TW201226561A - Submerged cultivation of pleurotus eryngii mycelia high in ergothioneine content - Google Patents

Abstract

The present invention discloses a submerged cultivation method of Pleurotus eryngii mycelia with high ergothioneine (EGT) content. The present invention further provides Pleurotus eryngii mycelia obtained by the cultivation method. The Pleurotus eryngii mycelia of the invention is useful in large-scaled liquid fermentation of ergothioneine, and can be used in the production of Pleurotus eryngii fruit body with high EGT content.

Description

201226561 VI. Description of the invention: [Technical field to which the invention pertains] The present invention relates to the use of a liquid hair _ method for cultivating amygdala sinensis, and cultivating high wheat = two lines. More particularly, the liquid cultured hyphae of the present invention body. Buy the ergot sulphur content of the fine base 1 ‘effective ^ apricot box body. [Prior Art]

The development of modern nutrition and medicine has made it clearer, and deeper understanding of things like = production has been widely loved by people = :; Γ ίί . j angry, yes, ι, nuclear _ and enzymes and medical Production of substances, such as antibiotics 9:2_; _ genus. Heterogeneous activity, Department of Food and Applied Biotechnology, Lizhongxing University ^文文~t 'Taiwan)' At present, the research on the axis mainly focuses on the efficacy of anti-U-lift, therefore, the research on physiological active substances = after rain Chun U is as big as 4 published. 'In the development of the chemical industry, the drug county has become the 20th century emerging = research ^ point (Wang Boche et al., 1998. Jianguan Axis and Gu. Food Development Research Hsinchu, Taiwan). The functional ingredients rich in beneficial substances, in addition to the well-known polysaccharides and the three brains, have also been discovered in recent years - some ingredients that are beneficial to human health, pure class II-human metabolites, including various A variety of compounds, proven to have good antioxidant properties (Ishikawa, Y. et al! 984. 〇 / plus (10) 〇 2 said. Noisy 61 (12): 1864-1868; Mail, JL and Ma, J. T, 2002. Fzmgfl/Sc(iv)ce 17 (1,2): 1-1〇). Dubost et al. pointed out in 2007 that there is a unique natural antioxidant in the [4 201226561 mushroom - ergothione (in Journal) Of Medicinal Mushrooms 9(2): 163-176a), which deals damage to oxidative stress in living organisms Prevention plays an important role (Chaudiere, J. and Ferrari-Iliou, R. 1999. Food and Chevnic Ql Toxicology 37 (9-10): 949-962; Hartman, PE 1990. Methods in Enzymology 186: 310-318). Ergothioneine (chemical name 2-mercapto histamine tridecyl betaine) is a metabolite of fungi; found in plant and animal tissues (Aruoma, 0. I. et al. 1999· 働^ 37) (11): 1043-53). Ergot sulfur can not be synthesized in the human body and can only be supplied by food (Dubost et al., 2007, as mentioned above), which is absorbed by mammals and will be present in liver and kidney. , heart, lung, central nervous system, eyes, bone muscles and other tissues (Bao et al. 2009. Such as wacw/iwre 295 (3-4): 243-249; Ey et al., 200 Ί. Journal of Agricultural and Food Chemistry 55(16).6466-74, Kawano 1982. Chemical & Pharmaceuticals 30(5): 1760-1765; Reglinski et al., 1988. Mag(10)zc 継6(2):217-223) 'In the human body The concentration is approximately 1 to 2 mM (Brummel, MC 1985. MWa? / Jiaqing 19:351-370; Dubost et al., 2007. 105(2): 727 -735). The content of ergot sulfur contained in mushrooms is much higher than that of other fruits and vegetables; Dubost et al. have pointed out that 'the mushroom is rich in ergot sulfur, and the mushroom is determined by the mushroom. The ergot has the highest sulfur content. In 2009, He Gongrui (in "solid-state fermentation of high ergot sulphur content of Pleurotus eryngii cereals and their taste properties and physiological activities." National Chung Hsing University Food and Applied Biotechnology Department Master thesis. Taichung 'Taiwan The study found that the content of ergot in the fruit body of Pleurotus eryngii was the highest in the 28 common medicinal medicinal mushrooms, which was 15216 mg/kg dw. Apricot Bautz (p/e (4) (10) machi feeding " (also Candolle: Fries) Quelet) belongs to the genus Agaricales, Agaricales, Tricholomataceae, Pleurotus (/> 仏 〇 ,), Alias snow velvet (Japan;), "201226561 Named Pleurotus" because of its taste of abalone, and has an almond flavor and force tiz豕ft: a white rot fungus 'has a strong decomposition of lignocellulose 26 (17? 4^ The side ear is less fragile, tastes delicious, tastes excellent, is the food is kind, so it is known as “Jingyiwang”. Xinglin and other, non-touch high, rich in multivitamins,

Fiber can reduce the absorption of calories and fat, and it can shorten the time of feces, and it is a healthy health food for obese people and patients with diabetes and hyperlipidemia. & continuous innovation and cultivation with the tube 'needs a lot of manpower' and the cost factor between the = and the preservation and transportation of the new sputum after harvesting, the heavy metal ions in the soil, J ^The use of = such as ^ worry, can get a large number of mycelium h physiologically active fermentation products in a short time (shi " people, Guan Xiaki 3 ΐ = Γ 439 Γ 85 · 793; Wang et al. Peng 7. Lung bowl Touch, submerged culture (submergedculture), is to micro-" ϊϊϊΐ ϊϊϊΐ ί 2 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当 适当'As mentioned above.) Changing the composition of the medium in the liquid culture and cultivating some of the self-financing and lying on the culture cake and the scales may stimulate the performance of its potential genes to obtain more diverse fermentation metabolites. & Support: Ming. 2002. 34: 32_35). In recent years, domestic and foreign profits (4) have been very common in training the riding body. This method can be used to cultivate a large number of silkworms ^ (Γ ζ 6 201226561 舄 very short time 'has several advantages · 1 ^ ^ ^ due to liquid culture can be fast growth and short growth cycle: preparation, not seasonal Restrictions:, Bp, 艮 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态 液态The composition and culture of the liquid medium of the cockroach are completed, and the sulphur content of the abalone sulphate of the ergot horn is obtained, and further, the invention provides a high ergot angle for the cultivation of a one-person one-factor shake flask. Sulphur furnace = the optimum culture condition of the silkworm body' and the analysis of the general composition, taste characteristics, physiological activity, = deuteration property, and the anti-inflammatory reaction for the prepared high ergot "々Sit." The method of cultivating a high ergot sulphur content of A. sinensis with a high ergo sulphur content, and a liquid sorrow culture method, which comprises inoculating the inoculated amount of the homogenized oyster mushroom liquid inoculum s 20% Contains glucose 20 muscle, yeast extract M =, _4) 2 § 〇 4 2 g / L, KH2P 〇 4 0 .5 g/L, Κ2ΗΡ04 0.5 g/L, 201226561' and the initial pH of the medium is about 6 3~6 8 10% (v/v). In another embodiment of the present invention, the culture is carried out. Three ===methionine, the ergothione content can be obtained - In one aspect, the invention relates to a

a culture of silk. ==================================================================================================== The use of the standard 1 or the sub-private 4 used in the embodiment for the convenience of the reader is not limited to the scope of the present invention. In addition, certain theories, which are disclosed and disclosed herein, but whether they are right or wrong, as long as the invention is implemented according to the present invention, without considering any particular theory or action, should be Limitations are within the scope of the invention. Other features and advantages of the present invention will be further exemplified and illustrated in the following examples, which are intended to be illustrative only. ' ^ The analysis items described in the following examples were tested in three replicates. The known data was statistically analyzed using the Statistical Analysis System (SAS Institute, Inc., 2000) software, and the variation analysis was performed using the AN〇VA program, and the model was determined by the multivariate domain of the α月氏氏α α 〇.〇5 The significant difference between the average values is compared. 201226561 Example 1: High ergot sulphur content Liquid culture condition of Pleurotus eryngii mycelium Apricot Baud strain (provided by Mr. Huang Shizheng from Zhongzhou Technical College, Changhua, Taiwan) In PDA solid medium (Peptone, potato dextrose agar (PDA) ), after activation in Difco, USA, 'cut four strains of about 5 mm χ 5 mm in a 250 mL flask containing 100 mL of liquid medium at 25.振 Incubation at 125 rpm for 7 days, homogenization at a low speed for 15 seconds by Waring blender, as a liquid inoculation source. Discussion on the amount of bacteria

The inoculation amount will affect the growth rate of mycelium. When the inoculum is low, the mycelium grows slowly; however, if the inoculum is too high, the mycelium grows faster but is prone to aging. In this experiment, 100% liquid medium was inoculated with 5%, '10% wide I5 %, 20% (v/v) Pleurotus eryngii strains and placed at 25. (In the incubator, cultured at 125 rpm for 14 days' to investigate the effect of different inoculum on the mycelium and sulphur content of Pleurotus eryngii. The optimal temperature was discussed during the microbial culture, and the culture temperature was affected by the bacteria. A very important factor in growth, which affects microbial growth, metabolic activity, and enzyme activity. Important components of micro-organisms, such as proteins and nucleic acids, are sensitive to temperature and may be irreversibly destroyed as temperature increases. It affects the length of microorganisms. Because of ^, in the fermentation system must be selected in a stable and suitable temperature environment, only in a certain temperature range, the metabolic activity and reproduction of the bacteria will rise with the temperature. Liquid medium (composition of 20 ^ glucose, 5 g / L yeast extract, 2 machine _4) 2 § 〇 4, 〇 5 == Κ 2 ΗΡ〇々〇. 5g / LMgS 〇 4.7H2 〇) ') 4 Pleurotus eryngii species, placed at 20 〇 c, 25 〇 c, 3 分别. 〇 Incubator was shaken for 14 days. In contrast, starting pH at 125 rpm explores one of the important factors in microbial fermentation. The pH value of the medium == long form, metabolite production, nutrient source consumption and by-products have a great influence. When the bacterial filaments are cultured in liquid form, the mycelium growth has a wide pH value of 201226561, and the optimum growth pH is about 5.0 to 7.0, but the optimum growth pH varies with different species. In this experiment, the initial pH value of 100 mL medium was adjusted to pH 6, pH 7, pH 8, pH 9, pH 10 and no adjustment (pH 6.3~6.8), and 10% of Pleurotus eryngii strains were added. Place in a 25 ° C incubator and incubate at 125 rpm for 14 days. The results are summarized in Table 1. 201226561

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Health, = ί, i warm i? high is not suitable for bacterial growth = dry weight is not significant: different: %, 15%, 20% when 'mycelium ^ due to inclusion, when the amount of bacteria is ω = in wheat, sulfur content of 2.79 mg (10) of the wheat liquid can produce 27.28 mg of ergot sulfur f ' in the mother 4 liters of culture 3 » / 0 - 15 〇 / 〇, 0 2 〇 Vf, i; :::rM°.18m• Therefore it is better to determine::: pH of the whole medium (starting 6: PH 8 ίίί-^^^5Η: Γσ^ ^ , 78 g/L) 0 i 21=adjusting the pH value At the time, the amount of ergot sulfur per liter of culture solution ί = the type of source of shouting source is very important for the growth of microorganisms, can supply the energy required for the mycelium, and can also be used as a component of the body. = Ben ' JR r. 12 201226561 π, 丨 为 为 心 心 心 to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to to Sucrose, the concentration of which is 2%. A 10% Pleurotus eryngii strain was placed in a 100 rnL liquid medium, placed in a 25 〇C incubator, and shaken at 125 rpm for η days. Discussion on the species of sputum source—the argon source types of sigma nutrient base are yeast extracts (^beef extract, acid hydrolyzed casein (casamin〇aicd), peptone (p^tone), pancreatization Protein (tiypt〇ne), the concentration of which is 〇5 0/(^ in 1〇〇mL liquid sad medium, access to 1〇% (v/v) Pleurotus eryngii strain, placed in 25〇c incubator The medium was cultured at 125 rpm for 14 days. The results were summarized in Table 2. 201226561 #礆》1^0痗4^1♦鮏0观4, !>Hdt蟠,«詖||漩®踹絜郑韧密"^鳑为4进_叛了 K-,<N< Ίε) Η93. (—-5? 菩) 103. (| 念旦鮏24 qce 3/00) 佑IT Weng 洄a17ld-Hct7>υ68ί S »ηΟΌ·ΉΖ.ΓΖ. S9.0 TZ-SZ-CN V90-1-Hse o inch ΟΌΐΓΙ H inch Γ0-Η66Ί S Ιοοΐ Ι8·Ι γοο0Ό-Η(5ο0·ε νΙΓΟ-Ηζ, inch·ε

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CQ9ro-H96oo V inch οΐ inch inch 01 υεΓΟΤ ΙΓ8 vz.ro-Hft7ol V6S-H9SI music-ffi coffee•©^缝渡^智荽4浒#盘(% S)ΊΊ/ Back HCU Huanwu w^ fin. (ςοο> CZ) 甽 甽 甽 蹑 砘 ^ ^ ^ ^ ^ ^ ^ ! ! ! ! ! ! 1! Long <(£=^氐-^Rewarding the cow 5«?令2娜_ 201226561 ^When using 2% glucose as the carbon source", and the 8% of the two people using fructose as the carbon source , and ^ is 9.73 g / L, , g / L. In terms of ergot sulfur content, ^ he =, 3.50 ~ apricot, body per gram dry weight wheat (four) fruit carbon source, dw, significantly higher than other When the carbon source is L27~i 991Λ 08 tons/g mother liters of apricot, _ broth of ergot sulphate is converted into household = less production, and it is cultivated per liter;

f mg/L ′ and when the carbon source is sucrose, the amount per liter of 33 is 33.72 mg/L, which is significantly higher than the ergothione content of fructose of the genus Pyrene mushroom. For the anti-source, the mother liter of apricot abalone as shown in Table 2 = shows that when the nitrogen source is α5% of the yeast extracter one, there is no significant difference in the dry weight of the mycelium, cattle = stroke = and 10.44 g / L, and significantly higher The acid weight of casein and kiss Weng. mycelium dry weight 8.11 and 8.96 g / L. Such as sound 1 - ^, for the mouse source Nitrogen source day 2, dry weight Pleurotus eryngii mycelium; sulfur; amount: = mother, matter and acid turn slightly from the nitrogen source s person W, H is the ergot sulphur of the parent literate culture solution 3=day, and the ergot sulphur content of the literate culture solution is the yttrium sulphur content of the sulphate culture solution. The type and proportion of amino acid added to the liquid culture of silky silky silkworms. The experiment examined the addition of 4 mM histidine after different days of culture, and the addition of cysteine, histidine and guanidine The effect of acid mixed amino acid on the mycelium of Pleurotus eryngii and the content of ergot sulfur. The amino acid addition ratio is calculated as the whole medium, and the amino acid addition concentration unit is mM. In the medium, 1% (v/v) was added. Apricot running: δ έ 种 ' was placed in an incubator at 25 ° C. The culture was shaken at 125 rpm for 14 days. The results are not shown in Figures 1 and 2. The experimental results shown in Fig. 1 show that after the culture for seven days, the histidine acid is added with ergot sulfur [201226561, the content can be as high as 33.7G mg/L, followed by the addition of nine days after the fifth day of culture, and the ergot sulphur is 31.4 respectively. G, 29 46 and the first were significantly higher than 14.73 mg / L when not added; in culture = amine acid, its body dry weight and _ body ergot =, plus time; regardless of the number of days of adding 4 mM histidine The weight did not significantly affect 10.19~10.85 g/L. _ silk body dry body ^ two culture, after the addition of 4 ^ histidine can increase the Pleurotus eryngii hyphae, the middle ergot sulphur content, continue to explore the addition of two 1 2 after the culture of cysteamine Acid and methionine on mycelium

ΛIn addition, in the addition of histidine, the addition of 4 fusantic histidine is best. The ergothione content can be as high as 44 88111 1 8 mM, 1 mM and 12 fuci sulphur sulphur content is 4〇. 91 :, 3t26 and 37.90 mg / L, the effect is less significant when added with 0.1 mM, / dry weight of mycelium and ergosterol content: the increase of the concentration of the tunnel, the dry weight of the mycelium And the ergosterol content is also added. When adding 12 mM histidine, the dry weight of mycelium and ergosterol are 13.97 g/L* 5.21 mg/g dw, regardless of the addition of histidine. 'The ergot sulfur and mycelium dry weight are higher than those without added day; the second mM histidine k, although the dry weight of the mycelium is not as good as the addition of I] mM, as a whole, After 7 days, the addition of 4 mM histidine per liter of apricot infusion culture yielded a erectile content of 44.88 mg/L, which was higher than the addition of i2 j$ (37.90 mg/L). As shown in Fig. 2, after adding five days, the mixed amino acid was added, and the ergosulfur content was as high as 48.771!^, which was 33.70 mg when the 41^1 group was added separately after seven days of culture. /L turns high. In the initial stage of culture, mixed ammonic acid was added, and the mycelium ergot sulfur content was higher than that in the latter stage of culture. After adding three days and five days, the mixed amino acid was added, and the ergot sulfur content was ^ Do not ^ 44.46 and 48.77 mg / L, and after seven days of culture, the content of mycelium ergot is 36.79 mg / L, added after nine days of cultivation, mycelium ergot sulfur content is 23.66 mg / L The reason is speculated that ergot sulfur is the second metabolite production of mycelium 201226561

The force/page is metabolized only after the cells have grown and matured, as shown in Fig. 2. On the fourth day, the growth of the body reaches the highest level of stationary phase, and after the hybrid growth matures, the amine synthesized by the ergothione A precursor of acid, 4 ώΜ group or mixed amino acid containing 4 mM bis, 1, 1 mM cysteine and mM methionine, can effectively extract ergot sulfur from mycelium Due to the content.

:曲虚f this 'from the results of Figure 1, 2 and Table 3, no matter the number of days of addition of histidine, 'can significantly increase the dry weight of the body and mycelium, i sulfur content' Adding methionine and a cysteine B with a concentration higher than 4 hits, the growth of Pleurotus eryngii mycelium, but the addition of three amino acids to the ancient '^Bao Rusi H ergotrope content and the addition of histamine alone When it comes to acid, it is speculated that if weaving acid is added in the culture stage, it will be able to increase the acidity and the methionine. It may be possible to increase the apricot. "201226561 (Ί/3ε) 103 B(l M/ 3S) 10H 2 Ice qa OQ inch Ί-Η10Γ6Ι a εζ/ι-Η9 inch·0(Ν CQV6 inch Ο-Ηο inch·(NCN vi-H6rs(N CQVU-I +6 inch·Ι(ΝPQAro-Hoor 卜CQ0060-H8901 CQ61 .0-H inch one 01 v I inch ·(Ν+ι09οε νζ-ο.ι Τ inch 67.CN mtN inch Ί-Η06·ζ, ε SO inch·Ι-Η16Ό inch V or I Τ 88, inch inch 036ζο- Η9Γ8ε υ(Ν9Ό+ι(ΝΓΙε

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The conversion rate of 'vitamin D' is the most (Koyyalamudi et al., 2009). Vitamin D can promote the body's absorption of calcium and phosphorus and bone formation, lack of vitamin d2, combined with bone calcification and collagen synthesis, a slight lack of vitamin 〇2, then two or more osteoporosis, resulting in rickets and adulthood in young children Human rickets (M; uet al. ' 1998b). The results in Table 4 show that the highest content of ergosterol in the apricot-boiled fruit body is 6.17 mg/g dw, and the ergosterol content in the mycelium of B. abalone and the foot of the abalone is 1.93 and 1.27 mg/g dw 〇201226561 ergot The ergothioneine is a fungus present in plant and animal tissues. Ergot sulfur can only be absorbed by food sources in humans. It is a low molecular weight cut-off that is absorbed by mammals and stored in specific tissues, including the liver, $3'. As shown in Table 4, the content of 'Xingbao fresh body and Yijia ergot sulphur content respectively & still and l, 74mg/gdw, while the optimum culture of Pleurotus eryngii mycelium has been adjusted, the mycelium The wheat stalk contains a higher than that of the apricot thief and the ergot sulphur content of 3.93 mg/g dw.

Polysaccharide content

Polysaccharide is a biological polymer rich in nature. It is made up of many monosaccharides (glucose, etc.). These polysaccharides will have a variety of sugars, chemical combinations, molecular size and properties. Differences may also vary depending on the strain or extraction method (Huang Lili. 2(8) 7. Master's thesis, Department of Food and Applied Biotechnology, National Chung Hsing University. Taichung, Taiwan). There are many anti-tumor activities, immune enhancement, anti-cancer, blood sugar lowering, blood pressure lowering and cholesterol lowering (J〇ng, SC et al. 1991. and λ^-7?ζ·νζ·shirt £)/ Tm fine "Ed Immunofarmacologia 11 (3): 115-122; Misaki et al. 1986. Agricuhura! and Biobgicd Chemistry 50 (9): 217. The physiologically active polysaccharides in medicinal fungi are mainly β-D-glucan (β-D-glucan), and β-D-glucan can stimulate macrophages, tau lymphocytes, b lymphocytes, and Natural killer cells, etc., regulate immune function to achieve anti-tumor effects. The contents of polysaccharides of apricot, body, mushroom and mycelium are as shown in Table 4, 89.50, 136.40 and 155.13 mg/gdw, respectively. The polysaccharide content of mycelium is the most south. 201226561 Sterol, ergothione And Table 4, Pleurotus eryngii fruiting body, mushroom foot and mycelium content a (mg / g dry weight) fruiting body foot ergosterol ergot thiophene

6.17 ±0.07 a 1.27±0.13C 2.05 ± 0.04 B 1.74 ± 0.05 C 89.50 ± 0.97 C 136.40 ± 0.23 B

Mycelium 1.93 ± 〇.〇3 B 3.93 ±〇.〇l A 155.13 ±0.74 A a ΪΪΪΓΓ^ΓΕ(η = 3) silk. The different letters in each block represent the fourth embodiment: high ergot sulphur content, apricot abalone, anti-oxidation preparation: sample powder... Water extraction heating method for 3 small

Whatman NcU I paper pumping filter ', the resulting sputum to deionized nucleus # to - find the degree, after riding the sputum U j and then stored at -20 t for use. Preparation of ethanol extracts from the east flank, place: 10 g of sample powder was accurately weighed into a 250 mL Erlenmeyer flask 'Add 200 mL of ethanol' at 25 ° C for 24 hours at 150 rpm, and then filtered with Whatman Ναΐ filter paper. The collected filtrate was concentrated to dryness under reduced pressure of 4 Torr, and then quantified to a certain concentration with ethanol, and the obtained extract was stored at -20 ° C until use. The ability to remove 1,1-diphenyl-2-picrylamine groups (dpph) can cause free radicals in the process of self-chemical process, resulting in rancidity of the oil. Antioxidants can be removed by providing hydrogen or free radicals. The lipid peroxide radicals during the chain reaction period, and thus the chain reaction of peroxidation. 1,1 -dipheny 1-2-picryl- hydrazyl ( DPPH C^H^l^O5) is a stable free radical with unpaired electrons. It is often used as a detector for antioxidants in 201226561 for the study of antioxidants. Ability. The DppH sterol solution has a strong absorption at 517 nm. Once it is reduced by antioxidant (AH) or free radical (R · ), its absorbance disappears or decreases (Bmnd-Williams, W. et al. 1995. Shape (10) Cheng Teng w

Now ^8:25-30) 'This method can test a large number of samples in a short time, and still has excellent sensitivity at low concentration. By measuring the wavelength absorbance, it can be judged that the sample is for the money to be cleaned 2 Base ability. In addition, DPPH methanol solution is stable and has appropriate absorption at pH 5.0~6.5, but it is less stable under alkaline conditions, and DppH sterol solution will deteriorate with storage time, so it is necessary to carry out experiments every time. Freshly prepared (Zeng Yuzhen. 2004. Ph.D. Thesis, Department of Food Science, National Chung Hsing University. Taichung, Taiwan). = The same concentration of the sample extract (4 mL) and the freshly prepared iOmMDppH methanol / gluten solution (1 mL) were uniformly mixed in a suction tube, left to stand in the dark for 3 minutes, and then in a Hitachi U-2001 spectrophotometer, The mixture was measured at 517 nm. · The absorption of light is low, and the ability of the sample to remove DppH free radicals is stronger. Clearance rate (%) = t1 - (A5nnm of the sample) / (A517nm of the control group)] X 1〇〇Ef5〇 value is 5〇% removal The effective concentration of DppH radical is obtained by interpolation of line I1 Count differently. In addition, a control group was used as a sample with anti-bad acid, butyl-sounding, and alpha-tocopherol. The results of comparing the sulphur content of prawn horn and the traditional culture of the buckwheat horn sulphate mycelium and the free radical scavenging ability are shown in Fig. 3, Fig. 4, and the extract is abalone. The removal of the entity, the foot and the mycelium by a force of the base increases with the increase in the sample. As shown in Fig. 3 = 'at a low concentration of 0.5 mg/mL, the ability of Pleurotus eryngii mycelium to clear DppH free radicals was ^54%, which was significantly higher than that of Pleurotus eryngii fruiting bodies (26·〇8 % :>, Mushroom foot (25.9^ /〇) and ascorbic acid (24.20%), the ability of these three to remove DPPH free radicals' and when the concentration is 5.0 mg/mL, the hot water extract of mycelium has cleared PH free radicals. More than 50%, and significantly higher than the mushroom foot (47 79%) ^' 201226561 Pleurotus eryngii fruiting body (45.43%) and ascorbic acid (33 31%). When the hydrophobic extract moisture is 20 mg / mL 'high The ergot sulphur content of Pleurotus eryngii is 57 ,, the ability of pots to remove DPPH free radicals is 57.58 %, which is higher than (he, 2_) apricot ^ = body (48.56 %), it is known that high ergot sulphur content Its ability to scavenge DPPH free radicals is better.

In terms of ethanol extract, the monthly b-force of DPPH free radicals, the garlic feet and the mycelium cleared DppH free radicals, and the sample concentration increased. As shown in Fig. 4, when the mycelium ethanol extraction concentration is 1 mg/mL, the _ppH free: the force has reached 7〇.H%; when the degree is increased to 5 mg/mL, the apricot Entity ^ Astragalus membranaceus has a DPPH free radical scavenging capacity of 9213 %

93.66 %, comparable to BHA (92.82 ° / 〇) and α-tocopherol (94 5 %) in scavenging DppH free radicals, and significantly higher than mushroom feet (4912%) and anti-ascorbic acid (33.31%) 〇Example 5: High ergot horn content Apricot Baogu silk body 1G L fermentation tank liquid culture g heart This experiment uses 10 L fermentation tank to culture high stone IL with apricot abalone 4 _ Silk body. The culture conditions used were μ. 〇, inoculation amount 1〇% (v/v), rotation speed 150rpm, aeration volume 1 vvm, culture volume 3~7L medium used for glucose, 20 g/L, yeast extract, 5, (NH4)2S04 , 2 g/L, KH2P〇4, 0.5 g/L, k2HP04, G.5 g/L and

MgS〇4.7H2〇, 0.5 g/L, and mM histidine was fed in the medium after seven days of culture. 4 [S] In terms of ventilation, when the fermentation of most mushroom strains is used, the ventilation densities used are 0.5~2, depending on the culture strain, culture stage and tank use. In the preliminary test, the ventilation used is 〇5~15 _, but when the ventilation is large, the amount of foam generated in the tank is relatively more than that of the tank. Although it can quickly sense and add foaming agent to the tank during rapid foaming, the amount of foaming is quite large due to the short length of the induction crucible and the % of the culture solution. After the foaming agent, the second section 201226561 can completely defoam. At this time, the defoaming sensor will be adhered by the substance in the medium to induce the effect, which leads to the continuous feeding of the defoaming agent into the tank body, causing the tank content to be too high and affecting. The growth of the cells, and the use of Iwm for the ventilation is not θ, this phenomenon occurs, so the fermentation ventilation is 1 yvm.觫5, no, 4 mM histamine was added to the medium after seven days of culture. Because I contained I, the highest point was 62.2 Gmg/L on the first day. There was no significant change in the content of 麦αα*3/f from the second day to the fourth day, which was 9.94~ 夭 夭 i i i i 四 四 四 四 四 四 四 ' ' ' ' ' ' ' ' People arrive at the highest point in time. The mycelium has a dry weight of ttiii ysis), which causes a decrease in mycelial concentration. On the fourteenth day ίίίί !' content is lower than ο.1 g / L, the mycelium also began to produce self, the bacteria (10) and ergot sulfur content continued to rise, presumably because the Martine silk body began to self-dissolve After the decomposition, the spring sputum produced, but the _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ The optimum energy level of ergot sulfur content in the mycelium of Pleurotus eryngii: 1 - the temperature is μ. . , 接 詈 is 10 〇 / rμ mouth condition 'including Peiyuan and 〇. Take yeast extract as nitrogen source, = raise = content of apricot _ bacteria 4: use: ί bacteria ^ i2 = bottle: ^ first 5.90 mg/g dw and 48.09 mg/L; with the same Λ 3 ς 刀 为 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Subsequent experiments revealed that 4 histidine, 1 mM cysteine and 5 were added after five days of culture! Fuza thiaminic acid, which is high in ergot sulfur due to the infusion of σ 4 mM histidine by the scorpion after seven days of culture.

The antioxidant activity of '8-new TEAC was significantly higher than that of Pleurotus eryngii and _, and its antioxidant activity, strength and ability to scavenge DppH free radicals were also superior to apricot, county fruiting bodies and purchases; The total age of the towel and the content of flavonoids were also significantly higher than the fruiting bodies and _. It can be seen that the addition of the amino acid can increase the content of the ergot horn of the apricot, and the obtained high ergot sulfur content of the mycelium of Pleurotus eryngii has good antioxidant properties. It is to be understood that the various embodiments of the present invention may be applied to the various embodiments of the invention, and the scope of the present invention as defined by the appended claims. It will be appreciated by those skilled in the art that the present invention may employ a number of modifications in many forms, structures and materials. Therefore, the present invention is to be considered as illustrative and not restrictive. The scope of the invention should be defined by the scope of the appended claims, and its legal equivalents are not limited to the foregoing description.

[Simple description of the diagram] Figure 1 shows the effect of adding 4 mM histidine on the dry weight, final pH value, ergosterol and ergothione content of Pleurotus eryngii mycelium after different days of culture. Figure 2 shows the mixed dry amino acid consisting of 4 mM histidine, 1 rnM cysteine and 1 mM guanidine thioglycol on the dry weight, final pH, ergot of Pleurotus eryngii mycelium after different days of culture. The effect of sterol and ergot sulfur content. Figure 3 is a comparison of the ability of the Pleurotus eryngii fruiting body, the mushroom foot and the mycelium hot water extract to remove 自由基, ^ 二笨基-2_ bitter 肼 group radicals. Fig. 4 is a graph showing the comparison results of the ability of the ethanol extract of Pleurotus eryngii fruit body, mushroom leg and mycelium to remove the radical of l5l_diphenyl-2-mercaptopurine group. Fig. 5 shows changes in the dry weight of the mycelium and the content of ergothione in the deep culture period of Pleurotus eryngii (25. 〇1 vvm, 150 rpm and 4 mM histidine) in a 10 L fermentation tank. 201226561 [Explanation of main component symbols]

Claims (1)

201226561 VII. Shen 5 Green Patent Range: 1. A liquid culture method for the mycelium of Pleurotus eryngii with high ergot sulphur content, including: (8) (b) (c) (6) Preparing a liquid inoculated source of homogenized Pleurotus eryngii; the inoculated source of the homogenized Pleurotus eryngii is inoculated with 5% to 2%, inoculated with glucose 20 g/L, yeast extract 5 Team, _4) 2S04 2 g / L, ΚΗ 2 Ρ 04 0.5 g / L, Κ 2 ΗΡ 04 0.5 g / L, MgSCV7H2 〇 0.5 g / L and at least one additional addition selected from histidine, methionine and cysteine, Add a liquid medium (starting pH 6~8) with a concentration of 〇1 to 12 mM amino acid; shake culture at a temperature of 21 to 25 °C for 125 days; and rpm A liquid culture of apricot thief _ body with high ergo sulphur content. 2. According to the method of claim 1, further comprising (e) the apricot abalone sulphur content of the high ergot sulphur content obtained by the poem _ tears: washing and lyophilization, after milling After the machine is crushed, the sieved content of Pleurotus eryngii mycelium powder.寸门麦角石』 3. The method according to item 1 of the patent application, wherein the temperature of the liquid is carried out. The culture is at 25 ° C. 4. According to the scope of the patent application! The method of the invention, wherein the inoculum (v/v), and the initial pH of the medium is about 6 3 to 6 8 . /〇 5. According to the method of claim 1, wherein the culture is carried out 3 to 4 mM histidine is added to the medium. After 7 days, the company will re-export 5 items according to the patent for the eye, in which 4 mM histidine is added to the medium. 〇6. 201226561 7. The method according to claim 1, wherein after 5 to 7 days of culture, 4 mM histidine, 1 mM cysteine and 1 mM guanidine thiocyanate are added to the medium. . 8. According to the method of claim 7 of the patent application, wherein after 5 days of culture, glutamic acid, 丨mM cysteine and 1 mM methionine are added to culture 9· having high content of methicin It is based on the method of the first method of Shen Qing patent scope. 1〇.申一具=角角=量法=Mycelium powder, which is a liquid culture of mycelium according to item 9 of U. ^^^ Fine culture, which is used to make a end, and the secret is to make high wheat as high wheat.