TWI550084B - Liquid culture method for improving the content of ergot sulfate in mycelia of - Google Patents

Description

Liquid culture method for increasing ergot sulfur content of mycelium of coral mushroom

The present invention relates to a liquid culture method for increasing the content of ergot sulfur in mycelium of coral mushroom. More particularly, the present invention relates to the use of a reaction surface method to determine the optimum concentration and ratio of amino acid precursors to produce a high ergot sulfur content of the coral mushroom mycelium, and to be used for optimal liquid culture conditions. A culture solution containing a high ergothione content is prepared.

Ergosine is a low molecular weight thiol compound isolated by Tanret in 1909 when it studied Claviceps purpurea . The ergot sulfur is present in plant and animal tissues because of the metabolites of fungi, but the human body Vertebrates cannot synthesize ergot sulfur, so they can only be absorbed by ingesting food. Genghof ( Journal of Bacteriology. 103 (2): 475-478, 1970) found that ergothione and its precursor, hiscynine, can be produced in fungi and actinomycetes. Physarum polycephalum also has the ability to synthesize ergot sulfur. Askari ( Journal of Biological Chemistry. 237 :1615-1618, 1962) pointed out that ergot can be synthesized by fungi and synthesized by amino acids: histidine, methionine and cysteine. (cysteine). Ergosine is the only amino acid that can occur 2-thio-imidazole, which exists in the natural form as a thioketone and a thiol type, and in an aqueous solution with a thioketone type (Franzoni et al, Biomedicine & Pharmacotherapy. 60 :453 -457, 2006).

Huang Lingyi et al. used deep culture to increase the ergot sulfur content of Pleurotus eryngii mycelium at a temperature of 25 ° C, 10% inoculation, 2% glucose as carbon source, and 0.5% yeast extract as nitrogen source. The flask was cultured at 125 rpm, and 4 mM histidine was added after 7 days of culture, and the obtained mycelium ergothione content was 5.05 mg/g dw, so the medium composition was adjusted or the amino acid precursor was added. Increasing the content of ergot sulfur in mushroom mycelium (Mao Zhenglun, Liang Zhihong, Huang Lingyi. 2013. Republic of China invention patent No. I-408226).

Pleurotus citrinopileatus Sing. is classified into Basidiomycetes, Agaricales, Pleurotaceae, and Pleurotus. Its fruit body is named after coral, and it is also known as the golden mushroom, the yellow mushroom and the jade mushroom in mainland China. In Taiwan, it is named after the golden ear, golden mushroom, corn mushroom or jade mushroom (Huang, 1998). ). Coral mushroom tastes delicious, sweet and delicious, rich in protein, amino acid and vitamins. It is one of the delicious edible fungi, and it is popular because of its golden color, beautiful appearance. In addition to its edible properties, according to the Chinese large-scale fungal primary color map (1998) compiled by Huang Nianlai, director of the Sanming Fungal Research Institute of Fujian Province, coral mushrooms have the effect of being used as medicine and nourishing and strengthening, and have kidney deficiency, impotence and dysentery. Certain help. According to "Chinese Materia Medica", the coral mushroom is sweet and tonic, and it is nourishing and strong. Main weakness, emphysema, dysentery.

The nutrient requirements of artificially cultivated coral mushrooms are generally provided by using wood chips, cottonseed hulls, bagasse and waste cotton as raw materials. Below pH 4, it is difficult to produce mushrooms above 8 or higher, and the optimum pH for mushrooming is 6.0 to 6.5. Coral mushroom is an edible fungus with strong ability to decompose cellulose and lignin. It needs abundant carbon and nitrogen sources when planting. Especially when the nitrogen source is rich, the mycelium grows fast and the fruit body yield is high (Huang Shizheng, Yang Meichun) Chen Jinchu. Jinding Pleurotus. Country Road. 11:45, 1999).

In recent years, it has been common to culture mycelium by deep fermentation at home and abroad. This method can culture mycelium in a large amount, and the time required is very short, and has several advantages: 1. The mycelium grows fast and the growth cycle is short: the liquid culture can control the ventilation, temperature, pH and other equipment. The optimal culture conditions proliferate a large amount of mycelium and physiologically active metabolites in a short time. 2. It can be factory-produced and has no seasonality: the edible fungus is liquid cultured in the fermentation equipment, and is not subject to seasonal restrictions. Using liquid fermentation culture, although the cultivation time of edible fungi can be shortened, how to control the production of mycelium, how to effectively control the production of useful secondary metabolites (such as anti-tumor substances or hypoglycemic substances), how to Separation of effective ingredients, as well as product differences in active ingredients, technology and quality control are issues that must be overcome, in order to improve the quality of edible mushroom products and contribute to human health (Wang Peiming. Food Industry. 34:32- 35, 2002; Cai Shuyao, Ph.D., Department of Food and Applied Biotechnology, National Chung Hsing University, 2006).

The medium for deep culture is the basic food for microbial growth and the source of reactants for anabolic products. Appropriate selection of nutrient source components and ratios can make full use of microorganisms to produce the desired metabolites. The source may make the fermentation result in unit time unable to meet the economic benefits, which will make the subsequent separation and purification difficult and increase the operation cost. Therefore, it is very important to efficiently select the ratio of nutrients to the medium. In this study, the growth conditions of mycelium liquid culture of Pleurotus ostreatus were firstly studied in a single factor method, and the relationship between the dry weight of mycelium, the content of ergot sulfur, the residual sugar of fermentation broth and the pH value were analyzed during the cultivation.

Based on the above object, the present invention finds out the growth conditions of the liquid culture of the mycelium of the coral mushroom by using the one-factor and the reaction surface method, and finds the optimal concentration ratio of the three amino acid precursors added with ergothione. Coral mushroom mycelium with high ergot sulphur content can be obtained.

Accordingly, one aspect of the present invention relates to a liquid culture method for increasing the content of ergot sulfur in mycelium of coral mushroom.

In some embodiments of the present invention, the method for preparing a high content of ergot sulfur mycelium mycelium comprises: vibrating the mycelium of the coral mushroom at 25 ° C for 7 days for activation; and culturing the mycelium The liquid was inoculated with 5% inoculum, and the culture was carried out at a temperature of 20-25 ° C at a temperature of 20-25 ° C at an initial pH of 10.0 or a medium containing no initial pH of the medium and containing 2% carbon source and 0.5% nitrogen source. Day, and on the 7th day of culture, add a comprehensive amino acid solution (containing 8 mM cysteine, 4 mM histidine and 0.5 mM methionine); and a high content of ergot sulfur mycelium mycelium .

In one embodiment of the invention, the carbon source is glucose. In another embodiment of the invention, the carbon source is tryptone.

In a specific embodiment of the invention, the nitrogen source is a yeast extract. In another embodiment of the invention, the nitrogen source is tryptone.

In a specific embodiment of the present invention, the method for preparing a high content of ergot sulfur mycelium mycelium further comprises the step of freeze-drying the collected mycelium.

Another aspect of the invention relates to a high content of ergoporphyrin mycelium mycelium produced by the liquid culture method of the present invention.

In some embodiments of the present invention, the high content of ergot sulfur is contained in the mycelium of the coral mushroom, and the ergot sulfur content is 5.0 mg/g dw or more. In other specific embodiments, the high content of ergot sulfur due to the mycelium content of the coral mushroom mycelium is 10-20 mg/g dw.

Still another aspect of the present invention relates to a food additive comprising a high content of ergothione-derived mycelium mycelium produced by the liquid culture method of the present invention.

The effect of the invention: the original ergot sulfur content of the strain used in the invention is only 1.81 mg/g dw (control group), and after adjusting the initial pH value of 10, the ergot sulfur content is 10.65. Mg/g dw was significantly increased by 5.88 times compared with the control group. Furthermore, in the present invention, a comprehensive amino acid solution was added on the 7th day of culture, and the ergot sulfur content was up to 14.57 mg/g dw at the 22nd day, which was 8.05 times higher than that of the control group. The mycelium content of the mycelium mycelium obtained by adding the integrated amino acid solution on the 10th day of culture showed that the content of ergothione in the mycelium (2.54 mg/g dw) had reached a mycelial culture similar to that without the addition of the precursor. The ergothione content of 22 days (2.87 mg/g dw) showed that the present invention specifically shortened the culture time.

Figure 1 shows the effect of adjusting the pH value to 10 after different days of liquid fermentation of coral mushroom, the dry weight of mycelium, the final pH value (A) and the content of ergot sulfur (B).

Figure 2 shows the dry weight (A) and wheat of mycelium mycelium fermented for 22 days during the liquid fermentation of coral mushroom by adding amino acid (cysteine, histidine and methionine) at a concentration of 4 mM. The effect of the sulphur content (B).

3 is fixed histidine (X ₂₎ concentration is 4.00mM, homocysteine (X ₁₎ and methionine _(X-3) of the submerged culture mycelium coral ergothioneine Quantity (mg / g dw) contour map (A) and surface map (B).

Fig. 4 is a graph showing changes in the dry weight of the mycelium, the amount of residual sugar, the final pH (A), and the ergosulfur content (B) of the liquid fermentation of the coral mushroom with Tryptone as a nitrogen source.

Fig. 5 is a graph showing the changes of dry weight, residual sugar amount, final pH value (A) and ergothione content (B) of coral mushroom mycelium in Figure 6-4 and the optimum conditions for adding RSM on the 7th day of culture. .

Figure 6 shows the antioxidant power of 70% ethanol extract of coral mushroom fruit bodies and mycelia.

Figure 7 shows the reducing power of the 70% ethanol extract of the coral mushroom fruiting body and mycelium.

The other features and advantages of the present invention are further exemplified and illustrated in the following examples, which are intended to be illustrative only and not to limit the scope of the invention.

The mycelium of Pleurotus citrinopileatus used in this study was subcultured from the coral mushroom fruit body purchased by Changcun County Dicun Biotechnology Co., Ltd. The coral mushroom strains were stored in PDA (potato dextrose agar) slant medium and stored in a refrigerator at 4 ° C. A small piece of the sterilized sterilized knife was used to cut into a PDA plate medium and placed in a 25 ° C incubator for activation. When inoculation, the strain is activated on the plate medium, and the inoculated knife which has been sterilized is also used to cut the hyphae with strong peripheral activity as the strain, the size is 5mm x 5mm, and the PDB (potato dextrose broth) liquid is connected. The culture medium was placed in a 25 ° C incubator for static growth, and the mycelial growth pattern was observed. The average colony diameter was 70-80 mm for 12 days, and the hyphae showed white color. The hyphae were collected and lyophilized.

Study on culture conditions of mycelium of coral oyster mushroom containing high ergot sulfur by one-factor method

In this embodiment, the effects of various culture conditions on the formation of ergot sulfur in the mycelium of Pleurotus ostreatus were studied by means of deep culture, and the dry weight of the mycelium, the content of ergot sulfur, and the residue of the fermentation broth were analyzed during the cultivation. The relationship between the amount of sugar and the change in pH. Mycelium of coral mushroom was preserved in PDA (potato dextrose agar) medium and stored at 4 °C. At the time of inoculation, the culture dish which was activated was taken, and the hyphae having strong peripheral activity was cut out as a strain by the inoculation knife after sterilization, and the size was about 5 mm×5 mm, and cultured and activated at 25 °C. In the aseptic table, the bacteria stored in the PDA were cut into 5 pieces of 5 mm × 5 mm size bacteria by a sterilizing scalpel, and the bacteria were inoculated into 100 mL of liquid medium (containing 20 g of glucose, 5 g of yeast extract, 2 g per liter). 250 mL grooved triangular cone bottle of (NH ₄ ) ₂ SO ₄ , 0.5 g KH ₂ PO ₄ , 0.5 g K ₂ HPO ₄ and 0.5 g MgSO ₄ .7 H ₂ O), shake culture at 125 rpm, 25 ° C 7 days as an inoculum. The obtained mycelium and medium were pulverized with a Waring blender for 15 seconds to obtain a mycelium homogenate, and then 5 mL of the homogenate was placed in a 250 mL triangular cone bottle containing 95 mL of liquid medium as an inoculum. In this experiment, the mycelium cultured in the shake flask shaking medium was circulated as an inoculum.

In this experiment, two kinds of mediums were used, which were basic medium and mineral medium. The composition of the two mediums is shown in Table 1 below. 95 mL of the basic medium and mineral medium were respectively placed in a 250 mL triangular cone type bottle, and after sterilization through a sterilization pot, 5 mL of the prepared inoculum (5%) was added, and shaking culture was carried out at 125 rpm at 25 °C. Analyze the dry weight of mycelium and the content of ergot sulfur, the pH value of the fermentation broth and the amount of residual sugar every 2 days during the culture period. The growth curve of the liquid fermentation of the basic medium and the mineral medium was used to select the suitable medium as the subsequent experimental medium, and further explored the optimum temperature (experimental temperature of 20, 25 and 30oC) and the inoculum amount (1, 5, 10). , 15 and 20% vaccination), initial pH (experimental group adjusted the pH of the medium to 2.0, 4.0, 8.0, 10.0, 12.0 and 14.0 with 0.1N HCl or 0.1N NaOH, while the control group did not adjust the pH value) Carbon sources (including: fructose, lactose, maltose, sucrose, and glucose (as control groups) at 2%), and nitrogen sources (including: corn extract, malt extract, peptone, tryptone, and yeast extract) (as a control group), the concentration of 0.5%) and other culture factors, in order to find the optimum medium composition and the best growth and harvest days for the liquid fermented coral mushroom to produce ergot sulfur, and carry out various tests in triple overlap.

^a composition and concentration per liter: 2.3 g MgSO ₄ . 7H ₂ O, 0.1g FeSO ₄ . 7H ₂ O, 0.023 g MnSO ₄ . H ₂ O, 0.06 g ZnSO ₄ . 7H ₂ O, 0.08g CaCl ₂ . 2H ₂ O and 0.03g CuSO ₄ . 5H ₂ O

The total results of the experiments were carried out in a one-factor, one-factor manner using different medium components and culture conditions. In the 5% inoculation amount, glucose was used as the carbon source and the yeast extract was used as the nitrogen source, and the pH value was adjusted to 10, The highest amount of mycelium (8.28 g/L) and ergothione (10.65 mg/g dw) were obtained by culturing at a temperature of 25 ° C for 22 days. In the early stage of culture, the basic medium and In the two culture media, the dry weight of mycelium of coral mushroom increased with the increase of culture time. When using the basic medium, the dry weight of mycelium reached the highest at 8.70g/L on the 14th day; In the case of the medium, the dry weight of the mycelium reached a maximum of 11.22 g/L on the sixth day.

Optimum culture conditions of mycelium of coral oyster mushroom containing high ergot sulfur by two-stage stimulation

Next, further explore the timing of adjusting the pH value of the liquid medium and adding the precursor, and observe the effects of adjusting the pH value or adding the precursor at different time points on the dry weight of the mycelium and the content of ergot sulfur. The experiment was designed to adjust the pH of the culture medium to 10 in 0.1N NaOH on the 0th, 7th and 14th day of the culture period, and to discuss the content of mycelium ergot in the second stage stimulation mode. In addition, it was also designed to add the three amino acid precursors of ergothione on the 0th, 7th and 14th days of culture: cysteine, histidine and methionine. . After 22 days of culture, the dry weight of the mycelium of the coral mushroom and the content of ergot sulfur and the pH of the fermentation broth were analyzed.

It is known that the optimum growth pH of mycelium of coral mushroom is 5.0 to 6.5. However, it is found that the ergot sulfur production of coral mushroom mycelium by one factor is as follows. Adjusting the pH value of the medium to 10 can effectively improve the mycelium. Ergot sulfur content.

The results shown in Fig. 1 showed that the mycelium of the coral mushroom mycelium obtained by adjusting the pH value of the culture medium on the 0th day of culture was the highest, which was on the 0th day (8.28g/L)> control group (7.59g/L). > Day 14 (6.20 g/L) > Day 7 (4.41 g/L). The content of mycelium ergot sulfur was also the highest on the 0th day of culture (10.65mg/g dw), and the other order was control group (1.81mg/g dw)> 14th day (1.43mg/g) Dw)> Day 7 (0.55 mg/g dw). Considering the conversion of the hyphae yield per liter, the ergot sulfur content of the mycelium per liter of fermentation broth is 0 day (88.19 mg/L)> control group (13.75 mg/L) > day 14 ( 8.46 mg/L) > Day 7 (2.40 mg/L). From the above results, it can be seen that the dry weight of the mycelium of the coral mushroom (8.28 g / L) and the highest content of ergot sulfur (10.65 mg / g dw) obtained by adjusting the pH value of the medium on the 0th day of culture, per liter The content of ergot sulfur in the mycelium of the fermentation broth was 88.19 mg/L, which was 6.4 times higher than that of the control group.

The addition of amino acid precursors at different growth stages affected the growth of mycelium of coral mushrooms (Fig. 2A), but all of them could effectively increase the content of mycelium ergot sulfur, which was also cultivated on the 7th day. The mycelium ergosulfide content of the mycelium obtained by adding 4 mM cysteine was 16.31 mg/g dw, which was nearly 9 times higher than that of the control group (Fig. 2B).

Study on optimum culture conditions of mycelium of coral mushroom containing high ergot sulfur by reaction surface methodology

The reaction surface method is often used to explore the optimal medium composition and improve the food production process. The experimental design can effectively reduce the number of experiments and understand the interaction between various factors. Therefore, the experiment further uses the reaction surface method to explore the interaction between the three precursors to find the optimal concentration ratio of the precursor.

From the results of the above-mentioned liquid culture one-factor test, the culture conditions which have significant differences in the liquid fermentation growth of the coral mushroom are selected. In this experiment, the three factors of cysteine ( X ₁ ), histidine ( X ₂ ) and methionine ( X ₃ ) were used as independent variables, and the selected concentrations ranged from 0.64 to 7.36 mM for the first batch of experiments. Through a three-factor central composite design (CCD), a combination of 20 different concentrations can be obtained. With three independent variables of cysteine, histidine and methionine, a three-factor and five-level central mixing design was carried out, and a total of 20 sets of tests were required. The concentration or conditions of each factor designed in the table were prepared in a liquid medium and cultured in an incubator for 22 days. After the freeze-dried mycelium of the coral mushroom was analyzed, the dry weight of the fungus and the content of ergot sulfur and the pH of the fermentation broth were analyzed. The experimental results were obtained by computer software SAS (version 8.0, SAS Institute Inc., Cary, NC) and SigmaPlot (version 12.0). , SPSS, Inc., Chicago, IL) Calculate and plot, respectively, can obtain the quadratic equation of mycelial dry weight and ergot sulfur content, and then draw into the reaction surface map and contour.

From the analysis of the variation of the second batch of mycelium ergot sulfur content, cysteine ( X ₁ ), histidine ( X ₂ ) and methionine ( X ₃ ), three independent variables for coral mushrooms The mycelium ergot sulfur content was significant at 0.1%. Moreover, as shown in Table 2 below, the ternary quadratic mathematical model for the content of ergot sulfur in three independent variables, cysteine ( X ₁ ), histidine ( X ₂ ) and methionine ( X ₃ ), is described. The P value is less than 0.0001, and there is a statistically significant difference, that is, the reliability of the reaction surface mode is as high as 99.99%; the P value of the Lack of fit is 0.6633, so this describes the ternary quadratic mathematical model of biomass. It was established. The coefficient of variation is 10.12%, indicating that the mathematical model describing biomass has high accuracy and reliability. The verification coefficient is 0.9880, which also means that the correlation coefficient is 0.9940.

The ternary quadratic equation for the ergot sulfur content of the mycelium of Pleurotus ostreatus fermented by three variables after deep analysis by ANOVA (analysis of variance):

Y _ergothione : the ergot sulfur content (mg/g) of the mycelium of the coral mushroom in the deep culture.

Table 3 series shows the results of different variables of the liquid fermented coral mushroom ergot sulfur obtained by the reaction surface method, which shows that the P value of the independent variable array of amine acid ( X ₂ ) is significantly different, while the independent variable cysteine ( X ₁ ), and methionine ( X ₃ ) and X ₁ X ₂ , X ₁ X ₃ , X ₂ X ₃ have no significant difference in interaction; in the secondary effect, with Both have significant differences. Therefore, the concentration of immobilized histidine ( X ₂ ) was 4 mM, and the interaction between cysteine ( X ₁ ) and methionine ( X ₃ ) was investigated, and the content of mycelium ergot sulfur was drawn. The contour map and the reaction surface map are shown in Fig. 3. The optimum points can be clearly seen in both figures.

Based on the above results, the ternary quadratic mathematical model is established, and the optimal point can be clearly seen in the contour map and the reaction surface map. It can be predicted from the contour map that when the normal value of cysteine, histidine and methionine is 0, the content of mycelium ergot can be highest, and the actual concentration is cysteine. 8 mM, histidine 4 mM and methionine 0.5 mM, with the normal value calculated in the equation, the predicted value of ergothione can be obtained, the value is 13.90 mg / g dw, and the actual addition of the precursor obtained coral mushroom mycelium The experimental value of ergot sulfur content is 14.57 mg/g dw, which shows that the actual experimental value is similar to the predicted value obtained by the equation, and the mycelium ergot sulfur content is even higher.

Effect of factors on the growth curve of mycelium mycelium of mycelium

From the previous one-factor condition, it was found that by adjusting the pH of the medium to 10, using tryptone as a nitrogen source and adding the precursor amino acid in time, the content of ergot sulfur in the mycelium of coral mushroom can be effectively improved. The number of days in the experiment is 22 days, and changing the concentration and proportion of the culture factor or the added precursor may affect the growth curve of the mycelium of the coral mushroom. Therefore, this experiment further explores the influence of the factor on the growth curve of the mycelium. Find out the factors that can effectively increase the ergot sulfur content and shorten the culture time of the deep fermentation of the coral mushroom mycelium.

According to the growth curve of mycelium of coral mushroom mycelium at pH 10, the mycelium content of mycelium reached the highest at 7.48 mg/g dw when cultured until the 20th day, but the medium produced similarly with the increase of culture time. The more the ammonia is, the more odor is. When tryptone was used as the nitrogen source, the content of ergot sulfur in the mycelium of the coral mushroom mycelium was up to the 20th day, and the content of ergot sulfur was the highest, and the value was 5.50 mg/g dw. The weight is 7.12g/L, and the converted ergot sulfur content per liter is 39.17mg/L, which is higher than 1.81mg/g dw and 18.17mg/L when the yeast extract is nitrogen source. Many, in addition to tryptone as a nitrogen source for deep culture of coral mushroom, ergot sulfur cultured to 12 days mycelium Because it can reach the content of 22 days when the yeast extract is used as a nitrogen source, it can effectively shorten the fermentation days (Fig. 4).

In view of the discussion of amino acid precursors, the hyalin mycelium gradually decreased with the increase of culture time after the 8th day when the comprehensive amino acid solution was added to the culture medium on the 7th day. Compared with the growth curve of mycelium mycelium in the mycelium, compared with the growth curve of the mycelium of the coral mushroom, without adding any amino acid precursor, the coral mushroom mycelium obtained by adding the integrated amino acid was also cultured on the 16th day of culture. The content of sulphur in the middle ergot was 34 times higher than that in the mycelium without the addition of precursors. The mycelium of the coral mushroom was added to the mycelium of the mycelium on the 10th day of culture. The sulphur content (2.54 mg/g dw) has reached an ergothione content similar to that of the mycelium culture without addition of the precursor for 22 days (2.87 mg/g dw) (see Figure 5). Compared with the growth curve of coral mushroom mycelium when adding a single amino acid precursor (4 mM cysteine), the mycelium ergothione content of only one single amino acid concentration was added on the 16th day of culture. When it was 8.04 mg/g dw, it was found that the mycelium ergosulfide content obtained by adding the integrated amino acid solution was better than when only a single amino acid was added. From the above results, it can be seen that the addition of a comprehensive amino acid solution to the culture medium of the deep-layered coral mushroom can effectively shorten the time of producing ergot sulfur in the mycelium of the coral mushroom, and can significantly increase the ergot sulfur in the mycelium of the coral mushroom. The content.

Active substances and antioxidation analysis of mycelium of coral oyster mushroom

According to the experimental results of the previous reaction surface method, during the liquid culture of the coral mushroom, the addition of the comprehensive amino acid solution in the medium can effectively increase the content of the mycelium ergot sulfur, and the obtained mycelium is abbreviated as high ergot sulfur. High ergothioneine Pleurotus citrinopileatus mycelia (HEPM). At present, the mushroom mycelium obtained by liquid fermentation has been used in the formulation of nutritious foods or functional foods. As a food raw material, it has a high degree of product acceptance in terms of chemical composition and non-volatile taste components. Correlation, however, during the literature review, it was found that there was little correlation between the coral mushroom fruiting bodies and the non-volatile taste components of the mycelium. Therefore, the purpose of this study is to understand the general composition of HEPM and low ergothioneine Pleurotus citrinopileatus mycelia (LEPM), non-volatile taste components including soluble sugars and sugar alcohols, free amino acids And the difference in the composition of 5'-nucleotides, etc., also explored the difference between the bioactive components of the two mycelium, such as ergosterol, adenosine and GABA, and compared with the coral mushroom fruiting body.

When the mycelium is cultured, 0.250L of the coral mushroom liquid strain is taken in a 10L table type fermentation tank containing 4.75L of liquid medium. The liquid medium used is composed of the same liquid species, the stirring speed is 125 rpm, and the aeration rate is 1 vvm. Cultured at 25oC, no amino acid precursor was added during the culture, the hyphae were recovered on the 14th day of culture, the mycelium was separated from the fermentation broth by a 100mesh sieve, the mycelium was washed with deionized water, and the mycelium was frozen. Dry, the obtained mycelium is a mycelium of coral oyster mushroom with low ergot sulfur, and the following is simply referred to as LEPM mycelium. Take 20 mL of liquid bacteria in a 500 mL flask containing 180 liquid medium and incubate at 25 ° C, 125 rpm. The medium used is the same liquid species, and the combined amino acid solution is added on the 7th day of culture, and the culture is carried out. The hyphae were recovered in the day, and the mycelium was separated from the fermentation broth by a 100 mesh sieve. The mycelium was washed with deionized water and then freeze-dried. The obtained mycelium was a coral mushroom mycelium containing high ergot sulfur. Referred to as HEPM (high ergothioneine Pleurotus citrinopileatus mycelia) mycelium. The synthetic amino acid solution was prepared by preparing a high-concentration amino acid solution, filtering it through a 0.22 μl filter, and adding it to a medium content of 8 mM cysteine, 4 mM histidine, and 0.5 mM methionine.

The water content of the mycelium of the coral mushroom fruit body and the two ergot sulphur content was not significantly different after lyophilization, and the moisture content ranged from 8.88 to 9.03%. One of the main ingredients of mushrooms is the carbohydrate of carbohydrates, which accounts for 40 to 70% of the components of mushrooms. Based on dry weight, HEPM contains a higher amount of carbohydrates, which is 53.47%. In terms of crude protein content, the protein content of the mushroom is about 19% to 35% of the dry weight. Although the protein content is lower than the animal food, it can be a good source of protein. The analysis results show that the coral mushroom fruit body and HEPM and LEPM two kinds of mycelium crude protein There is no significant difference in the quality content, and the protein range is 33.83~34.23%.

The soluble sugar and sugar alcohols contained in the coral mushroom fruit body are arabinose, lactose, mannitol, ribose and trehalose, and the highest mannitol content is 46.57 mg. /g, which is 50.5% of the total soluble sugar, followed by arabinose and trehalose, and the contents are 16.28 and 15.27 mg/g, respectively. The soluble sugars and sugar alcohols contained in HEPM are mannitol, ribose and trehalose. The highest content of ribose in HEPM was 18.58 mg/g, which accounted for 39.17% of the total soluble sugar, followed by mannitol and trehalose, which were 16.07 and 12.87 mg/g, respectively. From the above results, it was found that mannitol, ribose and trehalose were contained in both the coral mushroom fruit body and the HEPM mycelium.

The free amino acid content of the coral mushroom fruiting body is significantly higher than that of HEPM and LEPM mycelium; in addition, the content of amino acid precursor related to ergothione in the free amino acid composition of mycelium, methionine None of them were detected; since the high concentration of 4 mM histidine was added to the medium in which the HEPM mycelium was cultured, the HEPM mycelium was much higher than the LEPM mycelium in terms of histidine content. In terms of nucleotide content, the total 5'-nucleotide content in HEPM mycelia was 200.44 mg/g, which was significantly higher than that of fruiting bodies and LEPM mycelia. In terms of physiologically active substances, the ergosterol content and GABA content of HEPM mycelium were significantly higher than that of coral mushroom fruit body and LEPM mycelium. The adenosine content was higher in the fruiting body and its value was 5.53 mg/g, which was significantly higher than that of HEPM and LEPM mycelium.

The anti-oxidation properties and antioxidant components of 70% ethanol extracts were studied for the mycelium with different content of ergot sulfur in the coral mushroom fruit body, HEPM and LEPM. The coral mushroom fruit body, HEPM and LEPM mycelium were extracted with 70% ethanol, extracted and concentrated to dryness under reduced pressure, and directly diluted with antioxidants or 70% ethanol to different concentrations (0.1~10.0 mg/mL). Used as an analysis of antioxidant properties. The antioxidant properties analyzed in this study include: antioxidant capacity, reducing power, scavenging DPPH free radicals, chelated ferrous ions and TEAC antioxidant capacity, and determining the content of antioxidants in 70% ethanol extract. Further, the evaluation results of various antioxidant properties obtained by the experiment are converted by the internal and external difference method of linear regression, and expressed by EC ₅₀ (Effective concentration) value, and the coral mushroom fruit body, HEPM and LEPM can be better understood. The anti-oxidation properties of the 70% ethanol extract of the mycelium; and the conversion of 70% ethanol extraction rate will better understand how much the coral mushroom fruit body and mycelium powder can exhibit the EC ₅₀ effect. And using the correlation coefficient (r) to assess the correlation between antioxidants and EC ₅₀ values of antioxidant capacity.

The determination of antioxidant capacity is based on the formation of conjugated diene compounds. The principle of the experiment is to use unsaturated fatty acids (such as linoleic acid) to form free radicals due to dehydrogenation during the initial oxidation. The intramolecular rearrangement produces a conjugated diene bond, and the conjugated diene compound can be obtained by measuring the absorbance of the maximum absorption wavelength of 234 nm to determine whether the test sample binds to the radical and lowers the conjugated diene. When the compound is formed, the lower the absorbance value, the stronger the antioxidant capacity, or the percentage of inhibition of peroxidation, that is, the higher the inhibition rate, the better the oxidation resistance.

It can be seen from Fig. 6 that the antioxidant activity of the 70% ethanol extract of the coral mushroom fruit body, HEPM and LEPM mycelium increases with the concentration of the extract. When the concentration was 0.1mg/mL, there was no significant difference in the antioxidant power between the coral mushroom fruit body and the HEPM and LEPM mycelium. The antioxidant capacity was 45.75~50.65%, which was better than BHA (93.09%). And α-tocopherol (90.02%) is poor, but better than the ascorbic acid antioxidant capacity (3.02%). When the concentration was 0.5 mg/mL, the antioxidant capacity of the fruiting body was 77.91%, which was significantly higher than that of HEPM mycelium (63.11%) and LEPM (61.72%). When the concentration is 5.0mg/mL, the antioxidant capacity of HEPM mycelium (76.52%) is higher than that of LEPM mycelium (71.44%), while the antioxidant capacity of coral mushroom fruit body can be as high as 97.87% and standard BHA. (98.46%) is equivalent. When the concentration was increased to 10.0 mg/mL, the antioxidant capacity of HEPM mycelium was 82.47%, which was significantly higher than LEPM (79.17%).

The reduction power is measured by the amount of Prussian blue (Fe ₄ [Fe(CN) ₆ ] ₃ ). The main principle is to reduce the potassium ferricyanide (K ₃ Fe(CN) ₆ ) to yellow. After the blood salt (K ₄ Fe(CN) ₆ ), it reacts with ferric ions to form Prussian blue, and the Prussian blue content is measured by a spectrophotometer at a maximum absorption wavelength of 700 nm to detect the reducing power of the sample. If the sample reduces iron ions to ferrous ions and then generates Prussian blue with ferric iron to increase the absorbance, it means that the reducing power of the sample is stronger. It can be seen from Fig. 7 that the reducing power of the HEPM mycelium is optimal in terms of reducing power. When the sample concentration is 1.0mg/mL, the HEPM mycelium reducing power can reach 0.59, which is significantly higher than the fruiting body (0.34) and LEPM mycelium (0.35). When the concentration was increased to 5.0 mg/mL, the reducing power of HEPM mycelium was as high as 1.14, which was significantly higher than that of the fruiting body (1.02).

In addition, in the ability to scavenge DPPH free radicals, HEPM mycelium was the best to remove DPPH free radicals with an EC ₅₀ concentration of 0.29 mg/mL. In terms of the ability to chelate ferrous ions, the 70% ethanol extract of the coral mushroom fruiting body has an EC ₅₀ concentration of 0.29 mg/mL, which is significantly lower than that of HEPM and LEPM mycelium. In terms of TEAC antioxidant capacity, the 70% ethanol extract of the coral mushroom fruiting body had an EC ₅₀ concentration of 3.14 mg/mL, which was significantly lower than that of HEPM and LEPM mycelium. In terms of the antioxidant content of the extract, the content of ergot in the 70% ethanol extract of HEPM mycelium was 61.53 mg/g, which was significantly higher than that of the coral mushroom fruiting body (17.04 mg/g) and LEPM mycelium (3.84). Mg/g). Overall, the 70% ethanol extract of HEPM mycelium has better antioxidant properties than coral mushroom fruit body and LEPM mycelium. The integration results are shown in Table 4 below.

Each value is expressed as mean ± SE ( n = 3). Different letters indicate a significant difference P < 0.05.

The above results show that the 70% ethanol extract of HEPM mycelium has a good effect on antioxidant capacity, reducing power and DPPH free radical scavenging ability. When the concentration of 70% ethanol extract is 0.08 mg/mL, Up to 50% of the antioxidant power was not significantly different from the coral mushroom fruiting body (0.09 mg/mL). Overall, the 70% ethanol extract of HEPM mycelium has superior antioxidant properties to coral mushroom fruit bodies and LEPM mycelia.

In terms of antioxidant composition, the total polyphenol content in 70% ethanol extract of HEPM mycelium was 89.54 mg/g, which was significantly higher than that of coral mushroom fruit body (35.76 mg/g) and LEPM (30.38 mg/g). Powdered, total polyphenol content in HEPM mycelium samples (21.20 mg/g) > coral mushroom fruit body (8.17 mg/g) > LEPM mycelium (2.97 mg/g), see Table 5 below.

^a Each value is expressed as mean ± SE ( n = 3). Different letters indicate a significant difference P < 0.05.

Because of the metabolites of fungi, ergot sulfur is mainly found in plant and animal tissues. However, since humans and vertebrates cannot synthesize ergot sulfur, they can only be absorbed by ingesting food. Studies have shown that mushrooms (about 0.1-1 mg ergothione per gram dry weight) and cyanobacteria are the best food sources for ergot sulfur. Lin ( International Journal of Medicinal Mushrooms. 15 : 315-323, 2013) analyzed the content of ergot sulfur in 49 kinds of edible and medicinal mushrooms, including 9 seed entities, 39 mycelia and a vegetative cell, of which Pleurotus Pleurotus ) has a high content of ergot sulfur. The present invention discloses a HEPM mycelium containing high ergot sulfur obtained after culturing under optimum medium conditions, and the content of ergothione in the 70% ethanol extract of the HEPM mycelium obtained by the method of the invention can reach 61.53 mg/ g, significantly higher than the coral mushroom fruit body (17.04mg / g) and LEPM mycelium (3.84mg / g); converted to powder dry weight, HEPM mycelium sample ergot sulfur content (14.57mg / g dw > Coral mushroom fruiting body (3.89 mg/g dw) > LEPM mycelium (0.37 mg/g dw).

According to the above results, it can be seen that the ergot sulfur content in the mycelium of the coral mushroom can be increased by 39 times under the optimum culture condition and the addition of the amino acid precursor, so the deep liquid culture method of the present invention can obtain the high ergot angle. Sulfuric acid mycelium mycelium, and the mycelium has excellent antioxidant, reducing power and DPPH free radical scavenging ability, and can be used for producing fermenting bacteria of ergot sulfur. Furthermore, the mycelium obtained by the culture method of the invention not only has high content of ergothione, but also has good antioxidant and anti-oxidation components, and does not affect the original umami taste of the coral mushroom, so it can be used as High-nutrient food source or supplement.

Claims (5)

A liquid culture method for improving the content of ergot sulfur in mycelium of coral mushroom, characterized in that: the mycelium of coral mushroom is cultured at 25 ° C for 7 days for activation; the mycelial culture solution is inoculated with 5%. The inoculation amount was shake cultured at a temperature of 20-25 ° C for 10-20 days in a medium having an initial pH of 10.0 and containing a 2% carbon source and a 0.5% nitrogen source, and added by the cyst on the 7th day of culture. A complex amino acid solution consisting of 8 mM of amino acid, 4 mM of histidine and 0.5 mM of methionine; and a mycelium of coral mushroom with high content of ergothione. The method of claim 1, further comprising the step of freeze-drying the collected coral mushroom mycelium. The method of claim 1, wherein the carbon source is glucose. The method of claim 1, wherein the nitrogen source is a yeast extract. The method of claim 1, wherein the nitrogen source is tryptone.