WO2019130535A1 - Method for culturing labyrinthulea microorganism - Google Patents

Abstract

Provided is a method for culturing a Labyrinthulea microorganism, whereby a Labyrinthulea microorganism which is a heterotroph can be cultured at a low cost and the availability of the thus obtained culture composition is little lowered. The method for culturing a Labyrinthulea microorganism is characterized by comprising culturing the Labyrinthulea microorganism with the use of a medium containing milk serum. Preferably, the medium for culturing the Labyrinthulea microorganism has a composition which is free from any medium component comprising a protein digestion product, for example, a casein digestion product such as tryptone, peptone, a casamino acid, etc.

Description

Culture method of labyrinthulas

The present invention relates to a method of cultivating labyrinthus, which are heterotrophs.

In recent years, techniques for producing useful substances using microalgae have been actively developed. In order to accumulate a large amount of lipids in cells, some types of microalgae have been put into practical use for producing functional components, physiologically active substances, biofuels and the like using this ability. In addition, since microalgae such as Chlorella, Spirulina and Euglena are rich in nutrients, their use for food products such as health food and feed is expanding.

In recent years, attention has been focused on labyrinthuras, which are protists closely related to microalgae. Labyrinthulae is a heterotrophic marine eukaryotic microorganism that does not undergo photosynthesis, and is widely distributed in the subtropics and the tropics. Labyrinthulas have been reported to accumulate a large amount of omega-3 fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) and higher hydrocarbons such as squalene in the cell, and also to show proliferation rate Are fast and the amino acid score is high.

Conventionally, marine fish such as blue fish have been mainly used as sources of DHA and EPA. Fatty acids in fish oil collected from marine fish are transesterified and distilled for the purpose of removing oxidized products generated during transportation etc. and chemicals accumulated in the sea, and for enhancing the purity of DHA and EPA in fatty acid groups. And purification treatment such as hydrophobic chromatography. On the other hand, the use of labyrinthulas eliminates competition with fishery resources, enables stable supply, and is expected to reduce the deterioration due to oxidation and the inclusion of impurities, thus reducing the cost of DHA and EPA. Is expected to advance.

GTY medium is generally used as a culture medium for growing heterozygous labyrinthulas as described in Patent Document 1 and Patent Document 2. The GTY medium is a basic composition containing 20 g / L of glucose, 10 g / L of tryptone, and 5 g / L of yeast extract, and is prepared using natural seawater or artificial seawater.

JP 2006-304685 A International Publication No. 2012/077799

Heterotrophic organisms such as labyrinthulae can be grown in a medium containing an energy source, a nutrient source necessary for growth such as essential nutrients, and the like. Heretofore, casein decomposition products such as tryptone and peptone have been used in common culture media as part of essential nutrients. However, protein degradation products including casein degradation products are relatively expensive although they are excellent in utilization efficiency as nutrients. Therefore, there is a problem that it is difficult to make profit when conducting culture on a large scale of heterotrophic organisms such as labyrinthulas in a medium containing expensive protein hydrolyzate such as GTY medium which is generally used. .

To date, the present inventors have attempted to use fish-derived extracts as nutrients to replace tryptone. The fish-derived extract is a protein degradation product obtained by enzymatic treatment of fish meat, but it is also possible to prepare waste as a raw material, and it is possible to obtain it cheaply as compared with tryptone and the like. Moreover, it is also 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, while the fish-derived extract is low in cost, it has a strong odor, which causes a problem of causing odor transfer to cultured heterotrophs and useful substances extracted from the cultured heterotrophs. Fish odor is an odor that is not suitable for use when converting the cultured heterotroph itself into a food or using the extracted useful substance, so deodorization treatment is required, or the cultured heterotroph is Usage is restricted. In addition, the production amount of fish-derived extract is likely to be influenced by fishing restrictions and demand for food, which may make it difficult to procure in the future and the cost may not be met.

Then, this invention can culture | cultivate the heterotrophic organism Labyrinthulas at low cost, and it aims at providing the culture method of the Labyrinthulas which is hard to spoil the availability of the culture composition obtained by culture | cultivation I assume.

In order to solve the above-mentioned problems, the present inventor has intensively searched 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 main solids from milk is effective as a component of a culture medium for cultivating heterotrophic organisms such as labyrinthulas.

Whey derived from animals is known, for example, as a by-product in cheese production, and the solid content in the whey is functional as a food material, feed or the like, and a specific protein called whey protein is There is an example used as a component. In addition, soya whey (so-called "whey") may be referred to as soya whey, soya milk whey or the like. However, in Japan and the like, there are few cases in which the whole whey has been effectively used for both animal-derived whey and soy whey, and many are discarded. Therefore, whey can be stably obtained as a by-product, and is an inexpensive material as compared to protein degradation products such as tryptone.

In addition, cheese whey includes proteins such as β-lactoglobulin, α-lactalbumin, immunoglobulin, lactoferrin, free amino acids, non-protein nitrogen compounds such as urea, vitamins such as riboflavin, Ca, Na And minerals such as K, lactose, etc., are excellent as a nutrient source. Soy milk whey, which is a squeezed juice during tofu production, still contains the nutrients originally contained in soy milk, and its utility value is high. In addition, whey has a weak odor peculiar to dairy products but is characterized by having no strong odor and being hard to cause odor transfer.

The culture method of the labyrinthulas according to the present invention invented based on such findings is characterized in that the labyrinthulas are cultured using a medium containing whey.

According to the culture method of labyrinthulas according to the present invention, labyrinthulas which are heterotrophs can be cultured at low cost, and the availability of the culture composition obtained by culture can not be easily impaired, and the labyrinthulas can be Even when using itself as a food, it is difficult to restrict the use of the cultured Labyrinthula itself. The culture composition mainly composed of labyrinthulas obtained by culture has little odor transfer from the medium and has a weak odor, so it is subjected to deodorization treatment to remove odors, or the useful substance produced by labyrinthulas is extracted The labyrinthula itself or the produced useful substance can be used for any purpose, if not.

It is a figure which shows the evaluation result of the assimilability of the disaccharide by oranthio chutrium. It is a figure which shows the influence of the density | concentration of whey in culture | cultivation of the Orangiochytrium. It is a growth curve of alanthiochytrium cultured by changing the conditions of pre-culture. It is a growth curve of aurantiochytrium which added and cultured the feed liquid containing whey. It is a growth curve of alanthiochytrium which added and cultured glucose.

Hereafter, the culture | cultivation method of labyrinthula which concerns on one Embodiment of this invention is demonstrated in detail.

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

In this embodiment, whey is used as a culture medium component, ie, an essential nutrient that promotes the growth of labyrinthulas, when preparing the medium for culturing the labyrinthulas. As the culture medium, an appropriate culture medium such as a liquid culture medium, a solid culture medium, a semi-solid culture medium can be prepared, but it is preferable to prepare a liquid culture medium because large scale culture of labyrinthulas is possible.

Whey may be used in the liquid state or in the solid state. Liquid whey may be separated from protein sources such as milk and then diluted or concentrated to an appropriate solid concentration. In addition, solid whey can be used in an appropriate state such as powder or granules by spray drying or the like. Whey is particularly preferably used in liquid form. In the case of liquid whey, a large amount of culture medium can be efficiently prepared because the operation of dissolving the solid in the culture medium is unnecessary. Moreover, since the initial state of whey obtained as a by-product is liquid, it is also efficient from the viewpoint of by-product utilization.

As whey, at least one can be used among whey derived from an animal separated from milk and soy milk separated from beans which are vegetable protein sources. As whey, 1 type may be used independently for preparation of a culture medium, and multiple types from which an origin differs may be mixed and used.

Whey from animal origin can be obtained by separating the water soluble fraction from milk. For example, when producing cheese, milk may be curded by fermentation with lactic acid bacteria as a starter, addition of curdling enzymes such as chymosin, pepsin, rennet, or addition of acid, or lactic acid fermentation with milk when producing yogurt. By doing this, a curd mainly composed of protein and fat can be obtained. When such curds are pressed or drained, whey can be obtained as a water-soluble fraction.

Milk used as a raw material of animal-derived whey may have a defatted milk fat content or may not have a defatted milk fat content. Moreover, the whey derived from an animal may be an acid whey obtained by curdling milk at about pH 4.6, or a sweet whey obtained by curding milk with rennet or the like. May be Moreover, as for the whey derived from an animal, sodium, potassium, etc. may be desalted, and it is not necessary to desalt.

Specifically as milk, milk, buffalo milk, goat milk, sheep milk, yak milk, horse milk, camel milk etc. can be used. Among them, milk, buffalo milk, goat's milk or sheep's milk is preferred among these because milk is easy to obtain and inexpensive. Milk is particularly preferred.

Soy whey can be obtained by separating a water-soluble fraction from a vegetable protein source such as beans. For example, when beans are ground and immersed in water and treated under acidic conditions of pH 4.5 to 5.0 or less, heating conditions of about 80 ° C. or higher, etc. to separate and remove solid matter such as fiber, An extract containing mainly water-soluble components is obtained. In addition, at the time of production of processed soybean products such as tofu and deep-fried foods, soy milk can be obtained if the ground beans are boiled down and then the solid content is removed. When the insoluble protein contained in the extract and soymilk thus obtained is salted out with a coagulant such as magnesium chloride, soymilk can be obtained as a water-soluble fraction.

The vegetable protein source used as a raw material of soymilk may have a degreased oil content or may not have a degreased oil content. For example, soymilk may be separated from any of soymilk from which defatted processed beans are used as a raw material and soymilk from which unprocessed unrefined beans are used as a raw material.

Specifically, soybeans, green beans, black beans and the like can be used as the vegetable protein source. Among them, soybean is preferable as a vegetable protein source in view of its easy availability and low cost. Soy whey may be obtained as soybean whey, green bean whey, black bean whey or the like by directly separating it from soybean milk, green soybean milk, black soybean milk or the like whose solid concentration is adjusted.

Whey and animal milk derived from animals are rich in protein, peptide, nitrogen compounds such as amino acids, and water-soluble vitamins, minerals, saccharides and the like necessary for the culture medium. In addition, whey is often produced as a by-product at the time of production of a processed product and is often treated as a waste, so it can be obtained inexpensively as compared with protein degradation products such as tryptone. Therefore, by using whey as a component of the culture medium instead of protein degradation products such as tryptone, it is possible to reduce the cost of the culture medium and to achieve effective use of wastes.

In addition, since whey does not have a strong odor, when protozoa are cultured in a medium using whey, it is difficult for odor transfer to the culture to occur, and since it has a weak aroma unique to dairy products, the culture Odor may be masked. Furthermore, since the culture is originally derived from a food material, the cultured labyrinthula itself can be converted into a food, or when utilizing a useful substance produced by the labyrinthula, the odor-eliminating deodorizing treatment, the produced useful substance Thus, it is possible to simplify the extraction process of extracellularly extracting and the purification process of purifying useful substances from medium components and cell components.

When preparing the culture medium for cultivating labyrinthulas, it is preferable that pH is adjusted to four or more and eight or less, and also pH is six or more and eight or less. For Labyrinthula, the pH optimum for culture is near neutral. On the other hand, whey derived from animals may be acidified to around pH 4.6 by the addition of lactic acid bacteria fermentation or acid. In addition, soy whey such as soy whey may be adjusted to around pH 4 to 5 in order to solidify the solid content and to process isoelectric point precipitation. Therefore, adjustment of the pH of the culture medium using whey can be simplified by adjusting the pH of whey to near neutral in advance.

The culture medium for cultivating labyrinthulae is, in addition to whey, other common nutrients such as carbon source, nitrogen source, vitamins and minerals, various buffers such as phosphate, sodium chloride etc. It may contain a tonicity agent, bacteria for two-member culture, microorganisms such as yeast and diatoms, and medium components such as agar. Moreover, it may be prepared using natural seawater, and may be prepared using artificial seawater. However, from the viewpoint of reducing the odor, it is preferable that the culture medium for cultivating the labyrinthulas has a composition that does not contain the fish-derived extract.

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

Examples of seawater salt for preparing artificial seawater include 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 silicate, sodium fluoride, magnesium sulfate, cobalt sulfate, copper sulfate, zinc sulfate, sodium molybdate, potassium bromide, boronate An acid etc. can be used.

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

In the medium for cultivating labyrinthulae, whey is preferably formulated 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. In a general medium, casein decomposition products such as tryptone, peptone, casamino acid and the like are used as main amino acid sources. However, these protein degradation products are relatively expensive. On the other hand, whey is inexpensive as compared with tryptone etc. while containing nitrogen compounds such as proteins abundantly. Therefore, when whey is used so as to be a medium component having the highest amino acid concentration, the medium can be prepared at low cost as compared with the case of using tryptone or the like.

The culture medium for cultivating the labyrinthulae preferably comprises glucose, fructose, mannose or sucrose together with whey, and particularly preferably comprises glucose. These saccharides can be, for example, at a concentration of 10 g / L to 200 g / L, preferably at a concentration of 20 g / L to 100 g / L, in terms of solid content. With these saccharides, the labyrinthula can often be assimilated, and the labyrinthula can be grown at a high growth rate.

The culture medium for cultivating the labyrinthulas preferably contains yeast extract together with whey. The yeast extract may have, for example, a concentration of 1 g / L to 10 g / L, preferably 2 g / L to 5 g / L, in terms of solid content. When the yeast extract has such a concentration, the labyrinthula can be grown at a high growth rate, using the yeast extract as a nutrient source such as vitamin, mineral, nucleic acid and the like.

The culture medium for cultivating the labyrinthulas contains whey as an essential nutrient source necessary for growth, contains glucose as an energy source, and more preferably contains yeast extract as another necessary nutrient source. GTY medium, which is commonly used as a growth medium for labyrinthulae, 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 It is done. On the other hand, if whey is used instead of tryptone, components other than the amino acid source can also act to increase the growth rate and reduce the cost of the culture medium. The culture medium for cultivating the labyrinthulae preferably contains whey, glucose, yeast extract, glutamate such as sodium glutamate, and seawater salt such as sodium chloride.

The culture medium for cultivating the labyrinthulas may be a composition that does not contain a medium component which is a protein degradation product. That is, it is decomposed with enzymes, acids, heat, etc., animal-derived extracts such as tryptone, peptone, casamino acids and so forth, soybean degraded products, gelatin degraded products, meat derived extracts, meat derived extracts and fish derived extracts It is good also as composition which does not contain the protein degradation products obtained by Furthermore, the composition may contain whey as a sole amino acid source. That is, as an amino acid source other than whey, it may be prepared without blending casein degradation product, soybean degradation product, gelatin degradation product, other proteins, peptides, amino acids, ammonia and ammonium salts. In addition, animal derived extracts such as meat derived extracts and fish derived extracts and plant derived extracts such as corn steep liquor, corn meal, soybean meal and malt extract may not be blended. According to the composition which does not mix | blend protein decomposition products, and the composition which made whey the only amino acid source, a culture medium is cost-reduced more, and it becomes unnecessary to mix | blend another amino acid source, and preparation of a culture medium becomes easy . In addition, the restriction of amino acids may increase the amount of accumulated lipid.

In the culture medium for cultivating labyrinthula, whey may be contained as an amino acid source and an energy source. That is, whey may be contained as a culture medium component having the largest amino acid concentration in nitrogen conversion, and as a culture medium component having the largest concentration in carbon conversion. Animal-derived whey contains lactose at a concentration of greater than about 70% solids. In addition, soymilk contains various sugars. Therefore, if whey is used as a medium component having the highest amino acid concentration and a medium component having the highest carbon concentration, not only amino acid sources such as tryptone but also energy sources or carbon sources generally used. The cost of the culture medium can be further reduced because the amount of

The culture medium for cultivating the labyrinthulae may have a composition containing whey as a sole source of amino acids and as a sole source of energy. That is, organic nitrogen compounds and inorganic nitrogen compounds are not blended as an amino acid source other than whey, and organic compounds such as glucose, fructose, mannose, galactose, sucrose, maltose, glycerol, dextrin, starch, alcohol and organic acids It may be prepared without blending. If whey is the only amino acid source and only energy source, the cost of the culture medium is further reduced, and there is no need to incorporate other carbon and nitrogen sources, and preparation of the culture medium becomes easier.

As the organism to be cultured, labyrinthulas which are heterotrophs are preferable. Labyrinthula is a chemosynthetic heterotrophic marine eukaryotic microorganism belonging to stramenopile, and is classified into motile oomycetes such as gliding movement and migration cells. Labyrinthulas have a relatively high growth rate and have the ability to accumulate various lipids such as fatty acid esters, hydrocarbons, phospholipids and glycolipids produced by assimilation as oil droplets in cells, and therefore produce useful substances Are preferably used.

In general, the labyrinthulas are roughly classified into the family Labyrinthulidae (Labyrinthulidae) and the family Thraustochytriidae (Thuraustochytriidae), and the genus Labyrinthula (Labyrinthula), the genus Aurantiochytrium (Aurantiochytrium), and the genus Schizochytrium (Schizochytrium) And Thraustochytrium, Aplanochytrium, Oblongichytrium, Botryochytrium, Japonochytrium, and the like.

As the Labyrinthulae to be cultured, Auranthochytrium, Schizochytrium or Thraustochytrium is more preferable. These types have relatively high ability to produce lipids, etc., and can produce highly unsaturated fatty acids such as DHA and EPA, carotenoids such as astaxanthin and β-carotene, and hydrocarbons such as squalene, and thus are edible. It is suitably used for applications, applications of raw materials for biofuels, and the like.

Especially as a Labyrinthula to culture | cultivate, Aurantiochytrium genus is preferable. In addition to polyunsaturated fatty acids such as DHA and EPA, some of the genus Aurantiochytrium produce odd-numbered fatty acids that are said to be effective for ameliorating Alzheimer's disease and type 2 diabetes. Moreover, since it contains abundantly essential amino acids such as lysine and proline as a raw material of collagen, and about 90% of algal cells are composed of fatty acids and amino acids, they have a feature of high nutritional value.

The culture of the labyrinthulae may be performed by any culture method such as batch culture, continuous culture, fed-batch culture and the like. Moreover, culture | cultivation of labyrinthula can be performed by appropriate culture | cultivation methods, such as shaking culture, aeration culture, aeration stirring culture, airlift culture, stationary culture. Among these culture methods, aeration agitation culture or air lift culture is more preferable. The medium containing whey may be prepared by mixing whey with other medium components and then sterilized with appropriate methods such as heat sterilization, UV sterilization, gamma ray sterilization, filter sterilization etc. before seeding seed cells it can.

As a culture apparatus used to culture the labyrinthulae, for example, a mechanical agitation type reactor, an airlift type reactor, a packed bed type reactor, a fluidized bed type reactor, or the like can be used. As a culture container, various containers, such as a tank, jar fermenter, a flask, a dish, a culture bag, a tube, a test tube, can be used according to the objective of culture | cultivation, culture volume, etc. 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.

Cultivation of the labyrinthulas can be performed under appropriate temperature conditions, pH conditions, aeration conditions, and the like. The culture temperature is preferably 5 ° C. or more and 40 ° C. or less, more preferably 10 ° C. or more and 35 ° C. or less, and still more preferably 10 ° C. or more and 30 ° C. or less. The pH is preferably 2 or more and 11 or less, more preferably 4 or more and 9 or less, and still more preferably 6 or more and 8 or less.

The culture of the labyrinthulas can be carried out while passing at appropriate intervals depending on the genus and species of the labyrinthulas, the medium composition, the culture conditions and the like. For example, after start of culture, the labyrinthines end the logarithmic growth phase about 2 days and enter the death phase about 7 days. Therefore, passaging of the labyrinthulas is preferably performed at intervals of 1 day to 10 days, more preferably at intervals of 2 days to 7 days, and at intervals of 2 days to 5 days. Is more preferred. The culture time of the labyrinthulae can be determined as an appropriate time depending on the genus and species of the labyrinthulae, the composition of the medium, the culture conditions, the purpose of the culture and the like.

Next, a culture composition obtained by culture of labyrinthulae 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 method for cultivating labyrinthulas described above, and a process for cultivating labyrinthulas using a medium containing whey. And 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 labyrinthula itself as a main component is obtained.

As the Labyrinthula to be cultured, a type having the ability to produce useful substances is preferable. Useful substances include, for example, higher unsaturated fatty acids such as DHA and EPA, monoesters, diesters, triglycerides thereof, phospholipids, glycolipids, and higher hydrocarbons such as higher alkadienes, higher alkatrienes, triterpenes, tetraterpenes, etc. , Essential amino acids, proteins containing them, polysaccharides, pigments, vitamins, physiologically active substances and the like.

As the Labyrinthulae to be cultured, a type in which at least one of DHA and EPA is accumulated in cells is more preferable. Also, the genus Orangiochytrium, the genus Schizochytrium, or the genus Thraustochytrium is more preferable, and the genus Orangiochytrium is particularly preferable. With such a type, the produced DHA and EPA are accumulated in the cells which are difficult to oxidize, so that the oxidation of DHA and EPA can be suppressed and used for various applications. Further, compared with fish oil-derived DHA and EPA, since the odor is weak, the accumulation of chemical substances is small and the composition is obtained with a fatty acid close to natural, so that the purification treatment can be simplified.

The step of culturing the labyrinthulae can be performed under the same medium, culture method, culture conditions, etc. as the above-mentioned culture method. As the culture medium, it is preferable to prepare a liquid culture medium because large-scale culture of labyrinthulas is possible and a large amount of useful substance can be produced. 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 is more preferably contained 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 the labyrinthulae are usually present in the cells of the labyrinthulae and in the fat-soluble fraction separated from the labyrinthulae. Therefore, by concentrating or drying the medium, it is possible to recover the labyrinthulas themselves and useful substances produced by the labyrinthulas at a high concentration.

As a concentration method for concentrating the medium, for example, centrifugal concentration for centrifuging solid content, sedimentation concentration for spontaneously settling solid content, evaporation concentration for heating and evaporating the medium, concentration under reduced pressure for evaporating the medium under reduced pressure, medium The pressure can be applied by pressure and filtration, the medium can be filtered through a separation membrane, the membrane can be concentrated, and the medium can be frozen and removed by freezing. In addition, 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 which performs heat drying in a rotating drum, etc. may be used. it can.

When concentrated, the culture composition containing the labyrinthulas as a main component can have, for example, a water content of 80% or less, preferably a water content of 30% or more and 50% or less. When concentrated to such a moisture content, the culture composition is sufficiently reduced in volume, and the culture composition is in the form of a paste which is difficult to flow appropriately, so that the handleability of the culture composition is improved. Moreover, the culture composition which has labyrinthulas as a main component can be made into 10% or less of moisture content, for example, when making it dry.

The culture composition containing the labyrinthulas as a main component may contain a component derived from whey, along with the cultured labyrinthulas themselves. The whey-derived component may be a metabolite metabolized by labyrinthulae, or may be a non-metabolized non-metabolite. If whey-derived components remain, the odor of the culture composition may be masked by the aroma of the components. Whey has a sufficient eating experience, so even if whey-derived components remain in the culture composition, they can be used directly for food use. On the other hand, in the culture composition, it is preferable that odorous components such as trimethylamine-N-oxide, trimethylamine, and piperidine derived from a fish-derived extract have a concentration below the detection limit.

The culture composition containing the labyrinthula as a main component can be used, for example, in various applications such as food, feed, fertilizer, and industrial raw materials. Specific examples of uses for food include general foods, health foods, food materials, beverage materials and the like. Further, specific examples of feeds include livestock feeds, poultry feeds, aquaculture feeds, pet feeds and the like. Further, specific examples of the industrial raw materials include biofuel raw materials, feed raw materials, fertilizer raw materials, chemical raw materials, pharmaceutical raw materials and the like.

The culture composition containing the labyrinthulae as a main component may be subjected to a step of disrupting cells of the labyrinthulae after concentration or drying. For cell disruption, for example, agitation, pulverization, disruption using shear force such as ultrasonic waves, disruption using pressure change such as osmotic pressure, disruption using chemicals or enzymes, disruption using freeze-thaw It can carry out using various methods, such as a law. As an apparatus for disrupting cells, for example, various apparatuses such as a French press, a vibrating homogenizer, an ultrasonic homogenizer, a freezing homogenizer, and a bead mill can be used.

The culture composition containing the labyrinthulas as a main component may be used for various applications without extracting the ingredients contained in the labyrinthulas after concentration or drying, or the ingredients contained in the labyrinthulas You may use for the process of extracting. For example, since Labyrinthula is rich in essential amino acids and vitamins, has a high amino acid score, and accumulates a large amount of DHA and EPA in cells, algal cells themselves are suitably used for food use. On the other hand, useful substances such as lipids accumulated in the cells of labyrinthulae can be used for applications such as food materials, beverage materials, industrial materials and the like.

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

Specifically, lactose, sucrose, starch, dextrin and the like can be used as an excipient. As a binder, carmellose sodium, methyl cellulose, hydroxypropyl cellulose, gum arabic etc. 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.

Moreover, as a disintegrant, carmellose calcium, crystalline cellulose, hydroxypropyl cellulose, crospovidone etc. can be used. As a lubricant, magnesium stearate, talc, glycerin fatty acid ester, sucrose fatty acid ester, etc. can be used. As a brightener, shellac, paraffin, beeswax etc. can be used. As a suspending agent, carmellose sodium, methyl cellulose, hydroxypropyl cellulose, gum arabic, sodium alginate and the like can be used.

Moreover, calcium stearate, lecithin, casein sodium, glycerin fatty acid ester, sucrose fatty acid ester etc. can be used as an emulsifier. As the antioxidant, vitamin C, vitamin E, catechin, tocopherol and the like can be used. As a pH adjuster, phosphoric acid, sodium hydrogencarbonate, lactic acid, citric acid, gluconic acid, malic acid, tartaric acid, succinic acid, adipic acid and the like can be used. As a preservative, benzoic acid, sorbic acid, polylysine, nisin, alveolar protein etc. can be used.

In addition, as sweeteners, glucose, fructose, maltose, sucrose, trehalose, oligosaccharides, starch syrup, isomerized sugar, reduced starch syrup, xylitol, sorbitol, mannitol, maltitol, stevia, licorice, aspartame, acesulfame potassium, sucralose, etc. It 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 a coloring agent, caramel pigment, gardenia pigment, turmeric pigment, safflower pigment, carotene pigment etc. can be used.

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

The components contained in the culture composition may be extracted from cells and then purified by an appropriate method. Purification of the components can be carried out, 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 mainly composed of labyrinthulae and the extract thereof can be used particularly for health promotion applications when labyrinthulae produces DHA and EPA. Specific examples of health promotion applications include: reduction of health risks associated with arrhythmia, reduction of risk of cardiovascular disease, reduction of blood triglyceride levels, improvement of blood fluidity, enhancement of brain function, , Protection from oxidative damage of cells, 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 etc. are mentioned.

The culture composition mainly composed of labyrinthulae, and the extract thereof are particularly useful for promoting cell growth when labyrinthulae such as aurantiochytrium produce odd-numbered fatty acids such as pentadecanoic acid It can be used. Specific examples of uses for promoting cell growth include healing of damaged tissue, alleviation of pain, autoimmune diseases, neurodegenerative diseases, immune diseases, diseases related to metabolic syndrome, cancer related diseases, etc. These include reduction of wrinkles, promotion of skin metabolism, hair growth, reduction of allergic symptoms, reduction of muscle pain, and improvement of exercise function.

EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but the technical scope of the present invention is not limited thereto.

First, the labyrinthulae were cultured using a medium containing lactose, and the assimilability of lactose, which is the main carbohydrate contained in whey, was confirmed. The aurantiochytrium SA96 WH strain was cultured as a labyrinthula. Moreover, culture was similarly performed using sucrose and maltose as a control of lactose which is a disaccharide.

The medium was prepared using GTY medium as a basic composition and using lactose, sucrose or maltose instead of glucose contained as a carbon source. As the medium, a medium having a concentration of 2% of each disaccharide and a medium having a concentration of 4% were respectively 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 disaccharides having these concentrations.

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

FIG. 1 is a diagram showing the evaluation results of assimilability of disaccharides by orangiochytrium.
As shown in Table 1 and FIG. 1, the assimilability of the disaccharide by the alanthiochytrium strain SA96 WH was observed for all of lactose, sucrose, and maltose, but was lower than glucose. Therefore, it is possible to use lactose contained in whey, but from the viewpoint of securing a high growth rate, it may be preferable to use glucose together with whey.

Next, the labyrinthula was cultured using a medium containing animal-derived whey, and the influence of the concentration of whey was confirmed. The aurantiochytrium SA96 WH strain was cultured as a labyrinthula. The effect of the concentration of whey was confirmed at six concentrations of 5%, 10%, 20%, 40%, 60% and 80%.

The culture medium changes the concentration of whey to 5%, 10%, 20%, 40%, 60%, 80%, respectively, 3% glucose, 0.5% sodium glutamate, yeast extract (Difco) And 0.2% seawater salt at a concentration of 1%. As whey, cheese whey derived from an animal having a water content of 93.9%, a protein of 0.3%, a carbohydrate of 5.2%, an ash content of 0.6% and a lipid content of less than 0.1% was used.

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

FIG. 2 is a diagram showing the effect of whey concentration in the culture of aurantiochytrium.
As shown in Table 2 and FIG. 2, in the culture of the aurantiochytrium SA96WH strain, the cell number was almost maximal when the concentration of whey in the medium was around 40%. After 72 hours of culture time, the error increased as seen for the concentration of 40%, but within the culture time up to 48 hours, the contribution rate increased when the concentration of whey reached about 10%. Was confirmed. And as the concentration of whey increased further, the contribution rate tended to gradually increase. However, when the concentration of whey exceeded around 40%, the contribution rate gradually decreased and the increase in cell number decreased. Therefore, whey is preferably contained in the medium in the range of 5% by volume to 50% by volume (3 g / L to 30 g / L in terms of solid content), 10% to 40% by volume (solid content) It is more preferable that the medium is contained in the range of 6 g / L or more and 24 g / L or less).

Next, the labyrinthulae were cultured using a culture medium containing whey derived from an animal to confirm the growth rate of the labyrinthulae. The aurantiochytrium SA96 WH strain was cultured as a labyrinthula.

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

The growth rates of the Labyrinthulae were compared for strains pre-incubated with medium containing whey in advance and strains pre-incubated with GTY medium. The main culture was performed by shaking culture, and the amount of inoculation was compared at 1% and 2%. Cultivation of SA96 WH strain precultured in GTY medium at 1% inoculation volume (1% GTY culture medium), cultivation of SA96 WH strain precultured in medium containing whey at inoculation amount of 1% (1% milk Culture medium), culture with 2% inoculum weight of SA96 WH strain precultured in GTY medium (2% GTY medium), culture with SA96 WH strain precultured in medium containing whey with 2% inoculation volume The number of cells was measured over time for each of (2% whey medium). The results are shown in Table 3 and FIG.

FIG. 3 is a growth curve of Aurantiochytrium cultured by changing the preculture conditions.
As shown in Table 3 and FIG. 3, when the strain precultured in a medium containing whey and the strain precultured in GTY medium are compared, the strain precultured in GTY medium grows rapidly in a short period of time The log growth phase tended to end early. On the other hand, strains precultured in a medium containing whey tended to show higher growth rates even at the stage when the culture time had passed to some extent.

Next, the labyrinthulae were cultured using a culture medium containing whey derived from an animal, and the growth amount of the labyrinthulae, that is, the amount of the culture composition obtained by the culture and the odor of the culture composition were confirmed. The aurantiochytrium SA96 WH strain was cultured as a labyrinthula.

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

The growth amount of the labyrinthulae was confirmed by inoculating a strain precultured in a medium containing whey beforehand into 3 L of a whey culture medium at an inoculation weight of 2% and performing airlift culture. In airlift culture, the air aeration rate was 1.0% by volume / min. After 48 hours of culture time, either 3% equivalent glucose or 3% equivalent glucose, 10% equivalent whey, and 0.2% equivalent yeast extract are added. It added and compared the amount of growth after that. 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. Moreover, FIG. 5 is a growth curve of orangiochytrium cultured by adding glucose.
As shown in Tables 4 and 5 and FIGS. 4 and 5, the airlift culture enables large-scale culture of the aurantiochytrium SA96WH strain, and a culture composition exceeding 15 g / L on a dry weight basis was obtained. When a feeding solution containing 3% equivalent glucose, 10% equivalent whey and 0.2% equivalent yeast extract is added after 48 hours of culture time (see Table 4 and FIG. 4), Comparing with the case where glucose equivalent to 3% is added after 48 hours of culture time (see Table 5 and FIG. 5), the stage where 72 hours of culture time has passed when only glucose is added Tend to enter a stationary phase or death phase, but when the addition of a liquid containing whey is added, a further increase in the amount of growth is observed, and a larger amount of culture composition can be obtained. confirmed. The odor of the obtained culture composition was confirmed by a sensory test. As a result, although a weak raw milk odor was confirmed, no odor was observed.

Next, the labyrinthulae were cultured using a culture medium containing animal-derived whey and a culture medium containing soymilk whey, and the growth amount of the labyrinthulae was compared. The aurantiochytrium SA96 WH strain was cultured as a labyrinthula.

The medium containing whey from animal origin changes the concentration of cheese whey to 5%, 10%, 20%, 40%, 60%, 80% respectively, and 3% glucose, 0.5% sodium glutamate, The composition contained 0.2% of yeast extract (Estock sp-d) and 1% of seawater salt. Cheese whey is 93.9% water, 0.3% protein, 5.2% carbohydrates, 0.6% ash, less than 0.1% lipids, as in the previous test.

A medium containing soy milk was prepared using soy whey by-produced in the process of producing tofu and soy milk whey obtained by purifying soy milk. The medium containing soy whey changes the concentration of soy whey to 5%, 10%, 20%, 40%, 60%, 3% glucose, 0.5% sodium glutamate, yeast extract (Estock sp The composition contained -d) at a concentration of 0.2% and seawater at a concentration of 1% (Devora lake salt 0.9% + magnesium sulfate 0.1%). The medium containing soy milk whey changes the concentration of soy milk whey to 5%, 10%, 20%, 40%, respectively, and 3% glucose, 0.5% sodium glutamate, yeast extract (Estock sp-d) And 0.2% seawater salt at a concentration of 1% (deborah lake salt 0.9% + magnesium sulfate 0.1%).

As a result of the culture, in the case of cheese whey, the higher the concentration, the higher the cell number was. At a low concentration of 5% or 10%, the growth rate slowed down after 48 hours, but at a concentration of 40% or less, an increase in the number of cells depending on the concentration of cheese whey was observed, and the culture time was 72 hours High proliferation rates were also confirmed at the time of passage. Also, in the case of soy milk whey or soy whey, a marked increase in the number of cells was observed until the culture time was 48 hours. In the case of soy whey, precipitates were produced at low concentrations of culture, and the growth rate varied in the decreasing direction as compared to high concentration cultures, but in the case of soy milk whey, the difference in growth rate depending on concentration was It was small. When cheese whey was compared with soya milk whey or soy whey, it was confirmed that the increase in the number of cells according to the concentration was larger in cheese whey.

Claims (8)

1. A method for cultivating labyrinthulas, which comprises cultivating labyrinthulas using a medium containing whey.
2. The culture method of the labyrinthula of Claim 1 in which the said whey is contained in the said culture medium by the density | concentration of 3 g / L or more and 30 g / L or less in conversion of solid content.
3. The culture method of labyrinthulas according to claim 1, wherein the whey is whey derived from an animal.
4. The method for cultivating labyrinthulas according to claim 1, wherein the whey is soy milk.
5. The culture method of labyrinthulas according to claim 1, wherein the whey is adjusted to a pH of 4 or more and 8 or less.
6. The method of claim 1, wherein the medium comprises glucose and a yeast extract.
7. The method for cultivating labyrinthulas according to claim 1, wherein the medium has a composition not containing a medium component which is a protein degradation product.
8. The method for cultivating labyrinthulas according to claim 1, wherein the labyrinthulas are Auranthochytrium, Schizochytrium, or Thraustochytrium.

Images