oil-defatted or oil-free. For example, soy whey may be separated from either soy milk made from defatted processed beans as a raw material, or soy milk made from undefatted raw beans.

  As a vegetable protein source, specifically, soybean, mung bean, black bean, and the like can be used. Among these, soybean is preferred as a vegetable protein source because it is easily available and inexpensive. Soy whey may be directly separated from soy milk, green soy milk, black soy milk and the like whose solid content concentration is adjusted to obtain soy whey, mung bean whey, black soy whey and the like.

  Animal-derived whey and soy whey are rich in nitrogen compounds such as proteins, peptides, and amino acids, water-soluble vitamins, minerals, saccharides, and the like, which are necessary for a culture medium. In addition, whey is often produced as a by-product during the production of processed products and is often treated as waste. Therefore, whey can be obtained at a lower cost than protein decomposed products such as tryptone. Therefore, by using whey as a component of a medium instead of a protein decomposed product such as tryptone, the cost of the medium can be reduced, and the waste can be effectively used.

  In addition, whey has no strong odor, so when protozoa are cultured in a medium using whey, odor transfer to the culture is unlikely to occur, and it has a weak odor peculiar to dairy products. Odor may be masked. Furthermore, since it is originally derived from food raw materials, when cultivated Labyrinthula itself is made into food, or when a useful substance produced by Labyrinthura is used, deodorization treatment to remove odors, produced useful substance Extraction outside the cell, purification of a useful substance from a medium component or a cell component, and the like can be simplified.

  When preparing a medium for culturing labyrinthula, whey is preferably adjusted to a pH of 4 to 8 and more preferably to a pH of 6 to 8. Labyrinthula has a pH optimum for cultivation near neutrality. In contrast, animal-derived whey may be acidified to around pH 4.6 by lactic acid bacteria fermentation or addition of an acid. In addition, soy whey such as soy whey may be adjusted to a pH of about 4 to 5 for a process of coagulating solids or a process of isoelectric focusing. Therefore, by adjusting the pH of whey to near neutrality in advance, the adjustment of the pH of the medium using whey can be simplified.

  In addition to whey, the medium for culturing Labyrinthula is a common carbon source, nitrogen source, other nutrients such as vitamins and minerals, various buffers such as phosphates, sodium chloride, etc. May be contained, a microorganism for two-member culture such as bacteria, yeast, and diatoms, and a medium component such as agar. Moreover, it may be prepared using natural seawater or may be prepared using artificial seawater. However, it is preferable that the culture medium for culturing Labyrinthuras has a composition not containing a fish-derived extract from the viewpoint of reducing odor.

  Specific examples of general medium components include carbon sources such as glucose, fructose, mannose, galactose, sucrose, and maltose, glutamic acid, amino acids such as sodium glutamate, peptides, proteins, urea, ammonia, ammonium salts, and nitrates. Nitrogen sources, extracts such as yeast extract, vitamins such as thiamine, riboflavin, niacin, pantothenic acid, vitamin B6, biotin, folic acid, vitamin B12, sodium, potassium, calcium, magnesium, phosphorus, sulfur, iron, Minerals such as cobalt, copper, zinc, manganese, and molybdenum are exemplified.

  As seawater salts for preparing artificial seawater, for example, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, strontium chloride, ammonium chloride, iron chloride, manganese chloride, cobalt chloride, sodium dihydrogen phosphate, hydrogen phosphate Disodium, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium nitrate, sodium carbonate, sodium silicate, sodium fluoride, magnesium sulfate, cobalt sulfate, copper sulfate, zinc sulfate, sodium molybdate, potassium bromide, borane An acid or the like can be used.

  Whey is preferably contained in the medium at a concentration of 3 g / L or more and 30 g / L or less in terms of solid content of the components contained in the whey, and is contained in the medium at a concentration of 6 g / L or more and 24 g / L or less. More preferably, it is included. If the concentration of whey is 3 g / L or more, the labulinturula can be grown at a significantly higher growth rate using whey as a nutrient source. When the whey concentration is 6 g / L or more and 24 g / L or less, a higher growth rate can be obtained.

  In a medium for culturing Labyrinthuras, whey is preferably blended as an amino acid source containing essential amino acids, that is, as a medium component having the maximum amino acid concentration in terms of nitrogen. In a general medium, a casein hydrolyzate such as tryptone, peptone, and casamino acid is used as a main amino acid source. However, these proteolytic products are relatively expensive. On the other hand, whey is inexpensive compared to tryptone and the like, while being rich in nitrogen compounds such as proteins. Therefore, when whey is used so that the amino acid concentration becomes the largest medium component, the medium can be prepared at a lower cost than in the case where tryptone or the like is used.

  The culture medium for culturing Labyrinthulas preferably contains glucose, fructose, mannose or sucrose together with whey, particularly preferably glucose. These saccharides can have a concentration of, for example, 10 g / L or more and 200 g / L or less, preferably 20 g / L or more and 100 g / L or less in terms of solid content. If these saccharides are used, labulinturula can often be used, and labulinturula can be grown at a high growth rate.

  The medium for culturing Labyrinthulas preferably contains a yeast extract together with whey. The yeast extract can have, for example, a concentration of 1 g / L or more and 10 g / L or less, preferably 2 g / L or more and 5 g / L or less in terms of solid content. When the yeast extract has such a concentration, the rabulinturula can be grown at a high growth rate using the yeast extract as a nutrient source of vitamins, minerals, nucleic acids and the like.

  It is more preferable that the medium for culturing Labyrinthula contains whey as an essential nutrient necessary for growth, glucose as an energy source, and yeast extract as another necessary nutrient. GTY medium, which is generally used as a growth medium for Labyrinthula, contains tryptone as an amino acid source containing essential amino acids, glucose as an energy source, and yeast extract as an essential nutrient source such as vitamins, minerals and nucleic acids. Have been. On the other hand, when whey is used in place of tryptone, components other than the amino acid source also act to increase the growth rate and reduce the cost of the medium. It is preferable that the medium for culturing Labyrinthula contains whey, glucose, yeast extract, glutamate such as sodium glutamate, and seawater salt such as sodium chloride.

  The culture medium for culturing Labyrinthuras may have a composition that does not include a culture medium component that is a protein degradation product. That is, tryptone, peptone, casein hydrolyzate such as casamino acid, soybean hydrolyzate, gelatin hydrolyzate, meat-derived extract, animal-derived extract such as fish-derived extract and plant-derived extract are decomposed by enzymes, acids, heat, etc. The composition may not contain the proteolytic product obtained by the above method. Further, the composition may include whey as a sole amino acid source. That is, it may be prepared without compounding casein hydrolyzate, soybean hydrolyzate, gelatin hydrolyzate, other proteins, peptides, amino acids, ammonia and ammonium salts as amino acid sources other than whey. In addition, an animal-derived extract such as a meat-derived extract or a fish-derived extract, or a plant-derived extract such as corn steep liquor, corn meal, soybean meal, and malt extract may not be used. According to the composition that does not contain a protein degradation product or the composition that uses whey as the only amino acid source, the medium can be reduced in cost, and there is no need to mix other amino acid sources, which makes the preparation of the medium easier. . In addition, the amount of accumulated lipid may increase due to the restriction of amino acids.

  In a medium for culturing Labyrinthuras, whey may be included as an amino acid source and an energy source. That is, whey may be contained as a medium component having the largest amino acid concentration in terms of nitrogen and as a medium component having the largest concentration in terms of carbon. Animal-derived whey contains lactose at a concentration exceeding about 70% per solid content. Also, soy whey contains various sugars. Therefore, when whey is used as the medium component having the highest amino acid concentration and the medium component having the highest carbon concentration, not only amino acid sources such as tryptone but also commonly used energy sources or carbon sources can be used. Can be reduced, and the cost of the medium can be further reduced.

  The culture medium for culturing Labyrinthuras may have a composition containing whey as a sole amino acid source and a sole energy source. That is, as an amino acid source other than whey, an organic nitrogen compound or an inorganic nitrogen compound is not blended, and glucose, fructose, mannose, galactose, sucrose, maltose, glycerol, dextrin, starch, alcohol, organic compounds such as organic acids May be prepared without blending. When whey is used as the only amino acid source and only energy source, the cost of the medium is further reduced, and there is no need to mix other carbon sources and nitrogen sources, which makes the preparation of the medium easier.

  Labyrinthula, which is a heterotrophic organism, is preferable as the organism to be cultured. Labyrinthulas are chemoautotrophic marine eukaryotic microorganisms belonging to stramenopiles, and are classified as oomycetes having motility such as generating gliding movements and migratory cells. Labyrinthula has a relatively high growth rate and has the ability to accumulate various lipids such as fatty acid esters, hydrocarbons, phospholipids, and glycolipids produced by assimilation as oil droplets in cells. It is preferably used.

  In general, Labyrinthuras are roughly classified into Labyrinthulidae (Labyrinthulidae) and Thraustochytriaceae (Thraustochytriidae). Labyrinthula, Aurantiochytrium, and Schizochytrium ), Thraustochytrium, Aplanochytrium, oblongichytrium, Botryochytrium, Japonochytrium and the like.

  Labyrinthuras to be cultured are more preferably genus Aurantiochytrium, Schizochytrium or Thraustochytrium. These types have a relatively high ability to produce lipids and the like, and can produce highly unsaturated fatty acids such as DHA and EPA, carotenoids such as astaxanthin and β-carotene, and hydrocarbons such as squalene. It is suitably used for applications and for use as a raw material for biofuels.

  Labyrinthula to be cultured is particularly preferably Aurantiochitrium. Some of the genus Aurantiochytrium produce polyunsaturated fatty acids such as DHA and EPA, as well as odd-numbered fatty acids that are said to be effective in improving Alzheimer's disease and type 2 diabetes. In addition, it is rich in essential amino acids such as lysine and proline, which is a raw material of collagen, and has about 90% of algal bodies composed of fatty acids and amino acids.

  The cultivation of Labyrinthula may be performed by any culture method such as batch culture, continuous culture, and fed-batch culture. The culture of Labyrinthula can be performed by an appropriate culture method such as shaking culture, aeration culture, aeration stirring culture, air lift culture, and stationary culture. Among these culture methods, aeration stirring culture or air lift culture is more preferable. A medium containing whey may be prepared by mixing whey with other medium components, then sterilizing by an appropriate method such as heat sterilization, ultraviolet sterilization, gamma ray sterilization, and filtration sterilization, and then seeding the seed cells. it can.

  As a culture apparatus used for cultivation of Labyrinthula, for example, a mechanical stirring type reactor, an air lift type reactor, a packed bed type reactor, a fluidized bed type reactor and the like can be used. Various containers such as a tank, a jar fermenter, a flask, a dish, a culture bag, a tube, and a test tube can be used as the culture container depending on the purpose of culture and the culture volume. The culture vessel may be made of an appropriate material such as an inorganic material such as stainless steel or glass, or an organic material such as polystyrene, polyethylene terephthalate copolymer, or polypropylene.

  Culture of Labyrinthula can be performed under appropriate temperature conditions, pH conditions, aeration conditions, and the like. The culturing temperature is preferably 5 ° C or more and 40 ° C or less, more preferably 10 ° C or more and 35 ° C or less, and even more preferably 10 ° C or more and 30 ° C or less. Further, the pH is preferably 2 or more and 11 or less, more preferably 4 or more and 9 or less, and even more preferably 6 or more and 8 or less.

  The culture of Labyrinthula can be performed while subculturing at appropriate intervals according to the genus and species of Labyrinthula, medium composition, culture conditions, and the like. For example, Labyrinthuras end the logarithmic growth phase in about 2 days after the start of culture and enter the death phase in about 7 days. Therefore, the passage of Labyrinthulas is preferably performed at intervals of 1 day to 10 days, more preferably at intervals of 2 days to 7 days, and more preferably at intervals of 2 days to 5 days. Is more preferred. The culture time of Labyrinthula can be set to an appropriate time according to the genus or species of Labyrinthula, medium composition, culture conditions, culture purpose, and the like.

  Next, a culture composition obtained by culturing Labyrinthuras and a method for producing the culture composition will be described.

  The method for producing a culture composition according to the present embodiment is a method for producing a culture composition using the above-described method for cultivating labulinturula, and a step of culturing labulinturula using a medium containing whey. And a step of concentrating or drying the culture medium containing the cultured Labyrinthula to obtain a culture composition. According to this production method, a culture composition containing the rabirintula itself as a main component is obtained.

  Labyrinthula to be cultured is preferably of a type capable of producing a useful substance. Examples of useful substances include higher unsaturated fatty acids such as DHA and EPA, monoesters, diesters, and triglycerides thereof, phospholipids, glycolipids, higher hydrocarbons such as higher alkadienes, higher alkatrienes, triterpenes, and tetraterpenes. Essential amino acids, proteins containing the same, polysaccharides, pigments, vitamins, physiologically active substances, and the like.

  Labyrinthulas to be cultured are more preferably those that accumulate at least one of DHA and EPA in cells. Further, a genus of Aurantiochytrium, a genus of Schizochytrium, or a genus of Thraustochytrium is more preferable, and a genus of Aurantiochitrium is particularly preferable. With such a type, the produced DHA or EPA is accumulated in a cell that is hardly oxidized, so that the oxidation of DHA or EPA can be suppressed and used for various uses. Further, as compared with fish oil-derived DHA or EPA, the odor is weaker, the accumulation of chemical substances is less, and the composition is obtained with a fatty acid composition similar to that of nature, so that the purification process can be simplified.

  The step of culturing Labyrinthula can be performed under the same medium, culture method, culture conditions, and the like as in the above-described culture method. As a medium, it is preferable to prepare a liquid medium from the viewpoint that large-scale culture of Labyrinthula can be performed and a large amount of useful substances can be produced. In addition, whey is preferably contained as an amino acid source containing essential amino acids, that is, as a medium component having the largest amino acid concentration in terms of nitrogen content, and more preferably together with glucose and yeast extract.

  The step of concentrating or drying the medium can be performed using a general concentration method or drying method. Useful substances such as lipids produced by Labyrinthuras are usually present in the cells of Labyrinthuras or in fat-soluble fractions separated from Labyrinthulas. For this reason, by concentrating or drying the medium, it is possible to collect the labulinturula itself and useful substances produced by the labulinturula at a high concentration.

  Examples of the concentration method for concentrating the culture medium include centrifugal concentration for centrifuging solids, sedimentation concentration for spontaneous sedimentation of solids, evaporation concentration for heating and evaporating the medium, vacuum concentration for evaporating the medium under reduced pressure, and culture medium. , Pressure-concentration in which the medium is filtered, membrane concentration in which the medium is filtered through a separation membrane, freeze-concentration in which the medium is removed by freezing, and the like. Further, as a drying method for drying the culture medium, for example, hot air drying, cold air drying, reduced pressure drying, spray drying, freeze drying, infrared drying, natural drying, drum drying that performs heating drying in a rotating drum, and the like can be used. it can.

  When the culture composition containing Labyrinthuras as a main component is concentrated, for example, it can have a water content of 80% or less, preferably 30% to 50%. When concentrated to such a water content, the volume of the culture composition is sufficiently reduced and, at the same time, the culture composition becomes a paste that is difficult to flow appropriately, so that the handleability of the culture composition is improved. In addition, when the culture composition containing Labyrinthuras as a main component is dried, for example, the water content can be 10% or less.

  The culture composition containing labulinthuras as a main component may contain whey-derived components together with the cultivated labulinthuras themselves. The component derived from whey may be a metabolite metabolized by Labyrinthuras or an unmetabolized unmetabolite. If a whey-derived component remains, the odor of the component may mask the odor of the culture composition. Since whey has a sufficient dietary experience, even if whey-derived components remain in the culture composition, whey can be directly used for edible use. On the other hand, the culture composition preferably has a concentration of odor components such as trimethylamine-N-oxide, trimethylamine, piperidine and the like derived from a fish-derived extract at a concentration below the detection limit.

  The culture composition containing labyrintura as a main component can be used for various uses such as food, feed, fertilizer, and industrial raw materials. Specific examples of edible uses include general foods, health foods, food materials, beverage materials, and the like. Also, specific examples of feed include livestock feed, poultry feed, aquaculture feed, pet feed, and the like. Specific examples of industrial raw materials include raw materials for biofuels, raw materials for feed, raw materials for fertilizers, raw materials for chemical products, raw materials for pharmaceuticals, and the like.

  After the culture composition containing labyrintula as a main component may be concentrated or dried, it may be subjected to a step of disrupting the labyrintula cells. Cell crushing includes, for example, stirring, crushing, crushing using shear force such as ultrasonic waves, crushing using pressure changes such as osmotic pressure, crushing using chemicals and enzymes, crushing using freeze-thawing. It can be performed using various methods such as a method. As an apparatus for crushing cells, for example, various apparatuses such as a French press, a vibration homogenizer, an ultrasonic homogenizer, a freezing homogenizer, and a bead mill can be used.

  The culture composition containing rabirintula as a main component, after being concentrated or dried, without extracting the component contained in rabirintura, may be used for various applications, and the component contained in rabirintula May be subjected to the step of extracting For example, Labyrinthuras are rich in essential amino acids and vitamins, have a high amino acid score, and accumulate a large amount of DHA and EPA in cells, so that the alga itself is suitably used for food use. On the other hand, useful substances such as lipids accumulated in the cells of Labyrinthula can be used for applications such as food materials, beverage materials, and industrial raw materials.

  The culture composition containing Labyrinthuras as a main component can be used in the form of a paste, powder, granules, or the like after being concentrated or dried. The culture composition may be used after being formed into pellets, flakes, blocks, or the like, or may be used after being formulated into capsules, suspensions, emulsions, and the like. When the culture composition is used for edible use, for example, a binder, a thickener, an anti-caking agent, a disintegrant, a lubricant, a brightener, a suspending agent, an emulsifier, an antioxidant, a pH adjuster And various additives such as preservatives, sweeteners, acidulants, coloring agents, flavors, and the like, and nutrients such as amino acids, vitamins, and minerals.

  Specifically, lactose, sucrose, starch, dextrin and the like can be used as the excipient. As the binder, carmellose sodium, methylcellulose, hydroxypropylcellulose, gum arabic and the like can be used. As the thickener, starch, dextrin, pectin, guar gum, carrageenan, xanthan gum and the like can be used. As the anti-caking agent, calcium silicate, calcium carbonate, magnesium carbonate, magnesium chloride and the like can be used.

  As the disintegrant, carmellose calcium, crystalline cellulose, hydroxypropylcellulose, crospopidone and the like can be used. As the lubricant, magnesium stearate, talc, glycerin fatty acid ester, sucrose fatty acid ester and the like can be used. Shellac, paraffin, beeswax, and the like can be used as the brightener. As a suspending agent, carmellose sodium, methylcellulose, hydroxypropylcellulose, gum arabic, sodium alginate and the like can be used.

  Further, as the emulsifier, calcium stearate, lecithin, sodium caseinate, glycerin fatty acid ester, sucrose fatty acid ester and the like can be used. As the antioxidant, vitamin C, vitamin E, catechin, tocopherol and the like can be used. As the pH adjuster, phosphoric acid, sodium hydrogen carbonate, lactic acid, citric acid, gluconic acid, malic acid, tartaric acid, succinic acid, adipic acid, and the like can be used. As the preservative, benzoic acid, sorbic acid, polylysine, nisin, milt protein and the like can be used.

  Examples of sweeteners include glucose, fructose, maltose, sucrose, trehalose, oligosaccharides, syrup, isomerized sugar, reduced syrup, xylitol, sorbitol, mannitol, maltitol, stevia, licorice, aspartame, acesulfame potassium, sucralose, and the like. Can be used. As the acidulant, lactic acid, citric acid, gluconic acid, malic acid, tartaric acid, succinic acid, adipic acid, and the like can be used. As the coloring agent, a caramel pigment, a gardenia pigment, a turmeric pigment, a safflower pigment, a carotene pigment, or the like can be used.

  Components contained in the culture composition can be extracted using an appropriate solvent such as ethanol, methanol, diethyl ether, propylene glycol, propanol, isopropanol, acetone, chloroform, hexane, and cyclohexane. One of these solvents may be used alone, or two or more thereof may be used in combination. Further, a supercritical fluid such as carbon dioxide and ethylene may be used. For example, triglycerides such as DHA and EPA produced by Labyrinthuras can be extracted using a mixed solution of chloroform and methanol at a volume ratio of 2: 1 or hexane.

  After the components contained in the culture composition are extracted from the cells, they may be purified by an appropriate method. The components can be purified, for example, using hydrophobic chromatography, solvent fractionation, molecular distillation, membrane separation, and the like. As the carrier, for example, silica gel, acid clay, activated clay, activated carbon, alumina and the like can be used.

  The culture composition containing labyrinturas as a main component and the extract thereof can be used particularly for health promotion when the labulinthuras produce DHA or EPA. Specific examples of uses for promoting health include lowering the health risks associated with arrhythmias, lowering the risk of cardiovascular disease, lowering blood triglyceride levels, improving blood fluidity, enhancing brain function, Protection of cells from oxidative damage, inflammatory diseases, allergic diseases, autoimmune diseases, neurodegenerative diseases such as Alzheimer's disease, diseases related to metabolic syndrome such as type 2 diabetes, cancer-related diseases, etc. Relaxation and the like.

  Culture compositions or extracts containing rabirintula as a main component, and extracts thereof, when rabirinturas such as auranthiochitrium are producing odd-numbered fatty acids such as pentadecanoic acid, particularly for use in promoting cell growth. Can be used. Specific examples of uses for promoting cell proliferation include healing damaged tissues, alleviating pain, autoimmune diseases, neurodegenerative diseases, immune diseases, diseases related to metabolic syndrome, cancer-related diseases, etc. Reduction of wrinkles, promotion of skin metabolism, hair growth, reduction of allergic symptoms, reduction of muscle pain, improvement of motor function, and the like.

  Hereinafter, the present invention will be described specifically with reference to Examples, but the technical scope of the present invention is not limited thereto.

  First, Labyrinthula was cultured using a medium containing lactose, and the assimilation of lactose, which is a major carbohydrate contained in whey, was confirmed. Aurabiochitrium SA96WH strain was cultured as Labyrinthuras. In addition, sucrose and maltose were similarly cultured as a control for lactose, which is a disaccharide.

  The medium had a GTY medium as a basic composition, and was prepared using lactose, sucrose, or maltose instead of glucose contained as a carbon source. As a medium, a medium having a concentration of each disaccharide of 2% and a medium having a concentration of 4% were prepared. The medium had a composition containing 1% of tryptone (manufactured by Difco), 0.5% of yeast extract (manufactured by Difco), and 1% of seawater salt together with the disaccharide at these concentrations.

The assimilation (utilization efficiency) of a disaccharide is determined by measuring the number of cells by culturing in a medium using each disaccharide as an energy source, and measuring the number of cells in a culture without an energy or carbon source (sugar-free culture). Was quantified by the following equation (I) as a relative value to the result of the GTY medium using 2% or 4% glucose (Glc) as an energy source. In addition, the number of cells was measured at the time when the culture time passed 48 hours. The results are shown in Table 1 and FIG.
Use efficiency (%) = (number of cells by disaccharide-number of cells in sugar-free culture)
/ (Number of cells by Glc−number of cells in sugar-free culture) × 100 (I)

FIG. 1 is a diagram showing the results of evaluation of the assimilation of disaccharides by auranthiochitrium.
As shown in Table 1 and FIG. 1, the assimilation of disaccharides by the Aurantiochytrium SA96WH strain was observed for all of lactose, sucrose, and maltose, but was lower than that of glucose. Therefore, it is possible to use lactose contained in whey, but from the viewpoint of securing a high growth rate, it can be said that it is preferable to use glucose together with whey.

  Next, Labyrinthuras were cultured using a medium containing animal-derived whey, and the effect of whey concentration was confirmed. Aurabiochitrium SA96WH strain was cultured as Labyrinthuras. The effect of whey concentration was confirmed for six concentrations of 5%, 10%, 20%, 40%, 60% and 80%.

  In the medium, whey concentration was changed to 5%, 10%, 20%, 40%, 60%, and 80%, glucose was 3%, sodium glutamate was 0.5%, yeast extract (manufactured by Difco). In a concentration of 0.2% and seawater salt in a concentration of 1%. As whey, animal-derived cheese whey having 93.9% water, 0.3% protein, 5.2% carbohydrate, 0.6% ash, and less than 0.1% lipid was used.

The effect of whey concentration (contribution ratio) is determined by culturing whey in a medium adjusted to each concentration, measuring the number of cells, and culturing without whey (culture with 0% whey concentration). The increase in the number of cells was quantified by the following formula (II) as a relative value to the result of culture without whey (culture with 0% whey concentration). In addition, the number of cells was measured when the culture time passed 24 hours, 48 hours, and 72 hours, respectively. The results are shown in Table 2 and FIG.
Contribution ratio (%) = (number of cells by whey-number of cells in sugar-free culture)
/ Number of cells in sugar-free culture x 100 (II)

FIG. 2 is a diagram showing the influence of whey concentration on the culture of Aurantiochytrium.
As shown in Table 2 and FIG. 2, in the culture of the Aurantiochytrium SA96WH strain, the cell number generally reached a maximum when the concentration of whey in the medium was around 40%. When the culture time was 72 hours, the error increased as seen when the concentration was 40%, but when the culture time was up to 48 hours, the contribution ratio increased when the whey concentration reached about 10%. Was confirmed. As the whey concentration further increased, the contribution ratio tended to gradually increase. However, when the whey concentration exceeded about 40%, the contribution gradually decreased, and the increase in cell number was reduced. Therefore, whey is preferably contained in the medium in a range of 5% by volume or more and 50% by volume or less (3 g / L or more and 30 g / L or less in terms of solid content), preferably 10% by volume or more and 40% by volume or less (solid content It can be said that it is more preferable for the medium to be contained in the range of 6 g / L to 24 g / L in conversion).

  Next, Labyrinthuras were cultured using a medium containing animal-derived whey, and the growth rate of Labyrinthulas was confirmed. Aurabiochitrium SA96WH strain was cultured as Labyrinthuras.

  The medium containing whey contains 10% whey, 3% glucose, 0.5% sodium glutamate, 0.2% yeast extract (Estock sp-d), 1% seawater salt, magnesium sulfate aqueous solution. The composition contained the Japanese product at a concentration of 0.2%. As the whey, an animal-derived cheese having 93.9% water, 0.3% protein, 5.2% carbohydrate, 0.6% ash, and less than 0.1% lipid as in the above test Whey was used.

  The growth rate of Labyrinthula was compared between a strain pre-cultured in a medium containing whey and a strain pre-cultured in a GTY medium. The main culture was performed by shaking culture, and the inoculated amount was compared between 1% and 2%. Cultivation with 1% inoculation of the SA96WH strain pre-cultured in GTY medium (1% GTY medium), and cultivation with 1% inoculation of the SA96WH strain precultured in a medium containing whey (1% milk) Cultivation with a 2% inoculation amount of the SA96WH strain pre-cultured in the GTY medium (2% GTY medium), and cultivation with a 2% inoculation amount of the SA96WH strain pre-cultured in the whey-containing medium. (2% whey medium), the number of cells was measured over time. The results are shown in Table 3 and FIG.

FIG. 3 shows a growth curve of Aurantiochytrium cultured under different preculture conditions.
As shown in Table 3 and FIG. 3, when the strain pre-cultured in the medium containing whey is compared with the strain pre-cultured in the GTY medium, the strain pre-cultured in the GTY medium grows rapidly in a short period of time. The logarithmic growth phase tended to end early. On the other hand, the strain pre-cultured in the medium containing whey tended to show a higher growth rate even after a certain period of culture time.

  Next, Labyrinthuras were cultured using a medium containing animal-derived whey, and the growth amount of Labyrinthulas, that is, the amount of the culture composition obtained by the culture and the odor of the culture composition were confirmed. Aurabiochitrium SA96WH strain was cultured as Labyrinthuras.

  The medium containing whey contains 10% whey, 3% glucose, 0.5% sodium glutamate, 0.2% yeast extract (Estock sp-d), and 0.2% seawater salt in the same manner as in the above test. The composition contained 1% of magnesium sulfate hydrate at a concentration of 0.2%. As the whey, an animal-derived cheese having 93.9% water, 0.3% protein, 5.2% carbohydrate, 0.6% ash, and less than 0.1% lipid as in the above test Whey was used.

  The growth amount of Labyrinthuras was confirmed by inoculating a strain pre-cultured in a medium containing whey in advance into a 3 L whey medium with an inoculum amount of 2% and performing airlift culture. In the air lift culture, the air aeration rate was set to 1.0% by volume / min. When the cultivation time has passed 48 hours, either 3% of glucose or a feed solution containing 3% of glucose, 10% of whey and 0.2% of yeast extract is added. Additional growth was compared afterwards. The results are shown in Tables 4 and 5 and FIGS.

FIG. 4 is a growth curve of Aurantiochytrium cultured by adding a feed solution containing whey. FIG. 5 shows a growth curve of Aurantiochytrium cultured with the addition of glucose.
As shown in Tables 4 and 5, and FIGS. 4 and 5, mass culture of the Aurantiochytrium SA96WH strain was possible by air-lift culture, and a culture composition exceeding 15 g / L in terms of dry weight was obtained. When a culture time of 48 hours has passed and a feed solution containing 3% of glucose, 10% of whey and 0.2% of yeast extract was added (see Table 4, FIG. 4), Compared with the case where 3% of glucose was added at the time when the culture time passed for 48 hours (see Table 5 and FIG. 5), the stage where the culture time passed for 72 hours when only glucose was added. However, when a fed-batch solution containing whey was added, the growth amount was further increased, and a larger amount of the culture composition was obtained. confirmed. When the odor of the obtained culture composition was confirmed by a sensory test, a weak raw milk odor was confirmed, but no odor was recognized.

  Next, using a medium containing animal-derived whey and a medium containing soy whey, labulinturas were cultured, and the growth amounts of labulinturas were compared. Aurabiochitrium SA96WH strain was cultured as Labyrinthuras.

  The medium containing whey derived from animals changed the concentration of cheese whey to 5%, 10%, 20%, 40%, 60%, and 80%, respectively, and added glucose 3%, sodium glutamate 0.5%, A composition containing yeast extract (Estock sp-d) at a concentration of 0.2% and seawater salt at a concentration of 1% was used. Cheese whey has 93.9% moisture, 0.3% protein, 5.2% carbohydrates, 0.6% ash, and less than 0.1% fat, similar to the previous test.

  The medium containing soy whey was prepared using soy whey by-produced in the process of producing tofu and soy milk whey purified from soy milk. In the medium containing soy whey, the concentration of soy whey was changed to 5%, 10%, 20%, 40%, and 60%, glucose was 3%, sodium glutamate was 0.5%, and yeast extract (Estock sp.) Was used. -D) at a concentration of 0.2% and seawater salt at a concentration of 1% (0.9% of Deborah salt + 0.1% of magnesium sulfate). In the medium containing soymilk whey, the concentration of soymilk whey was changed to 5%, 10%, 20%, and 40%, glucose was 3%, sodium glutamate was 0.5%, and yeast extract (Estock sp-d) was used. At a concentration of 0.2%, and seawater salt at a concentration of 1% (0.9% of Deborah lake salt + 0.1% of magnesium sulfate).

  As a result of the culturing, in the case of cheese whey, a tendency was obtained that the higher the concentration, the higher the cell number. When the concentration was as low as 5% or 10%, the growth rate after 48 hours was slowed down. However, when the concentration was 40% or less, an increase in the number of cells generally depending on the concentration of cheese whey was observed, and the culture time was 72 hours. Even after the passage of time, a high growth rate was confirmed. Also, in the case of soy milk whey or soy whey, a remarkable increase in the cell number was observed until the culturing time passed for 48 hours. In the case of soy whey, precipitates were produced in low-concentration culture, and the growth rate was reduced in comparison with high-concentration culture, but in the case of soy milk whey, the difference in growth rate depending on the concentration was It was small. When cheese whey was compared with soy milk whey or soy whey, it was confirmed that cheese whey tended to increase the number of cells depending on the concentration.

Claims (8)

  A method for cultivating labyrinthulas using a medium containing whey.   The culture method of Labyrinthula according to claim 1, wherein the whey is contained in the medium at a concentration of 3 g / L or more and 30 g / L or less in terms of solid content.   The method for cultivating Labyrinthura according to claim 1, wherein the whey is animal-derived whey.   The method for cultivating Labyrinthura according to claim 1, wherein the whey is soy whey.   The method for culturing Labyrinthuras according to claim 1, wherein the whey has a pH adjusted to 4 or more and 8 or less.   The method for culturing Labyrinthura according to claim 1, wherein the medium contains glucose and yeast extract.   The method for cultivating Labyrinthura according to claim 1, wherein the medium has a composition not containing a medium component that is a protein degradation product.   The method for cultivating labyrintula according to claim 1, wherein the labulinthura is a genus Aurantiochytrium, Schizochytrium, or Thraustochytrium.

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