CN104990881A - Method for quickly detecting vigor of morchella esculenta L. hyphae - Google Patents

Method for quickly detecting vigor of morchella esculenta L. hyphae Download PDF

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Publication number
CN104990881A
CN104990881A CN201510401452.5A CN201510401452A CN104990881A CN 104990881 A CN104990881 A CN 104990881A CN 201510401452 A CN201510401452 A CN 201510401452A CN 104990881 A CN104990881 A CN 104990881A
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bacterium liquid
liquid
mycelia
bacterium
quick detection
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李红
李超
刘娜
李宏亮
何雨
赵春燕
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INSTITUTE OF VEGETABLE LIAONING ACADEMY OF AGRICULTURAL SCIENCES
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INSTITUTE OF VEGETABLE LIAONING ACADEMY OF AGRICULTURAL SCIENCES
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Abstract

The invention relates to a method for quickly detecting the vigor of morchella esculenta L. hyphae. The method includes the steps that after a bacterial strain to be detected is cultivated in mother strain flat plate culture media, a bacterium block on a flat plate is cut to be transferred into liquid deep layer culture media for shake culture, and accordingly bacterium liquid is obtained; the bacterium liquid is collected into a centrifuge tube for centrifuging, liquid supernatant is removed, the hyphae are extracted, and the hyphae are washed by normal saline; the hyphae are weighed and added with water to be matched into the bacterium liquid to be detected; the bacterium liquid to be detected, a Tris-HCl buffer solution and a TTC (2,3,5-triphenyltetrazolium chloride) solution are transferred into the centrifuge tube for reacting; after reacting is completed, reacting liquid is placed on ice immediately, centrifuging is conducted at the temperature of 4 DEG C, a cell sediment is obtained, and an extraction agent is added into the centrifuged cell sediment so that extract liquor can be obtained; the extract liquor is centrifuged, the liquid supernatant is collected, and the absorbance value is detected at the wave length of 485 nm. Compared with a traditional method that the vigor of strains is detected according to hypha characteristics, the method has the advantages of being stable and reliable in detection result, good in reproducibility, high in accuracy, convenient to operate, high in speed and the like.

Description

A kind of method of quick detection Morciiella Esculeuta Mycelia vigor
Technical field
The present invention relates to a kind of method detecting enzyme and live, be a kind of method detecting edible and medical fungi mycelia vigor in particular, belong to physiological activator detection field.
Background technology
Hickory chick (Morchella esculenta L.) has another name called delicious hickory chick, be commonly called as sheep sparrow bacterium, maize bacterium etc., be under the jurisdiction of Ascomycotina (Ascomycotina), discomycete (Discomycetes), Pezizale (Pezizales), Morchellaceae (Morchellaceae), morchella (Morchella) by Ainsworth categorizing system.Because its cap is a reticulate body being covered with depression and rib ridge, shape is gained the name like sheep tripe.Hickory chick has high nutritive value and medical value, is the large-scale edible medicinal fungi of holding concurrently of a class.According to surveying and determination, the protein content of hickory chick is 22.1%, containing various trace elements and Cobastabs such as 19 seed amino acids and zinc, selenium, iron, germanium, copper 1, B 2, B 12, nicotinic acid, pantothenic acid, the multivitamin such as biotin, comprising 8 kinds of essential amino acids, account for 47.5% (general edible fungi is 25% ~ 40%) of total amino acid content, higher than corn, wheat, soybean and meat product, the content of some nutritional labeling has exceeded " Cordyceps sinensis ".Modern medicine study shows, hickory chick has reducing blood lipid, immunity moderation function, antifatigue, radioresistance, antitumor action, and can alleviate the toxic and side effect that cancer patient's chemicotherapy causes.
Supply falls short of demand in the international market for hickory chick, and hold at high price.Therefore, artificial cultivation hickory chick is one of most interested problem of edible Mycota always.China successfully cultivates hickory chick at present, and Yunnan utilizes cultivation basswood hickory chick, and Sichuan utilizes the cultivation hickory chick that ploughs.
Hickory chick bacterial classification is the same with other edible and medical fungi, belongs to fungi, and hereditary variation is remarkable, and merit can not complete stability heredity, and easily aging or degenerate, and bringing greater risk to batch cultivation, is also compare stubborn problem.
Excellent production kind how is selected to be that cultivation is successfully crucial, traditional method is chosen seeds according to morphological feature, physiological and ecological characteristic and cultivation habit, but hickory chick bacterial classification requires that external condition is strict, affects obviously by extraneous factor, cause this method inaccurate, and waste time and energy.If spawn activity can be detected fast and accurately at Morciiella Esculeuta Mycelia growth phase will greatly improve seed selection efficiency, save cost simultaneously.
Summary of the invention
The object of the present invention is to provide a kind of method of quick detection Morciiella Esculeuta Mycelia vigor.
For achieving the above object, the technical solution used in the present invention is: a kind of method of quick detection Morciiella Esculeuta Mycelia vigor, comprises the steps:
1) cultural hypha: test strains mother Yu planted after cultivating in plating medium, cut the bacterium block on flat board, goes to concussion in Submerged liquid culturation base and cultivates, obtain bacterium liquid.
Preferably, test strains mother Yu planted in plating medium, after cultivating 10d, cut the bacterium block on flat board, go in Submerged liquid culturation base at 23 DEG C, at 23 DEG C, 6d is cultivated in 150rpm concussion, obtains bacterium liquid.
Preferably, described mother plants plating medium and is: by weight percentage, peeled potatoes 20%, glucose 2%, peptone 0.3%, KH 2pO 40.3%, MgSO 47H 2o 0.15%, pH value 7, water surplus.
Preferably, described Submerged liquid culturation base is: by weight percentage, peeled potatoes 20%, glucose 2%, peptone 0.3%, KH 2pO 40.3%, MgSO 47H 2o 0.15%, Cobastab 110mg/L, water surplus.
2) sample pre-treatments: get bacterium liquid in centrifuge tube, centrifugal, remove supernatant, extract mycelia, with normal saline flushing mycelia.
Preferably, get bacterium liquid in centrifuge tube, 4 DEG C of centrifugal 5min of 12000rpm, remove supernatant, get mycelia, with normal saline flushing mycelia.
3) bacterium liquid to be measured is prepared: weigh mycelia weight, add water, be made into bacterium liquid to be measured.
Preferably, the concentration of bacterium liquid to be measured is 0.1-0.2g/mL.Preferred, the concentration of bacterium liquid to be measured is 0.15g/mL.
4) react with TTC: accurately pipette in bacterium liquid to be measured, Tris-HCl damping fluid and TTC (2,3,5-triphenyltetrazolium chloride) solution to centrifuge tube and react.
Preferably, accurately pipette bacterium liquid to be measured, Tris-HCl damping fluid and TTC solution in centrifuge tube, at 37 DEG C, react 2-4h.
Preferably, the pH value of described Tris-HCl damping fluid is 8.4.
5) cessation reaction: after reaction, placed on ice by reactant liquor immediately, 4 DEG C centrifugal, obtains cell precipitation, adds extractant, obtain extract in the cell precipitation after centrifugal.
Preferably, described extractant is absolute ethyl alcohol.
6) extract OD is measured 485: extract is centrifugal, get supernatant, under 485nm wavelength, measure absorbance.
Preferably, by extract in the centrifugal 5min of 12000rpm, get supernatant, under 485nm wavelength, measure absorbance OD 485.
The invention has the beneficial effects as follows:
1. reliable and stable, the favorable reproducibility of measurement result of the present invention, accuracy are high.Enzyme is very effective and that specificity is high biocatalyst.Because enzyme is very responsive on the impact of environmental baseline, therefore the influence factor of enzyme assay is more, comprises the concentration of enzyme, buffer type, pH value, temperature, time etc., and the condition determination of the different enzymatic activity of different biosome inconsistent.The present invention, according to the characteristic of hickory chick, makes TTC not easily diffuse into feature in cell, and through repetition test, the best testing program of optimization, various condition is accurately controlled, and measurement result accuracy is high, favorable reproducibility, and reliable and stable.
2. the present invention's medicine used, instrument kind are few, and test procedure is simple, convenient operation, are easy to study and grasp.
3. reaction velocity of the present invention is fast, and measure absorbance simple, quick, the whole testing process time is short.
4. cheaply, consumption is few, and experimentation cost is low for the present invention's medicine used, reagent.
5. can be detected the quality of hickory chick bacterial classification by method of the present invention fast, then be applied in extension production, improve the high-quality productive rate of finished product.
Embodiment
The method of a kind of quick detection Morciiella Esculeuta Mycelia vigor of embodiment
(1) strains tested: strains tested used is in table 1.
Table 1 strains tested
(2) reagent:
Tris-HCl damping fluid: pH8.4; With with joining.
TTC solution: concentration expressed in percentage by weight is 0.5%; With with joining.
Physiological saline: concentration expressed in percentage by weight is 0.85%;
Female kind plating medium: by weight percentage, peeled potatoes 20%, glucose 2%, peptone 0.3%, KH 2pO 40.3%, MgSO 47H 2o 0.15%, pH value 7, water surplus.
Submerged liquid culturation base: by weight percentage, peeled potatoes 20%, glucose 2%, peptone 0.3%, KH 2pO 40.3%, MgSO 47H 2o 0.15%, Cobastab 110mg/L, water surplus.
(3) method of Morciiella Esculeuta Mycelia vigor is detected fast
1) cultural hypha
Test strains mother Yu planted in plating medium, after cultivating 10d, cut the bacterium block (1cm × 1cm) 2 pieces on flat board, go in Submerged liquid culturation base at 23 DEG C, at 23 DEG C, 6d is cultivated in 150rpm concussion, obtains bacterium liquid.
2) sample pre-treatments
Get 2mL bacterium liquid in centrifuge tube, 4 DEG C of centrifugal 5min of 12000rpm, remove supernatant, get mycelia, with 1ml normal saline flushing mycelia, 4 DEG C of centrifugal 5min of 12000rpm, remove supernatant, repeat 4 times.
3) bacterium liquid to be measured is prepared
Weigh centrifugal after mycelia weight, add water, be made into the bacterium liquid to be measured that concentration is 0.15g/mL.
4) react with TTC
Accurately pipette each 400 μ L of bacterium liquid to be measured, Tris-HCl damping fluid and TTC solution in centrifuge tube, at 37 DEG C, react 3h.
5) cessation reaction
After having reacted, placed on ice by reactant liquor immediately, 4 DEG C centrifugal washes 2 times again, obtains cell precipitation, adds 1mL absolute ethyl alcohol, obtain extract in the cell precipitation after centrifugal.Color is more deeply felt open-birth and is become the amount of TF larger, and the activity of dehydrogenasa is higher.
6) extract OD is measured 485
By extract in the centrifugal 5min of 12000rpm, get supernatant 200 μ L on 752N type ultraviolet spectrophotometer, under 485nm wavelength, measure absorbance OD 485.When there being dehydrogenation reaction in microbial cell, TTC just accepts hydrogen atom and is reduced into red San Ben Ji Jia Za (TF), re-use absolute ethyl alcohol and extract TF, its absorbance is surveyed under 485nm wavelength, with absorbance value reflection dehydrogenase activity, absorbance value is larger, and the activity of dehydrogenasa is higher.
7) measurement result is in table 2
The absorbance OD of table 2 different strains 485
From table 2, measured by method of the present invention, the TF growing amount of ' the distant hickory chick No. 1 ' at the microbial project center, academy of agricultural sciences of Liaoning Province of E1 representative is maximum, OD 485maximum, reflect that enzyme activity is the strongest, the TF growing amount of ' distant sheep tames and dociles-2 ' is minimum, OD 485minimum, reflect that enzyme activity is the most weak.
8) cultivation checking
8.1) cultivation season: hickory chick is a kind of low form mushroom.Most in October ~ cultivation in November, suitable mycelial growth is grown, and March in next year ~ temperature rise in April, fructification starts to be formed.
8.2) composition of compost is cultivated: by weight percentage, consist of: agricultural residues powder 74.5%, wheat bran 20%, calcium superphosphate l%, gypsum l%, lime 0.5%, vegetable mould 2%, peat 1%.
8.3) cultivate the making of compost: by above-mentioned proportioning, mixed by raw material, then add water and regulate moisture to about about 60%, load 17cm × 33cm polypropylene plastics pocket, every sacked material is about 500g, more than normal-pressure sterilization 20h.
8.4) cultural method: can cultivate after the sterilization of mushroom room, first at one block, every layer of bed surface upper berth plastic foil, then spreads the thick vegetable mould of 3cm, is arranged one by one in bed by the bacterium rod sloughing polybag after clapping; General lm bed surface can arrange 17cm × 33cm polybag 40.Drained bacterium rod is gently sprayed water afterwards and is got final product earthing 3 ~ 5cm 1 time, earthing rear surface covers the thick broad leaf tree fallen leaves of 2cm again, keep ground moistening, fructification can be grown after 1 month, now air humidity is 85 ~ 90%, hickory chick is unearthed rear 7 ~ 10d with regard to energy growth and maturity, and general color becomes light grey or isabelline by Dark grey and can gather.
Test totally 5 bacterial strains, each bacterial strain 200 bags.Send out bacterium in holding chamber after inoculation, periodic logging mycelial growth situation, send out bacterium number of days, after mycelia is covered with, de-bag swing-bed, carries out management of producing mushroom after fructification grows, and plucks, and surveys and produces.
8.5) result is cultivated: as can be seen from Table 3, E1 output is the highest, is 230 strains/m 2; Secondly be E4 and E3, reach 150 strains/m 2above; E2 and E5 output is minimum, less than 100 strains/m 2.
The impact that the Morchella esculenta (L.) Pers sporophore of table 3 different strains grows
Can be found out by experiment in cultivation, at Morciiella Esculeuta Mycelia growth phase, the enzyme activity adopting method of the present invention to measure is consistent with the result of experiment in cultivation, proves that method accuracy of the present invention is high, is conducive to batch cultivation.

Claims (10)

1. detect a method for Morciiella Esculeuta Mycelia vigor fast, it is characterized in that comprising the steps:
1) cultural hypha: test strains mother Yu planted after cultivating in plating medium, cut the bacterium block on flat board, goes to concussion in Submerged liquid culturation base and cultivates, obtain bacterium liquid;
2) sample pre-treatments: get bacterium liquid in centrifuge tube, centrifugal, remove supernatant, extract mycelia, with normal saline flushing mycelia;
3) bacterium liquid to be measured is prepared: weigh mycelia weight, add water, be made into bacterium liquid to be measured;
4) react with TTC: accurately pipette in bacterium liquid to be measured, Tris-HCl damping fluid and TTC solution to centrifuge tube and react;
5) cessation reaction: after reaction, placed on ice by reactant liquor immediately, 4 DEG C are centrifugal, obtain cell precipitation, add extractant, obtain extract in the cell precipitation after centrifugal;
6) extract OD is measured 485: extract is centrifugal, get supernatant, under 485nm wavelength, measure absorbance.
2. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1, it is characterized in that: described step 1) be: test strains mother Yu is planted in plating medium, after cultivating 10d at 23 DEG C, cut the bacterium block on flat board, go in Submerged liquid culturation base, at 23 DEG C, 6d is cultivated in 150rpm concussion, obtains bacterium liquid.
3. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1 and 2, is characterized in that: described mother plants plating medium and is: by weight percentage, peeled potatoes 20%, glucose 2%, peptone 0.3%, KH 2pO 40.3%, MgSO 47H 2o 0.15%, pH value 7, water surplus.
4. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1 and 2, is characterized in that: described Submerged liquid culturation base is: by weight percentage, peeled potatoes 20%, glucose 2%, peptone 0.3%, KH 2pO 40.3%, MgSO 47H 2o 0.15%, Cobastab 110mg/L, water surplus.
5. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1, is characterized in that: described step 2) be: get bacterium liquid in centrifuge tube, 4 DEG C, the centrifugal 5min of 12000rpm, remove supernatant, get mycelia, with normal saline flushing mycelia.
6. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1, is characterized in that: described step 3) in, the concentration of bacterium liquid to be measured is 0.1-0.2g/mL.
7. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1, is characterized in that: described step 4) be: accurately pipette bacterium liquid to be measured, Tris-HCl damping fluid and TTC solution in centrifuge tube, at 37 DEG C, react 2-4h.
8. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1 or 7, is characterized in that: the pH value of described Tris-HCl damping fluid is 8.4.
9. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1, is characterized in that: described step 5) in: described extractant is absolute ethyl alcohol.
10. the method for a kind of quick detection Morciiella Esculeuta Mycelia vigor according to claim 1, is characterized in that: described step 6) be: by extract in the centrifugal 5min of 12000rpm, get supernatant, under 485nm wavelength, measure absorbance OD 485.
CN201510401452.5A 2015-07-09 2015-07-09 Method for quickly detecting vigor of morchella esculenta L. hyphae Pending CN104990881A (en)

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Publication number Priority date Publication date Assignee Title
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US20140370546A1 (en) * 2012-01-05 2014-12-18 Novartis Ag Protease Deficient Filamentous Fungal Cells and Methods of Use Thereof

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102048770A (en) * 2009-11-06 2011-05-11 华中科技大学 Artificially-cultured cordceps militaris sporocarp extract and preparation process thereof
US20140370546A1 (en) * 2012-01-05 2014-12-18 Novartis Ag Protease Deficient Filamentous Fungal Cells and Methods of Use Thereof
CN103141302A (en) * 2013-03-19 2013-06-12 中国科学院昆明植物研究所 Production method for morchella importuna cultivars

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Title
周春生等: "《TTC-脱氢酶活性检测方法的研究》", 《吉林建筑工程学院学报》 *
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Application publication date: 20151021