CN112113921B - High-throughput detection method for ergothioneine content between samples to be detected and application thereof - Google Patents
High-throughput detection method for ergothioneine content between samples to be detected and application thereof Download PDFInfo
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Abstract
The invention discloses a high-throughput detection method for ergothioneine content in a sample to be detected and application thereof, belonging to the technical field of microbial breeding. Adjusting the pH value of a sample to be detected to be 1.9-2.0, adding an iron thiocyanate solution into an ELISA plate, adding the sample to be detected, and mixing by blowing; recording the absorbance by using an enzyme-linked immunosorbent assay, wherein the wavelength used by the enzyme-linked immunosorbent assay is 450-470 nm; the lower the absorbance, the higher the ergothioneine content. The method has the characteristics of high efficiency, rapidness, convenience and low cost, and is suitable for research activities which do not need to accurately determine the ergothioneine content of each sample; the defects of time consumption and high cost of the LC-MS detection method are overcome.
Description
Technical Field
The invention belongs to the technical field of microbial breeding, and particularly relates to a high-throughput detection method for Ergothioneine (EGT) content in a sample to be detected and application of the high-throughput detection method in Ganoderma lucidum (Ganoderma) detection.
Background
Ergothioneine (EGT), also known as 2-thio-L-histidine trimethyl inner salt, is a natural amino acid rich in animal and plant (Lepidota and Mongolian 2010) EGT is only synthesized in some microorganisms (actinomycetes, streptomyces), funguses, some cyanobacteria, cannot be synthesized by the animal body itself, and can only be taken in from food and absorbed and accumulated in tissues and cells (panhong Yu et al 2019). EGT is considered to be a strong antioxidant, and Serville et al have studied the antioxidant mechanism, and have suggested a unique redox mechanism, and proposed a unique antioxidant effect of EGT in cells (Serville et al. 2015). EGT exists in solution in the tautomeric state of thiol and thione, and at physiological pH values, the stability of thiocarbonyl is higher than that of mercapto, so EGT exists mainly in the form of thione (Cumming et al, 2018). The standard redox potential of EGT is-0.06V, and the potential of other thiols is generally between-0.2V and-0.32V, so that EGT is more stable than other antioxidants in physiological pH environment and is not easily oxidized spontaneously (Qi Liu et al 2015). EGT can effectively remove hydroxyl ions, inhibit oxidation of copper ion-dependent oxyhemoglobin, and strongly remove hypochlorous acid, thereby preventing alpha1Anti-protease inactivation (Linchenshui et al.2006). EGTs also protect cells, especially body red blood cells, from free radical damage. EGT has wide application prospect in the industries of medicine, food, cosmetics and the like.
In recent years, people are intensively studying antioxidant, EGT is widely concerned as a natural active substance with strong antioxidant capacity, the research on EGT in China is still in an exploration stage, and more people have studied and studied the synthesis route and the biological activity of EGT abroad. EGT is a secondary metabolite, and the preparation method mainly comprises 3 methods: chemical synthesis method, biological extraction method, biological synthesis method. For the chemical synthesis, EGT is a chiral amino acid, and the synthesis of levorotatory EGT is difficult, and various synthesis methods do not achieve the expected effect due to partial or total racemization. Although a commercial synthesis of EGT is now developed by a company in the United states, the raw materials are expensive and the synthesis cost is high. The biological extraction method has a great problem, in the nature, EGT is widely existed in organisms, Carolin and the like find that blue algae can produce high-level EGT (Pfeiffer and the like.2011), but the research and production requirements cannot be met. The EGT content in most biological raw materials is generally low, and the method has the defects of more impurities after extraction, complex extraction operation, high cost and the like. In order to overcome the disadvantage of the low yield of EGT in the natural world, Tanka et al transferred the egtBCDE gene in mycobacteria into E.coli and used a fermenter for fermentation production (Tanaka et al, 2019), which has questioned the safety of EGT although a large amount of EGT is available.
EGT is commonly contained in fruiting bodies and mycelia of edible fungi, and the edible fungi contain various bioactive factors, such as polyterpenes, polysaccharides, functional proteins, flavones and polyphenols, and have various health-care functions on human bodies, such as improving the immunoregulatory capacity, preventing the formation of cancer cells, resisting aging and the like (Zhangye and the like, 2016). According to statistics that edible fungi in China have 936 varieties, 23 varieties, 3 subspecies and 4 variants (Daiyuching et al, 2010), EGT is prepared by utilizing deep fermentation of the edible fungi, has the advantages of low production cost, capability of improving the yield of products by technical means such as metabolic regulation and control, easiness in large-scale production and the like, and can better ensure the safety of products (preserved plum et al, 2015). In the aspect of detecting the EGT content, Lee et al compare 28 kinds of mushroom EGT by using liquid chromatography (LC-MS) detection (Lee et al, 2009), Zhang Cui et al also detect the EGT content in 10 different mushrooms by using an LC-MS detection method, wherein the content of the hazel mushroom and the flammulina velutipes is higher, and the content of the white mushroom and the oyster mushroom is lower (Zhang Cui et al, 2013). According to the research, the EGT content of different edible fungi has larger difference.
When EGT is researched, the application method is generally an LC-MS (liquid chromatography-mass spectrometry) detection method, and the method for measuring the content of EGT among different mushroom varieties is also generally used. For this purpose, sanden et al establish a set of detection systems (sanden et al, 2018) for quantifying EGT in liquid fermentation products of fungi. Takusagawa et al established a reliable and practical EGT production system using Aspergillus oryzae, in which LC-MS detection was also used (Takusagawa et al, 2018).
The LC-MS detection method can accurately detect the content of EGT from a system with complex substances, and is a convenient and high-accuracy method for detecting a crude extract of fungal hyphae with complex components. However, this method also has a significant drawback, and when it is used, a detection condition with a good detection effect needs to be explored, which is time-consuming and costly when the amount of the sample to be detected is large. In addition to LC-MS detection, paper partition chromatography and other methods have been used, but these methods have not been used until now after LC-MS detection.
With the increasingly intensive research on EGT, the cultivation of strains with high EGT yield becomes increasingly important. The method for cultivating the high-yield EGT strain by applying the biological engineering is a feasible method, a large amount of screening work is required in the process of cultivating a new strain, 35 hybrid strains are obtained for cultivating the ganoderma lucidum strain rich in the active macromolecular polysaccharide by the super-culture, and then the hybrid strains are required to be identified, screened and compared (the super-culture, 2017), so that a large amount of time is consumed. The process also needs to be carried out in the process of cultivating the high-yield EGT strain, and in the process of screening and comparing the hybrid strains, although the EGT content of each hybrid strain can be accurately measured by using an LC-MS method so as to carry out comparison, a larger amount of time and energy are consumed in the process, and the cost and the process are more complicated when LC-MS is used for detection. The EGT content of each hybrid strain is not necessarily accurately measured in the process of cultivating the high-yield EGT strain, and at the moment, the result of EGT content difference among the hybrid strains and between the hybrid strains and parents is only needed to be compared, so that a new hybrid strain capable of highly yielding EGT is obtained. At present, a method which is simple to operate and low in cost and can detect the EGT content difference among samples is not available.
Reference documents:
Cumming,B.M.,K.C.Chinta,V.P.Reddy,A.J.C.Steyn.2018.Role of Ergothioneine in Microbial Physiology and Pathogenesis.ANTIOXIDANTS&REDOX SIGNALING 28(6):431-444.
Lee,W.Y.,E.Park,J.K.Ahn,K.Ka.2009.Ergothioneine Contents in Fruiting Bodies and Their Enhancement in Mycelial Cultures by the Addition of Methionine.Mycobiology 1(37):43-47.
Pfeiffer,C.,T.Bauer,B.Surek,E.
D.Gründemann.2011.Cyanobacteria produce high levels of ergothioneine.FOOD CHEMISTRY 129(4):1766-1769.
Servillo,L.,D.Castaldo,R.Casale,N.D Onofrio,A.Giovane,D.Cautela,M.L.Balestrieri.2015.An uncommon redox behavior sheds light on the cellular antioxidant properties of ergothioneine.FREE RADICAL BIOLOGY AND MEDICINE 79:228-236.
Takusagawa,S.,Y.Satoh,I.Ohtsu,T.Dairi.2018.Ergothioneine production with Aspergillus oryzae.Bioscience,biotechnology,and biochemistry 83(1):181-184.
Tanaka,N.,Y.Kawano,Y.Satoh,T.Dairi,I.Ohtsu.2019.Gram-scale fermentative production of ergothioneine driven by overproduction of cysteine in Escherichia coli.Scientific Reports 9(1).
2017, a breeding research of a new disease-resistant ganoderma lucidum strain rich in active macromolecular polysaccharide, Shanghai university of application technology.
Daiyehhicheng, Zhouliwei, Yanghuang, Wenhuan, Tulipoul, Litaihui, 2010, Chinese edible fungus List, fungus article journal 01(29), 1-21.
Leanecdotal group, cycle waves 2010, biological functions of ergothioneine and applications thereof food engineering (03) 26-28.
Forest old water, Pacific Saturn, Li Xiaojun, Zuoming 2006, a rare natural amino acid-ergothioneine, amino acids and biological resources (01):63-67.
Liu Qi, Zhang Wei ya, Jiang Wen Xia 2015, synthesis and degradation metabolism of ergothioneine 27(06) 1112-1117.
Meibaoli, Qi Liu, Jiang wenxia, Zhang Weiya, Yang Nu 2015, research on the biosynthesis of ergothioneine enhanced by nutritional factors, food research and development (15): 108-112).
Panhong, Guoliqiong, Linjunfang 2019, research on distribution and metabolism in the body of ergothioneine and action thereof in diseases, food science 1-11.
Hsing Cheng, Liu Qi, Jiang Wen Xian, Wan Yan, Zhouzi, 2018, high performance liquid chromatography analysis of Ergota Ting in fungus liquid fermentation products 18(39): 238-.
Zhang Cui, Zhao Yan Ming, Baishufang, Liudailin 2013 HPLC method determine the ergothioneine content in different varieties of mushrooms 307 and 310.
Zhangyun Ye, Jiangming, Sunwaibo, Gandehuan, Yangjie, Chengyu 2016. for review of health promotion effect of edible fungi, Shanghai agricultural science and technology (1):19-21.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention mainly aims to provide a high-throughput detection method for detecting the content of EGT (epigallocatechin gallate) between samples to be detected. The method is efficient and simple, and overcomes the defects of time consumption and high cost of the LC-MS detection method.
Another objective of the invention is to provide an effective application of the high-throughput detection method in screening of Ganoderma lucidum strains with high EGT yield.
Comparing strains: pleurotus eryngii (Pleurotus eryngii 390), Pleurotus nebrodensis (Pleurotus nebrodensis 6), Heiping 114, Heiping 130, Pleurotus citrinopileatus (Pleurotus citrinopileatus 1), Flammulina velutipes (gold 55), Grifola frondosa, Cordyceps militaris CM10, Ganoderma lucidum and Ganoderma lucidum (Ganoderma resinatum) FQ23, and detection shows that Ganoderma lucidum (Ganoderma) belongs to the highest EGT content, so the invention selects Ganoderma lucidum in edible and medicinal fungi as the strain for determination.
Wherein, the pleurotus eryngii (390), the pleurotus nebrodensis (6), the heiping 114, the heiping 130, the pleurotus citrinopileatus (1), the flammulina velutipes (golden 55) and the grifola frondosa are purchased from shou-light edible fungi research institute in Shandong province in China;
Cordyceps militaris CM10 is disclosed in the document "high-yield carotenoid Cordyceps militaris solid fermentation system and its product research [ D ]. southern China agricultural university, 2016";
ganoderma lucidum (Ganoderma lucidunm) Gal-0201 is disclosed in the literature "Xuehenhong, Junfang, Stangjie, Yuanhao, Guoliqiong, 2007," analysis of biological properties and active ingredients of Ganoderma lucidum Gal-0201 [ J ]. edible fungi, p.19-21 ".
Deposit information of Ganoderma lucidum (Ganoderma resinaceum) FQ 23: the preservation unit: china general microbiological culture Collection center (CGMCC), the preservation date is 12 months and 23 days in 2019, and the preservation address is as follows: the microbial research institute of the national academy of sciences No. 3, Xilu No.1, Beijing, Chaoyang, Beijing, with the preservation number: CGMCC NO. 19152.
The purpose of the invention is realized by the following technical scheme:
the invention provides a high-throughput detection method for EGT content between samples to be detected, which comprises the following steps:
(1) taking an iron thiocyanate reagent, and mixing the iron thiocyanate reagent with the iron thiocyanate reagent according to a solid-to-liquid ratio (g/mL) of 0.1-0.3: 10, adding absolute ethyl alcohol for dissolving, and passing through a membrane to prepare an iron thiocyanate solution;
(2) adjusting the pH value of a sample to be detected to be 1.9-2.0, adding an iron thiocyanate solution into an enzyme label plate, adding the sample to be detected, and mixing by blowing; recording the absorbance by using an enzyme-linked immunosorbent assay, wherein the wavelength used by the enzyme-linked immunosorbent assay is 450-470 nm; the lower the absorbance, the higher the EGT content.
Preferably, in the step (1), the ratio of solid to liquid (g/mL) is 0.1: 10 adding absolute ethyl alcohol to dissolve.
Preferably, the lamination in the step (1) is 0.22 μm.
Preferably, the ELISA plate in the step (2) is a 96-hole ELISA plate;
preferably, the volume ratio of the iron thiocyanate solution to the sample to be detected in the step (2) is 0.5-2.5: 10; further 0.7 to 1: 10; the addition amount of the iron thiocyanate solution can be adjusted according to actual conditions, and if the color development effect of the sample is not obviously changed, the addition amount can be reduced.
Preferably, the wavelength of the microplate reader in the step (2) is 450 nm;
preferably, the sample to be detected in the step (2) is edible and medicinal fungi mycelium extracting solution; further extracting Ganoderma mycelium;
the preparation method of the ganoderma lucidum mycelium extracting solution comprises the following steps:
(A) collecting ganoderma lucidum mycelia;
(B) preparing a ganoderma lucidum mycelium extracting solution: weighing the ganoderma lucidum mycelia in the step (A), adding 60-70% ethanol, carrying out water bath, then centrifuging at normal temperature, and collecting supernatant, namely ganoderma lucidum mycelia crude extract; and then, in the ganoderma lucidum mycelium crude extract, adding a solvent according to a solid-to-liquid ratio (g/mL) of 0.8-2: 10, adding pretreated macroporous resin NKA-9, and standing; centrifuging at normal temperature, and collecting supernatant, i.e. Ganoderma mycelium extractive solution.
Preferably, the preparation method of the ganoderma lucidum mycelia in the step (a) comprises the following steps:
inoculating Ganoderma in liquid fermentation culture medium according to inoculation amount of 5% (v/v), culturing, centrifuging to collect mycelium, lyophilizing, grinding into powder, and storing in drying oven.
The liquid fermentation medium is a PSB medium: 200g/L of potato, 25g/L of sucrose, 2g/L of ammonium chloride, 3g/L of monopotassium phosphate and 1.5g/L of magnesium sulfate heptahydrate.
The culture conditions are 25-28 ℃, the rotating speed is 150-200 rpm, and the culture lasts for 10-15 days; further culturing at 25 ℃ and 200rpm for 10-15 days.
The freeze-drying time is 24-36 h; further 36 h;
preferably, in the step (B), the ratio of the ganoderma lucidum mycelia to the 60-70% ethanol is 1 g: 100-200 mL; further, the ratio of the ganoderma lucidum mycelia to the 60-70% ethanol is 1 g: 100 mL; furthermore, the ratio of the ganoderma lucidum mycelium to the 62% ethanol is 1 g: 100 mL;
the water bath condition is 50-60 ℃ for 0.5-1.5 h; further carrying out water bath at 53 ℃ for 1 h;
preferably, the ratio of solid to liquid (g/mL) is 1: 10 adding pretreated macroporous resin NKA-9.
The normal temperature centrifugation condition is 8000rpm for 10 min;
The standing treatment conditions are that the temperature is 28-30 ℃, the rpm is 150-200, and the standing is carried out for 3-4 hours; further standing for 3 hours at 28 ℃ and 150-200 rpm;
in the step (B), the coarse extract of the ganoderma lucidum mycelia is subjected to impurity removal treatment by adopting the pretreated macroporous resin NKA-9, so that the influence of impurities on the detection result is reduced.
Preferably, the pretreatment method of the macroporous resin NKA-9 comprises the following steps:
washing the macroporous resin NKA-9 with three-level water until the water is clear and transparent and has no opalescence after standing, performing suction filtration, and soaking the macroporous resin with absolute ethyl alcohol; washing the macroporous resin with three-level water until no obvious alcohol smell exists, performing suction filtration, and soaking the macroporous resin with 3-5% of HCl (preferably 5% of HCl) by mass fraction; washing the macroporous resin with three-level water until the pH value is neutral, performing suction filtration, and soaking the macroporous resin with NaOH (preferably 5% NaOH) with the mass fraction of 3-5%; washing the macroporous resin with three-level water until the pH value is neutral, and performing suction filtration to obtain pretreated macroporous resin NKA-9; can be used for the impurity removal method. The treated macroporous resin can be stored in first-grade water and placed at 4 ℃.
Preferably, the soaking conditions are all 4-25 ℃ for 4-12 h; further soaking at 4 deg.C for 12 hr.
The invention also provides application of the high-throughput detection method for high and low EGT content among samples to be detected in screening of high-yield EGT edible and medicinal strains.
Preferably, the edible and medicinal fungi are ganoderma.
Compared with the prior art, the invention has the following advantages and effects:
(1) the invention provides a method for detecting the content of EGT in different samples, which has the characteristics of rapidness, convenience and low cost; suitable for research activities that do not require accurate determination of the EGT content of each sample.
(2) The invention also provides an application of the method for detecting the EGT content difference of the hypha extracting solution among the ganoderma lucidum strains, and a method for effectively reducing the influence of other substances in the ganoderma lucidum crude extract on a detection system. The method can provide a certain idea for finding a method for eliminating the influence of other substances on a detection system when different samples are detected.
Drawings
FIG. 1 shows the results of a system for detecting pure EGT (purity 99%).
FIG. 2 shows the result of optimizing the amount of iron thiocyanate added to a pure EGT product.
FIG. 3 shows the result of measurement of Ganoderma strain NH 18.
FIG. 4 shows the result of measurement of Ganoderma strain NH 17.
FIG. 5 shows the result of measurement of Ganoderma strain NH 8.
FIG. 6 shows the result of detection of Ganoderma strain NH10
FIG. 7 shows the result of detection of Ganoderma strain NH5
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.
Iron thiocyanate reagent information in the examples: CAS registry No. 4119-52-2, available from Ostwald Biotechnology, Inc., Hubei.
Macroporous NKA-9 resin, commercially available.
The strain name and source used in the embodiment of the invention are as follows: pleurotus eryngii (Pleurotus eryngii 390) and Pleurotus eryngii (Pleurotus eryngii) used in experiment
American Ganoderma lucidum, Ganoderma atrum, bonsai No. 3, and Ganoderma sinense are all purchased from Shouguanggu edible fungus institute of Shandong province, China;
ganoderma lucidum (Ganoderma lucidunm) Gal-0201 is disclosed in the literature "Xuehenhong, Junfang, Stangjie, Yuanhao, Guoliqiong, 2007," analysis of biological properties and active ingredients of Ganoderma lucidum Gal-0201 [ J ]. edible fungi, p.19-21 ".
EXAMPLE 1EGT assay
(1) The EGT pure product information used in the embodiment of the invention is as follows: purchased from Tianjin Noxin, purity: 99 percent. Dissolving EGT pure product in first-stage water to prepare gradient solution, and preparing 5 concentrations as follows: 0.022mg/mL, 0.011mg/mL, 0.0055mg/mL, 0.00275mg/mL, 0.0022 mg/mL.
(2) Iron thiocyanate reagent according to solid-to-liquid ratio (g/mL) of 0.1: 10, adding absolute ethyl alcohol to dissolve, and passing through a film by 0.22 mu m to prepare an iron thiocyanate solution.
(3) Adding 10 or 7 mu L of iron thiocyanate solution into a 96-pore plate, adding 100 mu L of sample to be detected, and mixing by blowing. Absorbance was recorded using a microplate reader, which was used at a wavelength of 450 nm.
The results obtained were as follows:
TABLE 1 EGT pure product test results (10. mu.L of iron thiocyanate solution)
| Content (mg/mL) | 0.022 | 0.011 | 0.0055 | 0.00275 | 0.0022 |
| Absorbance of the solution | 0.427 | 0.548 | 0.572 | 0.579 | 0.599 |
Note: the actual effect of the absorbance is shown in FIG. 1.
TABLE 2 EGT pure product test results (7. mu.L of iron thiocyanate solution)
| Content (mg/mL) | 0.022 | 0.011 | 0.0055 | 0.00275 | 0.0022 |
| Absorbance of the solution | 0.399 | 0.419 | 0.422 | 0.432 | 0.446 |
Note: the actual effect of absorbance is shown in FIG. 2.
As can be seen from tables 1 and 2, as the EGT concentration decreases, the absorbance increases, and the amount of the added color-developing agent iron thiocyanate does not affect the color-developing result, and can be adjusted according to the actual situation when in use.
Example 2 liquid culture of Ganoderma strains
(1) The selected strains of the invention are as follows: ganoderma lucidum (Ganoderma lucidunm) as NH 18; ganoderma lucidum (Ganoderma sessile) USA, marked as NH 17; black sesame (Ganoderma sine), as NH 10; bonsai No. 3 (Ganoderma resinaceum), marked as NH 8; the method comprises the steps of taking Ganoderma lucidum (Ganoderma lucidum) as NH5, inoculating strains into a liquid fermentation medium PSB according to the inoculation amount of 5% (v/v), culturing at the rotation speed of 200rpm in a shaker at the temperature of 25 ℃ for 10-15 days, centrifugally collecting mycelia when the strains start to generate EGT in a stationary phase, freeze-drying for 36h, grinding into powder, and storing in a drying box.
The liquid fermentation medium PSB (1L): potato 200g/L (decoction), cane sugar 20g/L, ammonium chloride 4g/L, KH2PO43g/L，MgSO4·7H2O 1.5g/L。
Example 3 pretreatment of macroporous resin NKA-9 and preparation of iron thiocyanate solution.
S1, washing the macroporous resin NKA-9 with three-level water until the water is clear and transparent and has no opalescence after standing, performing suction filtration, soaking the macroporous resin with absolute ethyl alcohol, and standing at 4 ℃ for 12 hours; washing the macroporous resin with three-stage water until no alcohol smell is evident, filtering, soaking the macroporous resin with 5% HCl by mass fraction, and standing at 4 deg.C for 12 h; washing macroporous resin with three-stage water until pH is neutral, vacuum filtering, soaking macroporous resin with NaOH with mass fraction of 5%, and standing at 4 deg.C for 12 hr; and (3) washing the macroporous resin by using three-level water until the pH value is neutral, and performing suction filtration to obtain the pretreated macroporous resin NKA-9.
S2, mixing the components according to a solid-liquid ratio (g/mL) of 0.1: 10 adding absolute ethyl alcohol to dissolve iron thiocyanate, and passing through a membrane by 0.22 mu m to prepare an iron thiocyanate solution.
Example 4 extraction and purification of Ganoderma lucidum hypha extract
S1, weighing 0.1g of mycelium powder in the embodiment 2 respectively, adding 20mL of 62% ethanol, carrying out water bath at 53 ℃ for 1h, centrifuging at room temperature for 8000rpm for 10min, and collecting supernatant (10 mL), namely the ganoderma lucidum mycelium crude extract.
S2, adding the supernatant collected in S1 into a mixture of the supernatant and a solvent according to a solid-to-liquid ratio (g/mL) of 1: 10, adding the pretreated macroporous resin NKA-9 obtained in the example 3, placing the mixture in a shaking table at the temperature of 28 ℃, rotating the shaking table at the speed of 150rpm, and placing the mixture for 3 hours. Centrifuging at 8000rpm for 10min at normal temperature, and collecting supernatant, i.e. Ganoderma mycelium extractive solution.
Example 5 detection of crude extract EGT of Ganoderma lucidum hyphae
The pH of the Ganoderma lucidum mycelium extract obtained in example 4 is adjusted to 1.9-2.0. Adding 10 μ L of the iron thiocyanate solution of example 3 into a 96-well plate, adding 100 μ L of the treated Ganoderma mycelium extract, mixing by pipetting, and recording absorbance with an ELISA reader with a wavelength of 450 nm. The absorbance results are shown in tables 3 and 5, and FIGS. 3 to 7. Meanwhile, in order to prove the effectiveness of the high-throughput detection method, the invention also refers to the literature (Zhaoyingmin, Leizidong, Liucheng, Liudailin. high performance liquid chromatography is used for measuring the ergothioneine content in various fungi, food research and development, 2016,37(02): 117-. The ganoderma lucidum strains are divided into 2 batches for testing, and the absorbance and the detection result are as follows.
Testing one:
TABLE 3 Absorbance of EGT content in first Ganoderma lucidum mycelium extract
TABLE 4 high Performance liquid chromatography assay of EGT content in first Ganoderma lucidum mycelium extract
Table 3 shows the absorbance obtained when hypha extract of strains NH18, NH17 and NH8 was tested by the method of the present invention, and each strain was repeated 3 times. Table 4 shows the results of HPLC detection of hypha extracts of strains NH18, NH17 and NH8, wherein the peak area is the EGT peak area, and the larger the peak area, the higher the EGT content. Combining tables 3 and 4, the larger the peak area, the smaller the absorbance.
And (2) testing:
TABLE 5 Absorbance of EGT content in second Ganoderma lucidum mycelium extract
TABLE 6 high performance liquid chromatography for EGT content of Ganoderma lucidum mycelium extractive solution for the second time
Table 5 shows the absorbance obtained when the extract of the bacterial strains NH10 and NH5 were tested by the method of the present invention, and each bacterial strain was repeated 3 times. Table 6 shows the result of HPLC analysis of the extract of NH5 hypha of strain NH10, where the peak area is the area of EGT peak and the larger the peak area is, the higher the EGT content is. Combining tables 5 and 6, the larger the peak area, the smaller the absorbance.
The above results demonstrate that the detection method is feasible. When the kit is used, if the color development is deep and is not beneficial to judgment, the addition amount of the color development agent or the testing wavelength of the microplate reader can be adjusted according to actual conditions.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (8)
1. A high-throughput detection method for ergothioneine content between samples to be detected is characterized by comprising the following steps:
(1) taking an iron thiocyanate reagent, and mixing the iron thiocyanate reagent with the solid-liquid ratio g/mL of 0.1-0.3: 10, adding absolute ethyl alcohol for dissolving, and passing through a membrane to prepare an iron thiocyanate solution;
(2) adjusting the pH value of a sample to be detected to be 1.9-2.0, adding an iron thiocyanate solution into an enzyme label plate, adding the sample to be detected, and mixing by blowing; recording the absorbance by using an enzyme-linked immunosorbent assay, wherein the wavelength used by the enzyme-linked immunosorbent assay is 450-470 nm; the lower the absorbance, the higher the ergothioneine content; the sample to be detected is a ganoderma lucidum mycelium extracting solution;
and (3) the volume ratio of the iron thiocyanate solution to the sample to be detected in the step (2) is 0.5-2.5.
2. The high-throughput detection method for ergothioneine content among samples to be detected, as recited in claim 1, wherein the detection method comprises the following steps:
in the step (1), according to the solid-liquid ratio g/mL of 0.1: 10 adding absolute ethyl alcohol for dissolving;
the film passing in the step (1) is 0.22 mu m;
the ELISA plate in the step (2) is a 96-hole ELISA plate;
the volume ratio of the iron thiocyanate solution to the sample to be detected in the step (2) is 0.7-1: 10;
the wavelength of the microplate reader in the step (2) is 450 nm.
3. The high-throughput detection method for ergothioneine content among samples to be detected, as recited in claim 1, wherein the detection method comprises the following steps:
the preparation method of the ganoderma lucidum mycelium extracting solution comprises the following steps:
(A) collecting ganoderma lucidum mycelia;
(B) preparing a ganoderma lucidum mycelium extracting solution: weighing the ganoderma lucidum mycelia in the step (A), adding 60-70% ethanol, carrying out water bath, then centrifuging at normal temperature, and collecting supernatant, namely ganoderma lucidum mycelia crude extract; and then, in the ganoderma lucidum mycelium crude extract, adding a solvent according to the solid-to-liquid ratio g/mL of 0.8-2: 10, adding pretreated macroporous resin NKA-9, and standing; centrifuging at normal temperature, and collecting supernatant, i.e. Ganoderma mycelium extractive solution.
4. The high-throughput detection method for ergothioneine content among samples to be detected, as recited in claim 3, wherein the detection method comprises the following steps:
in the step (B), the step (A),
the ratio of the ganoderma lucidum mycelia to 60-70% ethanol is 1 g: 100-200 mL;
the water bath condition is 50-60 ℃ for 0.5-1.5 h;
the placing treatment is carried out for 3-4 hours under the conditions of 28-30 ℃ and 150-200 rpm.
5. The high-throughput detection method for ergothioneine content between samples to be detected according to claim 3 or 4, wherein the detection method comprises the following steps:
In the step (B), the step (A),
the ratio of the ganoderma lucidum mycelia to 60-70% ethanol is 1 g: 100 mL;
the water bath condition is that the water bath is carried out for 1h at the temperature of 53 ℃;
according to the solid-liquid ratio g/mL 1: 10 adding pretreated macroporous resin NKA-9;
the standing treatment is carried out for 3 hours under the conditions of 28 ℃ and 150-200 rpm.
6. The high-throughput detection method for ergothioneine content among samples to be detected, as recited in claim 3, wherein the detection method comprises the following steps:
the preparation method of the ganoderma lucidum mycelia in the step (A) comprises the following steps:
inoculating Ganoderma in liquid fermentation culture medium according to inoculation amount of 5% v/v, culturing, centrifuging to collect mycelium, lyophilizing, grinding into powder, and storing in drying oven;
in the step (B), the pretreatment method of the macroporous resin NKA-9 comprises the following steps:
washing the macroporous resin NKA-9 with three-level water until the water is clear and transparent and has no opalescence after standing, performing suction filtration, and soaking the macroporous resin with absolute ethyl alcohol; washing the macroporous resin with three-level water until no obvious alcohol smell exists, performing suction filtration, and soaking the macroporous resin with HCl with the mass fraction of 3-5%; washing the macroporous resin with three-level water until the pH value is neutral, performing suction filtration, and soaking the macroporous resin with NaOH with the mass fraction of 3-5%; and (3) washing the macroporous resin by using three-level water until the pH value is neutral, and performing suction filtration to obtain the pretreated macroporous resin NKA-9.
7. The high-throughput detection method for ergothioneine content among samples to be detected, as recited in claim 6, wherein the detection method comprises the following steps:
the culture conditions are 25-28 ℃, the rotating speed is 150-200 rpm, and the culture lasts for 10-15 days;
the freeze-drying time is 24-36 h;
the soaking conditions are all 4-25 ℃ for 4-12 h.
8. The application of the high-throughput detection method for ergothioneine content among samples to be detected, which is used for high-yield ergothioneine, in screening of edible and medicinal strains with high ergothioneine yield, as claimed in any one of claims 1 to 7.
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