TWI521059B - A method for preparing mycelium fermentation broth with anti - UVA activity - Google Patents

Description

Method for preparing fermentation broth of mycelium with anti-UVA activity

The invention relates to a technical improvement of a preparation method for the anti-UVA of the fermentation liquid of the genus Lepista nuda . By controlling culture conditions, aeration volume and rotation speed, the anti-UVA active substance ergosulfide is secreted into the extracellular fermentation broth of syringa sinensis, without additional extraction, by increasing the content of ergot in the fermentation broth. Improve the anti-UVA ability of the fermentation liquid of the purple clove mushroom.

Blewit mushroom is a fungus (Eumycota), Basidiomycotina, Hymenomycetes, Homobasidiomycetes, Agaricales, Tricholomataceae, The genus fragrant mushroom, also known as naked mushroom, amethyst mushroom, also known as purple mushroom, mushroom body is bright purple, with a special aroma, mainly distributed in Europe, North America and Asia, Heilongjiang, Northeast China, the popularity of Europe and Songling and The name of the beef liver mushroom is considered to be the finest ingredient in France. Fresh clove mushrooms are not expensive in the European market, about NT$2,000 per kilogram. The clove mushroom is originally a rare edible mushroom. It is not only delicious and has nutritional value. It is rich in vitamin B1 and can prevent beriberi. According to the literature, its extract has an inhibition rate of 90% on mouse sarcoma 180. The inhibition rate is 100%. The clove mushroom is rich in protein, various amino acids, various vitamins and various trace elements in the human body. The ergot sulfur contained in it is a new type of natural antioxidant.

Ergothioneine (2-mercaptohistidine) Trimethylbetaine, EGT) was first discovered in the fungus Claviceps purpurea in 1909. It is found in some fungi, plants, cereals and animal tissues such as oatmeal (17mg/Kg), mushroom mites (100-1000mg/Kg), blood. (5-20mg/Kg), etc., is a common component of microbial cells, has anti-oxidation, lowering blood sugar and protecting brain nerve cells. Because ergot sulfur is stored in many foods, it is easily digested and absorbed. The human body can only be supplied by food and cannot be synthesized by itself, and it is mainly taken by edible mushrooms. After being digested and absorbed, ergot sulfur is stored in red blood cells, kidneys, semen, liver and brain. Human studies have found that ergot sulfur is not toxic.

Ergosine has been shown to play an important role in the prevention of oxidative stress in the human body. It has powerful antioxidant capacity and is superior to Q10 and Idebenone, which can directly scavenge reactive oxygen species (ROS). Protects cells from UV rays and activates antioxidant enzymes, inhibits superoxide enzymes, and prevents skin aging and wrinkles. Because the molecular weight is smaller than the commonly used antioxidant peptides on the market, it has better absorption and permeability. Therefore, ergot has been widely used in the maintenance of beauty products, cosmetics and health foods, in food health and medical care. The field is getting more and more attention, and it is very valuable for developing beauty and health food.

Prof. Xu Youzhang, Department of Medicinal Cosmetics, School of Pharmacy, China Medical University, conducted UVA irradiation experiments on HaCaT skin cells, and analyzed MTT assay, antioxidant marker HO-1 activity and antioxidant SOD activity. The results show that the addition of ergothione can increase the survival rate of HaCaT skin cells after UVA irradiation, and can effectively prevent UVA damage to skin cells.

At present, the Japanese lilac mushroom has been imported from France by the Agricultural Research Center of the Wufeng Agricultural Committee in 1994. After more than ten years of cross-breeding technology, it was transferred to the Tainan Tiger Mountain Mushroom Farm. Past research has shown that the extracts containing the anti-aging function in the lower leg of the mushroom contain a relatively high amount of ergothione, which is even better than the edible part of the mushroom; there are also reports that the mushroom is discarded. After the extract Contains ergot sulfur; and the study indicates that the fruit body of the lilac mushroom contains a large amount of ergothione, but there is little ergosulfur content produced by liquid fermentation of mycelium. It has been previously explored that the ergot sulfur content of the liquid culture of the lilac mushroom is up to 46.9 mg/Kg, and the extracellular is free of ergot sulfur. However, the fruiting body is expensive and difficult to cultivate, and the mycelium cultured in liquid state must be extracted to obtain intracellular ergot sulfur. Therefore, in order to overcome the shortcomings of the above conventional techniques, the inventor of the present invention improved the conventional technology for producing ergot sulfur, and developed a method for secreting the anti-UVA active ingredient ergothiocyanin in the clove mushroom into the extracellular fermentation broth. A fermentation broth of lilac mushroom having anti-UVA ability can be obtained without extraction.

The object of the present invention is to provide a method for preparing a fermentation broth of the genus Escherichia coli with anti-UVA activity, wherein the method comprises the following steps:

(1) inoculation of the mycelium of the spirulina mushroom on a plate medium, and culturing at a temperature of 20-30 ° C for 2-3 weeks; wherein the plate medium is a potato dextrin medium; wherein the strain of the genus Syringa (DSM) NO.8620) was purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany).

(2) inoculation of the mycelium of the sinensis mushroom in the step (1) for 2-3 weeks in a flask, and shaking culture for 7-10 days at 20-30 ° C, pH 4-6 and a rotation speed of 110-130 rpm; The shock culture system comprises 1-3% cereal or legume, 1-3% carbon source, 0.25-0.75% nitrogen source, 0.1-0.2% magnesium sulfate, 0.1-0.2% potassium dihydrogen phosphate and 0.1-0.2% amine. Medium culture medium.

(3) inoculation of the mycelium of the syringa mushroom in the step (2) for 7-10 days in the fermentation tank, and in the hair The fermentation tank is cultured for 14-21 days at a aeration rate of 0.1-1.5 VVM at 15-30 ° C, a groove pressure of 0.5-1.0 kg/cm 2 , a pH of 4-6 and a stirring speed of 10-100 rpm; The medium inside was the same as the medium used in the flask in the step (2).

(4) separating the mycelium of the geranium mushroom cultured in the fermentation tank of the step (3) for 14-21 days by a centrifugal method, thereby obtaining a culture liquid containing the active substance ergothione; It is an object of the invention to provide an anti-UVA composition, wherein the anti-UVA composition comprises an active substance ergot sulfur culture solution prepared by the above method, and a pharmaceutically acceptable carrier, excipient, diluent, auxiliary The anti-UVA composition is administered in the form of an emulsion, a lotion, an essence, a sunscreen lotion, a barrier cream, and a mask.

Another object of the present invention is to provide a method for preparing an anti-UVA composition, wherein the method comprises the active substance ergothione culture solution prepared by the above method, and a pharmaceutically acceptable carrier, excipient, and dilution. The anti-UVA composition is prepared by using an agent, an adjuvant, or the like; wherein the anti-UVA composition is administered in the form of an emulsion, a lotion, an essence, a sunscreen lotion, a barrier cream, and a mask.

The method for culturing a secretory anti-UVA active ingredient ergot sulfur to the extracellular fermentation liquid by the liquid fermentation of the mycelium of the genus Escherichia coli can improve the anti-UVA ability of the gerbera mushroom fermentation liquid. The culture condition can be controlled by high aeration and low rotation speed to secrete ergot sulfur to the extracellular, and the material with anti-UVA activity can be obtained from the mycelium of the genus Spirulina without extracting, and the production is anti-UVA. The method of active substance has the advantage of reducing production costs.

The first picture shows the liquid culture of the mycelium of the genus Syringa sinensis using a 200 liter fermentation tank (rotation speed 10 rpm) Gas 0.375 VVM, culture volume 160L, inoculum 1L), yield comparison of intracellular and extracellular ergothione (EGT, mg/kg).

Figure 2 is a comparison of the yield of intracellular and extracellular ergothione by the liquid culture of the mycelium of the genus Erythrobacter sinensis using a 200 liter fermentation tank (rotation 60 rpm 0.375 VVM, culture volume 160 L, inoculum 1 L). , mg/kg).

Figure 3 is a comparison of the yield of intracellular and extracellular ergothione by the liquid culture of the mycelium of the genus Syringa velutipes using a 200 liter fermentation tank (rotation speed of 10 rpm 0.375 VVM, culture volume 160 L, inoculum size 2 L). , mg/kg).

Figure 4 shows the intracellular and extracellular ergothiones obtained by liquid culture of the mycelium of Syringa sinensis in different culture volumes using a 500 ml triangular shake flask (25 ° C, pH 4-6, rotation speed 120 rpm, culture volume 200 mL). Yield comparison chart.

Fig. 5 is a comparison of the yields of intracellular and extracellular ergothiones obtained by liquid culture of mycelium of different culture volumes (25 ° C, pH 4-6, rotation speed 120 rpm) using a 500 ml triangular shake flask.

Figure 6 is a structural diagram of Ergothioneine.

Example 1. Mycelium liquid culture with mycelium of Syringa velutipes

(1) Plate culture: The mycelium is inoculated on a plate. The medium formula is as shown in Table 1. It contains potato dextrose Agar (PDA), carbon source and nitrogen source, and is cultured at 25 ° C for 2-3. Zhou; the strain of S. sinensis (DSM NO.8620) was purchased from the German DSMZ company biological resources Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH) (Braunschweig, Germany). .

(2) Shake flask culture: The hyphae on the plate were scraped and inoculated into a shake flask, and shake cultured at 25 ° C, pH 4-6, and a rotation speed of 110-130 rpm for about 7-10 days.

(3) Fermentation tank culture: the above flask culture is inoculated into a fermentation tank at a temperature of 15-30 ° C, a tank pressure of 0.5-1.0 kg/cm 2 , a pH of about 4-6, and a stirring speed of 10-100 rpm. 0.1-1.5 VVM (air volume/culture volume/min) aeration rate was introduced into the air, cultured for about 14-21 days, and the mycelium of the mushroom was separated from the culture solution by centrifugation, and the hydatid hyphae were obtained in the culture solution. Body anti-UVA active substance ergothione.

Analytical method: The cultured culture was centrifuged to separate the hyphae and the filtrate. The filtrate was added to an equal volume of acetonitrile (Acetonitrile, ACN). After the polysaccharide and protein were precipitated, the precipitate was precipitated by centrifugation, the supernatant was taken, and the supernatant was filtered through a 0.45 μm microporous membrane to obtain high performance. Analysis by liquid chromatography. The hyphae ball is washed with water, the filtrate on the residual hyphae ball is washed away, and heated by extraction with distilled water (100 ° C, 15 min). After cooling, the mycelium is separated by centrifugation. The extract is taken and an equal volume of acetonitrile is added to the extract. After the polysaccharide and the protein are precipitated, the precipitate is precipitated by centrifugation, the supernatant is taken, and the supernatant is filtered through a 0.45 μm microporous membrane filter, and the filtrate is efficiently charged. Can be analyzed by liquid chromatography.

Analytical conditions: Instrument used: HITACHI HPLC, chromatography column: COSMOSIL HILIC, 4.6 x 250 mm, detector: UV lamp, detection wavelength: 254 nm, mobile phase: A: 10 mM aqueous ammonia solution, B: acetonitrile, flow rate: 1 mL/min, injection volume: 10 μL.

The chromatographic time is shown in Table 2:

Experimental results:

In a 200-liter fermentation tank, the culture volume was 160L, and the culture conditions were as follows: the inoculum volume was 1L and the cultured for 21 days, the wet weight of the mycelium was 36.7 kg, and the inoculum volume was 2L, culture 14 The wet weight of the mycelium was 39 kg; the fermentation was carried out at a speed of 60 rpm and 0.375 VVM. The wet weight of the mycelium was 8.4 kg after 21 days of culture. As shown in Table 3, increasing the inoculum size by aeration of 0.375 VVM at 10 rpm can shorten the culture time, and the wet weight of the mycelium after cultivation is 39 kg; as shown in Table 4, the mycelium fermentation culture is carried out by aeration at a high speed of 60 rpm and 0.375 VVM. Poor growth, dry weight of hyphae and extracellular ergot sulphur due to low yield. It can be seen from Table 3 and Table 4 that proper control of the rotational speed can increase the difference between the intracellular and extracellular ergot sulfur production, and no matter what. According to the culture conditions, if the inoculum size is increased as shown in Table 5, the dry weight of hyphae and the extracellular ergot sulfur yield increase with time.

The same volume of 200 ml of different medium was prepared in a 500 ml triangular shake flask, and the culture days were increased to 10-20 days under the conditions of the above shake flask culture, and the extracellular ergot sulfur content was as shown in Table 6, regardless of the medium formula. The extracellular ergot sulphur production is higher than intracellular.

The same medium with different volumes of 50 ml, 100 ml, 200 ml and 400 ml was prepared in a 500 ml triangular shake flask and cultured for 14 days under the conditions of the shake flask culture. The content of ergot sulfur was controlled as shown in Table 7. Under the condition of ventilation and reducing the shear force caused by shock, the extracellular ergot sulfur production is higher than intracellular.

The above-mentioned embodiments and the drawings are only the preferred embodiments of the present invention, and the scope of the present invention is not limited thereto, that is, the equivalent variations and modifications of the scope of the present invention should be covered by the present invention. Within the scope.

In summary, the present invention provides a preparation of anti-UVA active lilac mushroom The method for the mycelium fermentation broth and the anti-UVA composition are innovative, and the preparation method is simpler than the conventional one, and can improve the content of the ergo sulphur-resistant substance of the anti-UVA active substance in the fermentation broth, and should fully meet the novelty and the progressiveness. The statutory invention patent requirements, 提出 apply in accordance with the law, and ask your bureau to approve the invention patent application, in order to invent invention.

Claims (8)

A method for preparing a fermentation broth of the genus Escherichia coli with anti-UVA activity, wherein the method comprises the following steps: (1) inoculating the mycelium of the genus Syringa sinensis on a plate medium and culturing for 2-3 weeks; (2) Inoculation of the mycelium of the syringa edulis cultured in the step (1) for 2-3 weeks in the flask, and shaking culture for 7-10 days; (3) cultivating the lilac for 7-10 days in the step (2) Mushroom mycelium is inoculated into the fermentation tank and cultured in the fermentation tank for 14-21 days. The culture conditions in the fermentation tank are at 15-30 ° C, the tank pressure is 0.5-1.0 kg / cm ^ 2, pH 4-6 and stirring. At a speed of 10-100 rpm, culture at aeration rate of 0.1-1.5 VVM; (4) separate the mycelium of the geranium mushroom cultured in the fermentation tank of step (3) for 14-21 days by centrifugation, ie A fermentation broth containing an anti-UVA active substance; the anti-UVA active substance is ergothione. The method of claim 1, wherein the plate medium in the step (1) is a potato dextrin medium. The method of claim 1, wherein the culturing in the step (1) means culturing at a temperature of 20 to 30 °C. The method of claim 1, wherein the oscillating culture in the step (2) is oscillated at 20-30 ° C, pH 4-6, and a rotational speed of 110-130 rpm. The method of claim 4, wherein the oscillating culture in the step (2) comprises 1-3% cereal or legume, 1-3% carbon source, 0.25-0.75% nitrogen source, 0.1-0.2% Magnesium sulfate, 0.1-0.2% The medium was cultured with potassium dihydrogen phosphate and 0.1-0.2% amino acid. The method of claim 1, wherein the fermentation tank in the step (3) is cultured in a 1-3% cereal or legume, a 1-3% carbon source, a 0.25-0.75% nitrogen source, and a 0.1-0.2 Medium of magnesium sulfate, 0.1-0.2% potassium dihydrogen phosphate and 0.1-0.2% amino acid. A method for preparing an anti-UVA composition, wherein the method comprises the ergot sulfur fermentation broth containing the anti-UVA active substance prepared by the method of claim 1 and a pharmaceutically acceptable carrier, an excipient, An anti-UVA composition is prepared from a diluent, an adjuvant, and the like. The method of claim 9, wherein the anti-UVA composition is administered in the form of an emulsion, a lotion, an essence, a sunscreen lotion, a barrier cream, and a mask.