KR20030018959A - Red pigment by submerged culture of paecilomyces sinclairii and method for preparing the same - Google Patents

Abstract

PURPOSE: Provided are red pigment by submerged culture of Paecilomyces sinclairii and a method for preparing the same. The manufactured red pigment has excellent stability and is thus applied to foods, cosmetics, medicaments, etc. CONSTITUTION: The red pigment is manufactured by submerged culture of Paecilomyces sinclairii characteristically under the following conditions: inoculum age is 3 days; culture temperature is 25deg. C; initial pH is 6.0; carbon source is 1.5%(w/v) of soluble starch; and nitrogen source is 1.5%(w/v) of meat peptone.

Description

Red Pigment by Submerged Culture of Paecilomyces Sinclairii and Method for Preparing the Same}

The present invention relates to a red pigment produced from Cordyceps sinensis and a method for producing the same. More specifically, the present invention is a condition of three days of inoculum age, temperature 25 ℃, initial pH 6.0, 1.5% (w / v) soluble starch as a carbon source, 1.5% (w / v) meat peptone as a nitrogen source It relates to a red pigment produced from Cordyceps sinensis cultured with and a method for producing the same.

Because of the stability of many kinds of artificial synthetic pigments widely used in food, cosmetics, and pharmaceuticals, methods for producing pigments from natural products have been studied. It is already known that various kinds of plants, animals and microorganisms produce pigments. There are many types of natural pigments, but only a few of them are sufficient for industrial use because they are extracted from plants. Therefore, it is beneficial to produce natural pigments from microorganisms. There are several kinds of microorganisms that can produce pigments in high yields such as Monascus, Serratia and Streptomyces. Among these, many species of Monascus are attracting much attention because they have the ability to produce various pigments exhibiting high chemical stability. Red pigments, produced in solid-state cultures of several varieties of Monascus, have traditionally been used in many Asian countries to color and store many fermented foods. Moreover, the therapeutic properties and relatively high stability in relation to pH and temperature are properties of interest that are preferred as substitutes for synthetic colorants. Monascus production by solid state cultures was submerged in complex mediators. There is a need for research on possible industrial applications for microbial pigments, high yield pigment production and chemical optical stability. During the immersion culture of several kinds of edible mushrooms for the production of bioactive compounds, the inventors have found that several kinds of edible mushrooms have the ability to produce relatively high yields of pigments. In particular, according to the inventor's prior investigation, the red pigment production from Cordyceps sinensis was extremely stable in natural light (more than 30 days), which is one of the disadvantages of natural pigment.

There are a number of reports that pigment production in immersion culture is affected by several environmental factors, in particular the nature of the nitrogen source and mediator pH. There are no reports of pigments in other studies of the same species, particularly in medical mushroom research.

In the present invention, the appropriate culture conditions for the production of red pigment by Cordyceps sinensis were investigated in a vibration flask and batch fermenter.

Accordingly, it is an object of the present invention to provide a method capable of producing pigments from natural products. Another object of the present invention to provide a suitable culture conditions for the production of red pigment by Cordyceps sinensis.

The object of the present invention is 3 days inoculum age, temperature 25 ℃, initial pH 6.0, 1.5% (w / v) soluble starch as carbon source, 1.5% (w / v) meat peptone as nitrogen source It was achieved by providing a red pigment produced from the Cordyceps sinensis cultured and its manufacturing method.

Hereinafter, the configuration and operation of the present invention.

The present invention relates to a red pigment produced from Cordyceps sinensis (Cordyceps sinensis) and a method of manufacturing the same.

Using the immersion culture of edible mushrooms, high pigment-producing fungi Paecilomyces (P. sinclairii) was selected and its proper culture conditions were investigated. Proper culture conditions are 3 days of inoculum age, temperature 25 ° C., initial pH 6.0, carbon source 1.5% (w / v) soluble starch, nitrogen source 1.5% (w / v) meat peptone. Although addition of 10 mM CaCl _{2 to} the culture medium slightly increases pigmentation, most of the bio-components examined have no decisive effect on pigmentation. Under the titration conditions obtained in the flask incubator, nine-fold pigmentation (4.4 g / l) was achieved using a 5 L batch fermenter. Cordyceps sinensis secreted water-soluble red pigment into the culture medium. The pigment color is strongly dependent on the pH of the solution, such as pH 3-4 red, pH 5-9 purple, pH 10-12 pink.

Hereinafter, the specific method of the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1: Microorganisms and Culture Media

Cordyceps sinensis was collected from mountainous areas in Korea and collected in a laboratory. Accumulated cultures were stored in potato dextrose agar (PDA). The slants were inoculated, incubated at 25 ° C. for 7 days and incubated at 4 ° C. Inoculation culture medium is yeast malt extract (YME) containing 3g yeast extract, 3g malt extract, 5g peptone, 10g glucose in 1L of distilled water. The basic medium is mushroom culture medium containing 20 g glucose, 2 g yeast extract, 2 g peptone, 0.5 g MgSO ₄ · 7H ₂ O, 0.46 g KH ₂ PO ₄ , 1 g K ₂ HPO ₄ in 1 L of distilled water. MCM).

Example 2: Culture Conditions

Cordyceps sinensis was grown in PDA medium in petridish, cut agar plate culture into 5 mm with a sterile cutter and transferred to seed culture medium. Seed cultures were grown in 250 ml flasks containing 50 ml of spawn culture medium at 25 ° C. in a rotary shaker at 150 rpm for 2 days. Fermentation was carried out at 250 rpm in a rotary shaker at 50 rpm for 3 days in a 250 ml flask containing 50 ml of basic medium, and at temperature 25 ℃ for 2 days, aeration rate 2vvm, stirring speed 150 rpm, initial pH 6.0 conditions. After inoculation with% (v / v) spawn culture was performed in a 5 L fermenter containing 3 L of culture medium. All experiments were conducted at least twice for reliable reproducibility.

Example 3: Separation of Red Pigment

The red pigment was adjusted to pH 3.0 with 2N HCl and successfully extracted from the culture filtrate (1 L) with ethyl acetate (1 L), and the ethyl acetate extract was evaporated under vacuum at 50 ° C. The dried pigment was dissolved in distilled water (200 mL) and filtered through Whatman filter paper. The water-soluble pigment was dried at 50 ° C, dissolved in 200 ml of anhydrous ethanol, and filtered. The filtrate containing the red pigment was crystallized by adding 600 ml of chloroform / n-heptane (1: 2, v / v) and dried at 50 ° C.

Example 4: Analysis Method

Cultures were centrifuged at 10,000 x g for 20 minutes. The supernatant was filtered with Whatman filter paper (No. 2). Microreal biomass yield was measured by washing with deionized water and dried at 50 ° C. for 48 hours. The concentration of red pigment was measured by measuring the absorbance of the culture filtrate at 500 nm (Bio-Tek Kontron Uvikon Spectrophotometer, Italy). The residual sugar concentration was analyzed by liquid chromatography using an Aminex HPX-42C column (0.78 × 30 cm, Bio-rad) equipped with a refractive index detector (Shimadzu Co., Kyoto, Japan).

Example 5: Characteristic of Red Pigment

The absorbance of the dye was measured at 400, 470, 500 nm. These wavelengths represent the maximum absorption for the yellow, orange, and red pigments, respectively. The isolated pigments of the present invention exhibited unique light absorption characteristics at 500 nm indicating red pigments. Furthermore, the color of the separated pigment changed with the change of pH of the aqueous solution. The results are shown in Table 1.

pH Pigment color Absorption wavelength (nm) 3.0 to 4.0 Red 500,507 5.0 to 7.0 purple 522, 526, 529 8.0-9.0 Pale purple 536, 537 10.0 to 12.0 Pale pink 540, 541, 542

As the pH of the aqueous solution shifts to the alkaline region, the maximum absorption wavelength is alkaline solution, pale purple (537-540 nm); Neutral solution, purple (529-536 nm); Increased in association with color changes such as acid solution, red (500-507 nm). Similar observations were observed in pigments of anthocyanins, which are widely used as food colorants in that they are most vivid at pH 4-6 and almost colorless at high pH.

Example 6 Effect of Carbon and Nitrogen Sources on Red Pigmentation

To select an appropriate carbon source for red pigment production, Cordyceps sinensis was cultured in a basal medium containing several carbon sources (2%). Of the 11 carbon sources examined, glucose, fructose, manose, sucrose and maltose were relatively adequate for mycelial growth of Cordyceps sinensis. On the other hand, pigmentation was low. Maximum mycelial growth (3.9 g / l) was obtained in sucrose medium. On the other hand, maximum pigment production (467 mg / l) was obtained in aqueous starch medium. Table 2 shows the effect of carbon source on mycelial growth and pigment production in Cordyceps sinensis.

Carbon source (2%, w / v) Dry cell weight (g / ℓ) Pigment (mg / l) Final pH Glucose 3.5 334 4.12 Fructose 3.1 315 4.53 Galactose 2.2 239 5.02 Xylose 1.6 229 4.86 Arabinos 1.9 280 5.52 Manos 3.2 199 4.24 Lactose 1.8 298 5.51 Sucrose 3.9 250 4.09 Maltose 3.4 155 4.25 Water soluble starch 3.6 467 4.25 Glycerol 2.7 387 4.24

The stimulatory effects of nitrogen sources on pigmentation have been reported. In the present invention, the organic nitrogen source showed higher mycelial growth than the inorganic nitrogen source. Meat peptone, casein peptone, peptone-yeast extract mixture and corn steep powder had a positive effect on pigmentation, while soy peptone and malt extract strongly inhibited red pigment synthesis. Of all the nitrogen sources tested, meat peptone showed the highest yield in red pigment production. Table 3 shows the effect of carbon sources on mycelial growth and pigment production in Cordyceps sinensis.

Nitrogen source (0.4%, w / v) Dry cell weight (g / ℓ) Pigment (mg / l) Final pH Peptone-Yeast Extract (1: 1) 3.8 357 4.27 Meat peptone 2.8 428 4.30 Yeast extract 3.1 235 4.36 Malt Extract 1.8 14 4.55 Meat extract 1.6 244 4.15 Bakto peptone 1.5 157 4.28 Soy Peptone 2.0 20 4.58 Casein peptone 2.9 366 4.05 Polypeptone 2.8 190 4.38 Bacto trypton 3.3 295 4.24 Malton 3.7 273 4.37 Cone stew powder 3.4 342 4.20 (NH ₄ ) ₂ SO ₄ 1.4 nd * 3.36 NaNO ₃ 1.2 nd 6.09 KNO ₃ 1.2 nd 6.05 NH ₄ NO ₃ 1.4 nd 5.12

Different types of peptone supplied more pigment production in many types of pigment-producing fungi.

Example 7: Effect of C / N Ratio on Red Pigmentation

Although the C / N ratio affects the biosynthesis rate of many metabolites, the effect of mycelial growth and pigmentation on fungi is rarely explained. The effect of C / N ratio on pigmentation was investigated using a water soluble starch-meat peptone medium. The effect of the C / N ratio on mycelial growth and pigmentation in Cordyceps sinensis is shown in Table 4 below. As shown in Table 4, mycelial growth (6.2 g / l) and pigmentation (1881 mg / l) showed the maximum value at C / N ratio 1: 1. Increasing the C / N ratio higher or lower than 1: 1 resulted in a reduction of the red pigment. This result is compared with Nam and Lee (1991) 's report that the pink pigment carotenoid content decreases with increasing C / N ratio.

Meat Peptone (%, w / v) Water Soluble Starch (%, w / v) 0.5 One 1.5 2 2.5 0.5 (final pH) * 735 / 2.4 (4.13) 717 / 3.9 (4.2) 562 / 2.9 (4.44) 632 / 2.7 (4.35) 667 / 3.3 (4.31) One 1209 / 5.3 (4.59) 1256 / 4.0 (4.37) 1214 / 4.5 (4.63) 1107 / 3.3 (4.45) 923 / 3.8 (4.48) 1.5 1188 / 2.4 (4.69) 1819 / 4.2 (4.40) 1881 / 6.2 (4.42) 1247 / 2.3 (4.64) 1394 / 2.4 (4.55) 2 425 / 1.7 (5.42) 464 / 2.3 (5.55) 827 / 1.8 (5.15) 227 / 0.8 (5.27) 387 / 2.6 (5.51) 2.5 223 / 0.4 (5.33) 233 / 0.2 (5.32) 250 / 0.9 (5.32) 249 / 0.3 (5.27) 250/1 (5.17)

Example 8 Effect of Bio-Components on Red Pigmentation

Bio-components are one of the important factors affecting pigmentation in many microorganisms. Furthermore, an important finding has been reported that there is an interaction between pigment and iron ions. To investigate the effect of bio-components on mycelial growth and pigmentation, Cordyceps sinensis was cultured in the optimized culture medium. Each tracer was added to culture the medium at a concentration value of 1 mM. Maximal pigmentation (1528 mg / l) was achieved when supplemented with calcium chloride. The optimum concentration of calcium chloride was 10 mM. Other bio-components have been shown to have no decisive effect on both mycelial growth and pigmentation. The effects of bio-components on mycelial growth and pigmentation in Cordyceps sinensis are shown in Table 5 below. Bau and Wong (1979) reported the critical effect of zinc ions on Monascus pigmentation. The negative effects of iron and cobalt ions on red pigment production can be explained by the indirect contribution of metabolites to energy production in cells. An et al. (2001) observed that iron produced estaxanthin and blood. The composition in P. rhodozyma was reduced.

Iron ion (1 mM) Dry cell weight (g / ℓ) Pigment (mg / ℓ) Final pH - 2.2 1498 4.45 Ca ²⁺ 3.9 1528 4.42 Mg ²⁺ 2.0 1378 4.49 Fe ²⁺ 0.02 279 5.05 Cu ²⁺ 1.4 806 5.36 Zn ²⁺ 2.8 312 4.73 Mn ²⁺ 2.4 806 4.41 Co ²⁺ 1.0 188 5.39 K ⁺ 2.4 1287 4.53 Na ⁺ 3.0 1483 4.49

Example 9: Effect of pH and Temperature on Red Pigmentation

To investigate the effect of pH on mycelial growth and pigmentation, Cordyceps sinensis was incubated at different initial pH 3.0-10.0 in a flask incubator. Titration initial pH for both mycelial growth and pigmentation was observed at 6.0. Different types of fungi have an optimal pH during immersion culture for pigmentation. To find the optimal temperature for mycelial growth and pigmentation, Cordyceps sinensis was incubated under various temperatures (15-35 ° C). As a result, the optimum temperature for both mycelial growth and pigmentation was 25 ° C. Because mycelia always require long periods for immersion cultures that are exposed to the risk of infection, the optimal temperature is considered an appropriate physiological characteristic of Cordyceps sinensis.

Example 10: Influence of inoculum age on red pigment production

Among the fungi's physiological characteristics, inacuum age plays an important role in fungal development. To investigate the effect of incubation age on pigmentation, Cordyceps sinensis was incubated with optimal incubation medium using another incubation age at 25 ° C. for 2-6 days in a flask incubator. Proper incubation age for pigmentation was three days, and increasing incubation age decreased mycelial growth. Similar results were observed in the submerged culture of the Pesylomyces japonica for our biopolymer production.

Example 11: Red Pigmentation in a Batch Bioreactor

Red pigment was produced through fermentation and reached a concentration of 4.4 g / l on day 7, while mycelial growth reached a maximum of 17.2 g / l. Microbial growth increased rapidly during the first six days at the depletion of sugar concentration and slowly reached plateau. As the fermentation proceeds, the initial pH of the fermentation broth slowly drops from 6 to 4 and then returns to pH 6. Residual sugar concentrations were drastically reduced by five days, and the initial carbon source was almost consumed. Improved production of the pigment appears to be possible by overcoming the depletion of the carbon source using a fed-batch culture mode. Observations show that pigmentation in Cordyceps is related to growth.

As described above, the present invention provides a method for producing natural pigments from microorganisms that can be used in food, cosmetics, and pharmaceuticals, and thus is a very useful invention for the dye industry, which drastically improves the stability at issue in artificial synthetic pigments. will be.

Claims (3)

Incubation age 3 days, temperature 25 ℃, initial pH 6.0, soluble starch 1.5% (w / v) as a carbon source, meat peptone 1.5% (w / v) as a nitrogen source characterized in that the culture Cordyceps sinensis culture method. The mixture was adjusted to pH 3.0 with 2N HCl, extracted from the culture filtrate (1 L) with ethyl acetate (1 L), and the ethyl acetate extract was evaporated under vacuum at 50 ° C., and the dried pigment was dissolved in distilled water (200 mL). Filter by Whatman filter paper, the water-soluble pigment is dried at 50 ℃, dissolved in 200 ml of anhydrous ethanol and filtered, the filtrate containing a red pigment is chloroform / n-heptane (1: 2, v / v) 600 Red pigment production method produced from Cordyceps sinensis, characterized in that the crystallization by adding ㎖ and dried at 50 ℃. Red pigment, which is prepared by the method of claim 2.