JPH044865B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION Field and Object of the Invention The present invention relates to the cultivation of the protozoan Euglena.
By aerating high concentration oxygen, waxy material,
The present invention relates to a method for cultivating Euglena, the purpose of which is to suppress the production and secretion of mucilage and promote the synthesis of nutritional components such as unsaturated fatty acids and vitamin E.

Euglena contains chlorophyll and has the plant-like property of growing autotrophically through photosynthesis, and has a flexible proteinaceous outer cell membrane.
It also exhibits animal-like characteristics such as actively swimming with flagella, and is a unique single-celled organism that is taxonomically classified as both animals. Its cellular components are rich in protein, and its nutritional value is of extremely high quality.Also, because its outer cell membrane is proteinaceous, it is highly digestible in the gastrointestinal tract of higher animals, and can serve as an excellent protein source for humans and livestock. has already been reported (Kitaoka et al., Noka, Vol. 57, p. 477, 1977; Hosoya et al., Noka, Vol. 57, p. 483, 1977). In addition, the content of vitamin C, vitamin E, and highly unsaturated fatty acids is high, and when taken together, Euglena cells are expected to be developed as an excellent health nutritional food.

However, when Euglena is cultured, especially when cultured in large quantities, it often secretes mucilage to the outside of the cells and accumulates waxy substances (wax esters) within the cells, which affect Euglena's flavor and oral texture. Animal experiments have confirmed that rats, chickens, silkworms, etc. avoid it. The mucilage of Euglena is a substance whose main components are protein and polysaccharide, and the waxy substance is
Esters mainly composed of _C14 fatty acids and fatty alcohols (Inui et al., Agric.Biol.Chem., Vol. 47,
2667, 1983), most of it can be removed by physical separation of cells after culturing, treatment with organic solvents, etc., but it is more desirable to suppress its production by taking care during culturing.

The production of mucilage and waxy substances is influenced by the medium composition and culture conditions, but they are most likely to be produced when Euglena is placed in anaerobic conditions.

Therefore, it should be possible to avoid the production of these undesirable substances by optimally adjusting other factors during Euglena culture and providing aerobic conditions such as mechanical stirring and aeration.

However, because Euglena cells have a flexible outer cell membrane, they are extremely susceptible to shock, and excessive agitation or aeration can cause cell rupture due to collisions between cells, cell walls, or the stirring device, and the cell contents may be ejected. The substance is instead converted into a sticky substance, which contaminates the entire culture solution.

Structure and Effects of the Invention In view of the above-mentioned situation, the present inventor has made a
After elucidating the formation mechanism of waxy substances, we cultivated Euglena by aerating oxygen containing 30% or more oxygen or oxygen-enriched air with gentle agitation or without agitation to prevent cell damage. figure,
The present invention was completed by successfully producing high-quality cells in high yield.

It was also found that under the culture conditions of the present invention, Euglena cells are exposed to a moderate aerobic environment, which has the effect of promoting the production of reducing substances by stimulating intracellular antioxidant function. Therefore, cells containing a large amount of vitamin C, vitamin E, highly unsaturated fatty acids, etc. can be obtained, which is an extremely advantageous effect when Euglena is used as a nutritional food.

The Euglena used in the present invention may be any species of the genus Euglena, such as Euglena gracilis and Euglena viridis. In addition to various wild strains containing chlorophyll, any mutant strains including chlorophyll-deficient strains formed by antibiotics, ultraviolet rays, high temperature treatment, etc. can also be targeted. The composition of the medium used in the present invention includes a carbon source, a nitrogen source,
Any combination of growth promoting substances such as inorganic compounds and vitamins may be used, and the culture conditions are not particularly limited. In other words, the initial pH of the culture is
2.5 to 8.0, preferably PH3.0 to 5.5, culture temperature
The temperature is 10°C to 35°C, preferably 26°C to 31°C, with or without light irradiation. Cultivation can be done outdoors in an open system,
Any type of indoor culturing device may be used.

The oxygen used in the present invention may be oxygen filled in a normal commercially available cylinder, and by mixing this with air, any high concentration oxygen-containing air can be prepared. By using a gas mixer, the mixing ratio can be specified accurately and arbitrarily. Oxygen or high-concentration oxygen-containing air may be passed into the culture medium through a normal ventilation pipe, but the air bubbles can be made smaller by making the tip of the ventilation pipe porous or using multiple ventilation pipes.
It can also promote the contact between cells and air bubbles or the diffusion of oxygen into the culture medium. Suppression of waxy substance formation due to wax ester fermentation only requires an oxygen concentration of approximately 10 ^-5 M or higher, so if the purpose is simply to maintain the culture solution under aerobic conditions, the amount of aeration required should be determined depending on the culture solution volume and aeration amount. can determine the oxygen concentration of
In addition, if you intend to stir the culture solution only by aeration, it is necessary to increase the amount of aeration depending on the shape and volume of the culture vessel and the volume of the culture solution, and in this case, it is also possible to reduce the oxygen concentration. It is possible. When culturing Euglena, excessive agitation can lead to cell damage, but insufficient agitation can cause cells to sink and allow the production of impurities, so it is important to agitate to the extent that proper cell movement occurs. is desirable, and therefore it may be appropriate to use a stirring device that provides gentle stirring in addition to aeration. The amount of aeration and the oxygen concentration during aeration cannot be determined unconditionally depending on the amount of culture solution, the shape of the culture apparatus, and the presence or absence of stirring and its intensity, but the oxygen concentration during aeration is generally 30% or more.
You can choose from a range of 100%. 100% means ventilation of the oxygen gas directly from the cylinder. Furthermore, if the aeration rate is approximately 0.05 to 30/min per culture solution, sufficient oxygen can be supplied. If it is less than 0.05/min, the cells may sink, and if it exceeds 30/min, the cells may be damaged, which is not preferable.

Euglena cells cultured with aeration of oxygen or oxygen-enriched air are cultured in the same way as in normal culture.
It can be collected by centrifugation, filtration, etc., and dried by freeze drying, hot air drying, spray drying, etc. to obtain a powdered product. This product can be used alone in the form of powder, granules, or tablets, or can be used in powder or paste form mixed with other ingredients.

Examples Hereinafter, the present invention will be explained in more detail with reference to Examples.

Example 1 The following medium 8 was placed in a 10 glass jar fermenter, and Euglena gracilis Z strain was cultured at 28° C. for 5 days under light irradiation (2000 lux). Aeration was performed using air containing 60% oxygen by mixing equal amounts of oxygen and air from a cylinder, and the aeration rate was 4 per minute through a normal aeration pipe. Stirring was carried out using a conventional stirrer at 80 revolutions per minute. The initial pH was 3.5.

Medium composition KH ₂ PO ₄ 0.4g MgSO ₄・7H ₂ O 0.5g CaCO ₃ 0.2g Glucose 12.0g Malic acid 7.0g Glutamic acid 5.0g ZnSO ₄・7H ₂ O 25mg MnCl ₂・4H ₂ O 20mg Fe ₂ (SO ₄ ) ₃・xH ₂ O 1mg NaMoO ₄・2H ₂ O 1mg CuSO ₄・5H ₂ O 1mg CoSO ₄・7H ₂ O 1mg H ₃ BO ₃ 1mg NiSO ₄・6H ₂ O 0.5mg Vitamin B ₁ Hydrochloride 2.5mg Vitamin B ₁₂ 10 μg distilled water 1000 ml The cell yield was 50 g after lyophilization, the protein content was 54% and the amino acid value was 84, which was not different from that when aerated with normal air. Vitamin C content is
50mg/100g, vitamin E content is 60mg/100g, and polyunsaturated fatty acid content is 510mg/100g, which are 10-20% higher than in the case of normal aeration. No mucilage was formed in the culture solution, and the wax content of the cells was less than 0.2%. These Euglena cells were ingested by rats, chickens, and chicks without being repelled.

Example 2 Euglena gracilis SM-ZK (chlorophyll-deficient strain) was cultured in the dark at 30°C for 7 days in a 200° stainless steel jar fermentor with 160ml of the following medium. Ventilation was carried out at a rate of 20 per minute by connecting oxygen directly from the bottom of the vessel through a total of four vent pipes installed in two sets on the wall of the vessel at opposing positions.
The culture solution was stirred at 60 revolutions per minute using a stirring rod with a small diaphragm plate. The initial pH was 4.0.

Culture medium composition: Molasses 40g Ammonium phosphate 5.5g Vitamin B _{monohydrochloride} 2.5mg Vitamin B ₁₂ 5μg Tap water 1000ml After culturing, the cells were collected by centrifugation, washed with water, and dried with hot air.The yield of cells was 0.9Kg. ) The yield was slightly lower than the yield under normal conditions in the culture medium, but this was due to the use of blackstrap molasses as a carbon source. The protein content was 52%, as high as in the case of normal culture, and the amino acid composition was also the same as that of cells in normal culture. No mucilaginous matter was produced in the culture solution, and the waxy matter content of the cells was less than 0.1%. The contents of vitamin C, vitamin E, and highly unsaturated fatty acids were 20-30% higher than those cultured in Koren-Huttner medium with normal aeration. When these Euglena cells were given to mice as a protein source, they ate well and showed the same growth as when casein was given.

Example 3 The same medium 8 as in Example 1 was placed in 10 glass jar fermenters, and Euglena gracilis Z strain was incubated at 28°C under light irradiation (2000 lux) for 50 minutes.
Cultured for 1 day. Ventilation was carried out using air containing 30% oxygen, which was a mixture of oxygen and air from a cylinder, and the aeration rate was 6 minutes per minute, using a normal vent pipe.
Stirring was carried out using a conventional stirrer at 80 revolutions per minute. The initial pH was 3.5.

The cell yield was 52 g after lyophilization, with a protein content of 54% and an amino acid value of 84, which was not different from that when aerated with normal air. Vitamin C content is
64mg/100g, vitamin E content 86mg/100g, and polyunsaturated fatty acid content 510mg/100g, which are 10-20% higher than in the case of normal aeration. There was no formation of mucilage in the culture solution, and the wax content of the cells was less than 0.2%. These Euglena cells were ingested by rats, chickens, and chicks without being repelled.

Claims (1)

[Claims] 1. A Euglena cultivation method characterized by aerating oxygen or oxygen-enriched air containing 30% or more oxygen.