KR101283149B1 - Development of medium and culture condition for mixed culture Lyophyllum cinerascens, Pleurotus ostreatus, Flammulina velutipes, Pleurotus eryngii etc with phaffia rhodozyma and Method for production of feed additive with astaxanthin having antioxidant effect. - Google Patents

Abstract

In the modern mushroom farm that mass-produces mushrooms such as Enoki mushroom, Pleurotus eryngii, Pleurotus eryngii, and Mandang mushroom, it develops a production medium that allows P. rhodozyma and mushrooms to grow together. The tin content was about 50 ppm produced. In addition, the use of sawdust fermented for 60 days or more in the composition of the medium is reduced by 10% (w / w), corn steep powder 2% (w / w) and rice bran 8% (w / w). By increasing the number of mixed cultures, the growth of the mushrooms was prevented. The composition of the mushroom producing medium was developed as a material that can be used as a feed additive.
By developing a culture medium and process for mixing P. rhodozyma strains and mushrooms together in liquid form, the mushroom production process was improved by the liquid seed inoculation method, so that the seed concentration of yeast is high, mushroom mycelium growth rate is high, and contamination is low. It was.
After harvesting the mushrooms, the remaining parts with yeast were developed as feed additives, and the developed feed additives were fed to laying hens. Increased by 2 grades.

Description

Development of Mixed Culture Technology of Pleurotus eryngii Mushroom and Papia Rhodozaima Yeast and Development of Antioxidant Probiotics with Antioxidant Containing Astaxanthin {Development of medium and culture condition for mixed culture Lyophyllum cinerascens , Pleurotus ostreatus, Flammulina velutipes, Pleurotus eryngii etc with phaffia rhodozyma and Method for production of feed additive with astaxanthin having antioxidant effect.}

Astaxanthin is a carotenoid-based pigment that is abundant in the marine environment and biosynthesized from several microalgae, molds, or small crustaceans, and the pigments migrate and accumulate along the food chain to large animals such as flamingos, algae and fish, including salmon. .

In the poultry industry and fish farming including salmon and trout, the egg yolk and red meat such as salmon and trout increase the value of the commodity.Astaxanthin is not biosynthesized in the body, so artificially Must be added and used. Astaxanthin, which has been of constant interest for its anti-oxidative effects and its unique red pigment, with antioxidant activity 10 times stronger than other carotenoids and more than 100 times stronger than alpha-tocopherol, is a source of pigments for microorganisms, marine It can be divided into microalgae, natural pigments extracted from plant biosynthesis, and artificial dyes chemically synthesized. Typical artificial pigments include all-trans-Astaxanthin produced by F. Hoffman-LoRoche, Switzerland. However, artificial pigment is a safety issue as a food additive, so synthetic astaxanthin and canthaxanthin have not been approved as an additive by the US Food and Drug Administration. In addition, the preference for natural pigments is gradually increasing all over the world, and researches to obtain natural pigments in large quantities inexpensively continue.

The way to get astaxanthin pigment naturally

1. Shrimp ( Pandalus) borealis ) is used as a by-product, but its quality is limited due to the large amount of chitin and ash present in the crustacean shell.

2. Mycobacterium lacticola , Brevibacterium sp. Bacteria and Peniophora sp. It is reported that fungi can produce, but commercial production is low.

3. Haematococcus , marine microalgae In pluvialis green algae, astaxanthin can be produced as a primary carotenoid by controlling the cultivation method, but almost all (approximately 87%) are ester forms, which can affect accumulation and metabolism depending on animals, and due to the thick wall of algae Algae-fed salmon have low astsxanthin concentrations due to low utilization (Johnson et al., 1991). Microalgae are difficult to artificially cultivate in fermentation tanks. It is cultivated in the form of pond by blocking the seawater in the place.

4. Yeast Phaffia rhodozyma is relatively easy to cultivate compared to microalgae or fungi, and has abundant proteins and unsaturated fatty acids, vitamins, and unique flavors that promote pigment absorption. come.

Phaffia rhodozyma yeasts were separated from deciduous sap in mountainous regions of Japan, Alaska and Russia in the early 1970s and called Rhodozyma montanae , and were changed to Phaffia by Latin notation (Johnson et al., 1989), Yoneyama et al. (1976) Phaffia genus is only one species, P. rhdozyma , and it is a coliform yeast that produces carotenoids containing more than 80% of astaxanthin. Therefore, in order to increase the pigment content of the yeast cells industrially, many studies on the improvement of the strain, the culture and the fermentation have been conducted. At the same time, Grau et al. (1980) tested whether pigments accumulated in P. rhdozyma yeast cells accumulate in the body of salmon and trout, lobster shells, or egg yolk of eggs. There has been a great deal of commercial interest as the availability of Phaffia's yeast has been raised in the artificial culture of salmon. The global market for astaxanthin is expected to range from $ 120 million to $ 140 million, with feeds, dietary supplements, and medicines.

Efforts to use astaxanthin in Korea have already begun decades ago, but domestic production is rare due to the difficult culture methods and low price competitiveness of imported products. Nevertheless, market formation is rarely performed.

In the poultry industry, which produces eggs, the marketability of egg yolk is increased. For this purpose, canthaxanthin, a synthetic pigment that is not used in other countries, is added to feed. However, astaxanthin in the current market pursuing well-being is one of the important products recognized by the food industry and the livestock industry as a natural pigment.In spite of its high price, the astaxanthin is a high quality functional livestock product. Increasingly, usability is gradually increasing, such as being recognized and distributed at a high price.

Many studies have been conducted in Korea to produce Astaxanthin for decades ago, and there are many papers and patents. Most of the content

1. In order to produce astaxanthin in large quantities, the strains for production of P. rhodozyma , a chemical and physical method

2. It relates to the composition of the culture medium and the amount of dissolved oxygen or to culture conditions using light. However, P. rhodozyma culture is a metabolism in which the growth of cells and the production of pigments are opposite to each other, so that a small number of cells produces a lot of pigments, and a large amount of cells produces a small amount of pigments, thereby limiting the yield.

Haematococuss , algae, in Korea There have been many studies on the culture method using pluvialis . However, this method is inexpensive compared to the method of producing natural pigments by cultivating green algae naturally by using natural natural conditions in Hawaii, etc. (CYANOTECH, Aquasearch, etc.).

In conclusion, the method of producing astaxanthin is the only commercial and practical technology currently using liquid fermentation using P. rhodozyma in Korea, and the production method using green algae is made mainly by preventing seawater.

To produce astaxanthin in Korea, green algae or yeast must be cultured, and the yield is so low that the production price is higher than that of imported products. In particular, P. rhodozyma yeast, which grows at low temperatures, cannot be cultured in the summer, so many studies have been conducted in Korea for decades, and many patents have been registered.

As mentioned earlier, in order to make good use of astaxanthin, which is essential for the poultry and aquaculture business, it must be supplied at low prices. In other words, the production costs should be reduced. The domestic research direction has been focused on the composition of medium, culture conditions and strains for the culture of green algae or yeast. The cost could not be reduced.

By the necessity of such a development, the development focus of the present invention was tailored to reduce production costs. If P. rhodozyma , which produces astaxanthin, is mixed with mushrooms using a mushroom cultivator operating at a low temperature around 20 ° C per year, even if the yield of astaxanthin is low, mass production based on low production cost Will lower the selling price of astaxanthin, which will increase its domestic utilization.

As a development method, ① the existing mushroom production medium is improved so that P. rhodozyma and mushrooms can be grown together in modern mushroom farms that are mass-produced by growing mushrooms such as top mushrooms, oyster mushrooms, matsutake mushrooms, and mandrel mushrooms. Develop a yeast inoculation form that can mass produce astaxanthin without disrupting the production process of mushrooms, and develop culture conditions for mushroom and P. rhodozyma to grow together. Finally, after ④ mushroom harvest, develop the remaining part of yeast as a feed additive.

    Although it is not easy to approach the mixed solid fermentation method, it is expected to be fully developed and have a great ripple effect upon success.

Developing a composition of a production medium in which yeast P. rhodozyma and mushrooms grow together to produce astaxanthin , a natural additive for feed, which plays a role in enhancing the quality of eggs and eggs, while growing mushrooms The low temperature allows the production of expensive pigments at low cost without the need for extra energy, so that products with excessively high production costs can be produced at competitive prices.

Developing spawn spawn inoculation methods using liquid spawn, and shortening the process by spawning spawn medium so that P. rhodozyma and fungi grow together, reduces mushroom growth and contamination rate. Liquid seed vaccination is a method that many mushroom farms feel the need to improve from the current solid seed vaccination method, and also has a positive effect of increasing the astaxanthin productivity even higher because of the high content of yeast in the mixed liquid seed.

The waste badges of mushrooms have been very difficult to process, and efforts have been made to develop them into livestock feeds, but they have not been activated due to low commodity value and concerns about deterioration. However, the present invention was developed to reduce the content of tannin and nigrin in sawdust and the like, and to increase the source of protein, such as rice bran and corn steep powder, so that after harvesting mushrooms, the astaxanthin-produced mushroom medium can be used as feed. The nutritional value and fibrinolytic enzymes of the mushroom mycelium in the mushroom medium are present together to increase the feed value. Since the medium produced after the production of mushrooms was developed as a feed additive having a high commercial value, it is possible to solve functional feed additives and environmental improvement problems at once.

The present invention, which can produce high value-added well-being livestock products, has high import substitution effect, and solves resource recycling, is convinced that the present invention is a very valuable technology that enhances corporate value and economic performance.

Figure 1 is the result of the antagonistic test of mushrooms and yeast,
Figure 2 shows the results of the antagonistic test of the oyster mushroom and yeast
Figure 3 results of the antagonistic test of the enoki mushroom and yeast
Figure 4 shows the results of antagonistic test of mushrooms and yeast
5 is a microbial observation of the culture of liquid bacteria of cultured fungi and yeast No 1
Figure 6 is a microbial observation of the culture and liquid culture of mushroom and yeast No 2
7 is a microscopic observation of liquid culture of cultured mushrooms and yeast No. 3
8 is a microscopic observation of liquid culture of cultured mushrooms and yeast No. 4
9 is a mixed culture culture No 1 of yeast and oyster mushroom
10 is a mixed culture culture No. 2 of the yeast and oyster mushroom.
11 is a specification test result of the produced product

The following describes the details of the present invention. This embodiment shows an example of the present invention and does not limit the technical scope of the present invention.

One. Used strain

As the strain used in the present invention, a yeast Phaffia rhodozyma producing astaxanthin was used. The mushrooms were Flammulina velutipes , Pleurotus ostreatus , Pleurotus eryngii , and Lyophyllum cinerascens strains. Strains were obtained. Phaffia rhodozyma used YPDA (Yeast extract 1%, Peptone 1%, Glucose 1% agar 1.5%) medium to maintain the strain, and mushroom strains were PDA (Potato dextrose agar: Potato infusion solid 0.4%, Dextrose 2%, agar 1.5%) medium was used. The culture temperature of all strains was incubated at 21 ℃.

In order to maintain the strain, yeast strains were passaged by dropping a relatively dark red strain from colonies grown in YPDA medium using sterilized toothpicks. Using this method has the advantage of easily selecting strains that produce better pigments than simply taking colonies and subcultured. In addition, the colonies with excellent pigment production could be deposited many times, so that the balance lines of the excellent strains could be well maintained.

2. Astaxanthin extraction and analysis (Total carotenoid)

5ml of the cultured yeast culture solution was added to a centrifuge tube, 15ml of distilled water was added, centrifuged to wash the precipitate, and water was removed by a vacuum dryer. Add 10 ml of DMSO (Dimethyl sulfuroxide) solution preheated to 55 ° C, further react at 55 ° C for 5 minutes, and shake it very vigorously. Add 1 ml of 1M phosphate buffer (pH 7.0), add 25 ml of solvent mixed with hexane and ethyl-acetate 1: 1, and shake gently to mix the pigment into the solvent layer. After centrifugation at 10,000 rpm for 5 s to remove impurities in the solvent layer, the absorbance of the upper solvent layer from which the dye was extracted was measured at 474 nm. The solvent which does not contain a pigment shall be blank.

  Total carotenoids were quantified by the following equation using 1% extinction coefficient = 2100 (E 1% / 1cm = 2100) using the absorbance value of the dye and the dry weight value of the cells (Johnson et al., 1989).

Total carotenoid (㎍ / g of dried yeast) =

Yeast Culture Characteristics and Antagonistic Test with Mushrooms

The culture characteristics of the yeast cultured in the fermenter using YPD medium consumed 1% of sugar after about 60 to 70 hours, and the yeast showed a yield of about 3 g / L in dry weight, and the pigment was initially 20 ppm. It was increased to 1000ppm at, and the culture characteristics were maintained because no special strain was improved.

It was confirmed whether yeast and mushroom mycelia inhibit the growth of each other. Since rice bran is used for PDA, YPDA, and mushroom bottle media, RBA 1 (1% rice, 1.5% agar) made from rice and agar only, RBA 2 (rice bran 2) %, agar 1.5%) was used to make a medium. The antagonism test also included growth comparisons in each medium.

Characteristics of each medium, contents of inoculation and comparative viewpoint after strain growth badge Badge characteristics Inoculation contents Strain Growth Comparison Perspective YPDA Yeast Cultivation
badge Yeast is smeared on the medium and then inoculated with the tops, zelkovas, birds and mushrooms 1. Observe the growth of yeast and mushrooms 2. Mixed culture and growth of RBA medium
Comparative observation PDA Mushroom Mycelium
Culture medium Only mushroom culture 1. By growing only mushrooms,
Comparison of Mushroom Growth with Mixed Cultures RBA 1 Mushroom production medium (1% rice bran) After spreading yeast on the medium, spinning top, zelkova, bird
Inoculates all strands of mushrooms 1. Observe whether yeast and mushrooms grow together 2. Mixed culture and growth comparison of YPDA 3. Growth and Comparative Observation of Mushroom Fungus in PDA 4. Compare RBA2 and all items RBA 2 Mushroom production medium (2% rice bran) After spreading yeast on the medium, spinning top, zelkova, bird
Inoculates all strands of mushrooms 1. 2. 3-same as RBA1 4. Compared to RBA 1

After incubation in each medium, the comparison results are as follows.

  ① Yeast strains grew very well in YPDA. Therefore, in the mixed inoculation medium, yeast grows first covering the medium, the growth of the mushroom was inhibited. Compared to the growth of each mushroom grown on PDA inoculated with mushrooms only, it was significantly slower.

  ② RBA1, RBA2 was good growth of yeast, mushrooms also grew well together. Yeast growth was slower than YPDA, but the yeast cells were good enough to cover the surface of the medium even though only the rice bran was supplied as a nutrient source. It doesn't seem to be. All strains grew well, and later, the yeast and mushroom mycelium covered the entire RBA medium.

  ③ RBA1 and RBA2 was not enough to see the difference in the growth rate of each strain or the concentration of cells.

As a result of the antagonistic test, it was judged that the antagonism between P. rhodozyma and each mushroom strain was small, and depending on the composition of the medium, it could be possible to cultivate the mushroom spawn and yeast used for mushroom production.

Liquid spawn  Badge development

Most mushroom farms that produce mushrooms inoculate spawns using solid spawns. The lack of microbiological knowledge is convenient and easy to handle visually. However, there is a disadvantage that the incidence of contamination is rather high, and mushroom culture period is slightly longer after inoculation. In order to overcome these disadvantages, some bottle mushroom farms may introduce a liquid spawn system, but the replacement speed is slow due to the burden of replacing existing equipment.

In the present invention, it is more efficient to use mushroom spawn as the solid spawn and yeast as the liquid spawn. However, as the inoculation process was increased, mushroom production was cumbersome, so it was determined that mushroom farms would not be inoculated with yeast. Therefore, a system for inoculating yeast and mushroom spawns at one time was necessary. In the antagonistic test results, it was possible to incubate the two strains together and then inoculate them at once with the liquid spawn, which was judged to be the most efficient method. And it was necessary to develop a medium capable of culturing two strains together to make a mixed seed.

    Molasses is the most commonly used industrial medium. Molasses is a by-product of sugar production and has the advantages of low price, high sugar content, and easy to obtain.

    ① YPD broth as a control, mixed strain culture using molasses instead of glucose, both yeast growth was so fast that the growth of mushroom bacteria was slow. And since the sugar consumption was fast, about 90% of the sugar was consumed after 2 days of culture. Of course, a large amount of sugar may be initially supplied to the liquid spawn medium, but if the residue remaining in the medium during the liquid spawn inoculation is supplied to the mushroom producing medium, the culture rate of the mushroom is changed, and thus stable mushroom production cannot be performed. Molasses, which is a very good raw material industrially, could not be used.

    ② Rice bran was used as carbon source. In the antagonistic test, both yeast and fungi were well grown and sugars were relatively easily decomposed by microorganisms, but both fungi and yeast were judged to be long-term cultivated raw materials without rapid growth. In particular, rice bran was an ideal carbon source that did not affect the growth rate of mushrooms even when mixed together in a bottle medium for mushroom production when inoculated with liquid spawn. YPD broth was inoculated with yeast and each of four mushrooms in a medium prepared with 2% concentration of rice bran as a control, and then cultured at 21 ° C. As a result, the yeast also grew red, mushroom fungi grew in the form of pellets. As a result of microscopic observation, it was confirmed that the mycelium of yeast and mushrooms grew together. In addition, even without using a separate nitrogen source was incubated in a state suitable for use as spawn, the medium of the mixed liquid spawn was determined to use more than 2% of rice bran.

Optimized medium composition for mushroom production

In the mushroom mushroom medium, sawdust fermented for 60 days or more is used as a main ingredient. It is intended to make mushrooms easier to use by decomposing tannins and nignin first by the action of microorganisms. Bottle composition consists of 90% (w / w) of sawdust, 10% (w / w) of rice bran and a few additional additives for each mushroom farm, such as beet pulp, and then sterilized. However, in the present invention, since the mushroom must be used after harvesting the remaining medium for the feed, if the content of the sawdust is high, the feed value is somewhat reduced. Therefore, it was necessary to form a medium that can increase the value as a feed while growing yeast well.

    ① The content of sawdust was reduced from 90% of the total medium to 80%. This was because the pores needed to increase the feed value and to grow mushroom and yeast were necessary. I wish to reduce the amount of sawdust a little more, but too low sawdust content of mushroom mycelium was lowered because it may be a deterrent to the growth of the mushrooms could not help it.

    ② It was necessary to supply nitrogen source to help the yeast grow in the bottle medium. Replenishing rice bran by the reduced amount of sawdust would be helpful for yeast growth, but supplying an appropriate amount of nitrogen would be helpful for yeast growth without affecting mushroom mycelium culture. Corn steep powder (CSP) was supplied 2%. This is because it is easy to supply and low price, and it is easy to store because it is powder form, not liquid, and it is easily mixed with sawdust and rice bran during bottle badge manufacturing.

    ③ 80% sawdust, 18% rice bran, and 2% CSP, 45% water moisture content after sterilization of the medium was inoculated with liquid seedlings cultured with rice bran medium cultured yeast and mushrooms in a bottle medium. The mushrooms were incubated with the top, oysters, birds and mushrooms. After incubating at 21 ° C. for 20 days, the bottles were cut, and the interior of the mushrooms was taken as a sample, and the number of viable cells of yeast was measured.

A significant amount of media had to be used, and on average about 1 × 10 ⁷ cfu / g (wet weight) of yeast was detected.

   ④ The total carotenoid content was measured in the sample inside the cultured medium. Astaxanthin was very susceptible to heat, making it difficult to determine the dry weight of the media sample. The medium inside the bottle after the mushroom mycelium grew was about 25% more water on average, but in actual mushroom growers, the humidity is higher than the laboratory value because it thoroughly maintains humidification and ventilation to grow the fruiting body of the mushroom well. It was judged. As a result of many trials and errors, the average pigment content was found to be around 50 ppm.

Pilot production

Fermented Sawdust 80% (w / w), Rice Bran 18% (w / w), CSP2% (w / w) and Water 45% (w / w) Insert the medium to 550g to 650g, press firmly, and then drilled the center of the center vertically using a round bar with a diameter of 1.5 ~ 2 cm. The liquid mixed spawn prepared with rice bran was inoculated in a sterile zone using a sterilized pipette of 15-20 ml per bottle. The inoculated bottle was incubated with mycelium at 21 ° C in a thermo-hygrostat, and after the incubation was finished, the bottle was scratched to stimulate it, followed by a so-called 'scratch' operation to supply water, and humidified by relative humidity of 90 Fruiting body growth was induced to be at least%.

After the test production, all of the remaining medium except for the mushroom was scraped and evenly crushed, and then the total carotenoid content was measured. As a result, 50 ppm was detected. A lot of contaminated medium is generated, and it is very difficult to repeatedly scrape and measure all the bottle media.

Prototype Manufacturing and Specification Experiment

After the mushroom production, all the bottle media were scraped and crushed, collected, and then refrigerated to prevent contamination. After a certain amount was collected, after low-temperature drying for 50 hours at 36 ℃ using a drying apparatus, the dried product was finely ground using a grinder. Finely ground powder was mixed with the probiotic product at a ratio of 1: 1 to complete the prototype.

The prepared prototypes were prepared by laying hens and subjected to specification experiments to determine whether the egg color was increased compared to the control. In order to experiment with existing laying hens, the amount of prototype should be large, and there should be no pigment product in the feed, because it can not be confirmed, after obtaining the laying hen, mix 2% (w / w) of this product in general feed and feed it to chicken. It was. As a control group, general feed was used as a pay group. Both control and test groups received 150 g of feed per day per animal. After 15 days of the specification test, the color of egg yolk was measured by ROCHE color pen, and the test sphere showed grade 8 chromaticity higher than grade 6 of ordinary sphere. If the production of astaxanthin stabilizes with full-scale mushroom production in the future, it is expected to be an excellent product to replace synthetic products.

Claims (2)

It consists of sawdust 80% (w / w), rice bran 18% (w / w) and corn steep powder 2% (w / w), sterilizing after adjusting the water to 45% (w / w). A method for producing astaxanthin, characterized by inoculating a seed culture obtained by mixing and cultivating a mushroom culture medium consisting of an enoki mushroom, an oyster mushroom, a fungus mushroom, a fungus and a yeast Phaffia rhodozyma , respectively.


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