CN109337895A - A kind of production method of good quality and high output selenium-enriched hericium erinaceus strain - Google Patents

A kind of production method of good quality and high output selenium-enriched hericium erinaceus strain Download PDF

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CN109337895A
CN109337895A CN201811422844.XA CN201811422844A CN109337895A CN 109337895 A CN109337895 A CN 109337895A CN 201811422844 A CN201811422844 A CN 201811422844A CN 109337895 A CN109337895 A CN 109337895A
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hericium erinaceus
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沙淼
高雪
谷玉秋
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Shanghai Zijing Biotechnology Co.,Ltd.
Wu Zitao
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Jiangsu Gao Hang Agricultural Science And Technology Co Ltd
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Abstract

The present invention provides a kind of production methods of good quality and high output selenium-enriched hericium erinaceus strain, belong to Edible Fungi field.The process flow of this method includes the following steps: (1) production parent species；Step 2, production shaking flask strain；Step 3, shaking flask strain electric field magnetic field are dealt with altogether；Step 4, production Selenium-enriched fermentation strain.The present invention produces hedgehog fungus bacterial using liquid fermentation technology, both the solids manufacture method of traditional hedgehog fungus bacterial had been changed, shorten the strain production cycle, improve strain quality and yield, it reduces costs, selenium element is added during liquid fermentation simultaneously, has obtained the selenium-enriched hericium erinaceus strain that organic selenium content is.Bacterium germination is fast, growing way is good, yield is high after strain inoculation, and organic selenium content is up in the hericium erinaceus fruiting body of output, has great market value.

Description

A kind of production method of good quality and high output selenium-enriched hericium erinaceus strain
Technical field
The invention belongs to Edible Fungi fields, are related to a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain, especially It is related to a kind of method using liquid fermentation technology production selenium-enriched hericium erinaceus strain.
Background technique
Hericium erinaceus (Hericium erinaceus) it is called Hericium erinaceus, hedgehog bacterium, cauliflower bacterium or mountain volt bacterium etc., belong to basidiomycetes Door, Hymenomycetes, Aphyllophorales, tooth bacterium section, hedgehog hydnum category is thick due to its fructification circle, is often suspended from trunk, is covered with needle-shaped bacterium Thorn, the pole of figure is like the head of monkey, and hence obtain one's name Hericium erinaceus.Hericium erinaceus bacterial context is fresh and tender, aromatic palatable, there is the title of " meat or fish in element ", with Bear's paw, sea cucumber, shark's fin and the referred to as big famous dish of Chinese tradition four.Hericium erinaceus is not only delicious food materials, while being also a kind of medicinal material. Chinese medicine thinks that Hericium erinaceus is sweet natured, enters stomach meridian, helps to digest, and relieving the five internal organs are strengthened the body resistance to consolidate the constitution, and has effects that brain refreshing, Modern medicine proves that Hericium erinaceus has good medical value, can be used for treating indigestion, gastric ulcer, antral gastritis, stomachache, gasteremphraxis And the diseases such as neurasthenia.
Hericium erinaceus has very high nutritive value, and every 100g Hericium erinaceus is 2 times of mushroom, rouge containing about protein 23.6g Fat 4.9g is qualified high protein, low-fat food.Furthermore Hericium erinaceus be also rich in carbohydrate, various vitamins and The inorganic salts such as calcium, iron, magnesium, zinc.Hericium erinaceus is not only full of nutrition, also has strengthen immunity, and antitumor, hypoglycemic, blood lipid resists Oxidation, anti-aging, anti-radiation, anti-mutation, antibacterial, liver protection improve body's hypoxia tolerance, the functions such as antifatigue.These functions Mainly albumen, polypeptide, polysaccharide, terpene, sterols isoreactivity ingredient effect result.Hericium erinaceum polysaccharide is current most study One of Hericium erinaceus active constituent.Largely studies have shown that hericium erinaceum polysaccharide can remove free radical, have significant anti-oxidant Function is a kind of ideal natural resource.Hericium erinaceum polysaccharide can also enhance human immunologic function, inhibit cancer cell Proliferation.Hericium erinaceus albumen and polypeptide also have an immunoregulation effect and anti-oxidation function, and with needle mushroom, mushroom, double spore mushrooms The common mushroom such as mushroom, coprinus comatus is compared, and the content of Hericium erinaceus protein is higher under phase homogenous quantities, thus its immunoregulation effect and Anti-oxidation function is also stronger.
Hericium erinaceus is the rare kind in edible mushroom.Wild Hericium erinaceus more be grown in remote, thickly forested mountains, it is saprophytic it is withered tree or The withered part of tree living, it is rare in Plain and knob.However wild Hericium erinaceus rare numbers, in order to meet the market demand, The cultivation industry development of Hericium erinaceus is very rapid, and development situation at home and abroad is all expected.The artificial cultivation of China Hericium erinaceus It starts from 1959, starts to promote and apply to the 1970s, at present northeast each province and Henan, Hebei, Tibet, Shanxi, sweet There are output in the provinces such as respectful, Shaanxi, the Inner Mongol, Sichuan, Hubei, Guangxi, Zhejiang.China Hericium erinaceus mainly uses traditional cultivation mould Formula, by being inoculated with solid spawn fruiting under field conditions (factors), but this planting type is by factors such as natural climate and seasonalities Limitation, and mechanization degree is low, planting density is low, production of hybrid seeds technique is cumbersome, low efficiency, can not achieve the production of anniversary metaplasia, Wu Faman The growing consumption demand of foot.In recent years, the factory culture development of the common edible mushroom such as oyster mushroom, needle mushroom, seafood mushroom is fast Speed, technology also gradually mature, and scale increasingly also expands, and liquid fermentation technology is used to produce liquid spawn, are used to replace tradition Solid kind is applied in these edible fungus industrial cultivations.Liquid strain have it is with short production cycle, hyphal development point is more, Sprout the advantages that fast, yield is high, mycelia sprawling is rapidly after inoculation, cell age is neat, at low cost, moreover it is possible to pollution is effectively reduced, with The improvement of people's living standards and the raising recognized edible fungus health-care effect, the batch production which is applied to Hericium erinaceus is planted Training will also become following development trend.
Selenium is the indispensable microelement of human body, and is a kind of very capable antioxidant, can effectively be removed Carcinogen-free radical has the good reputation of " king of anticancer ".Selenium can improve human immunity, promote the proliferation and antibody of lymphocyte With the synthesis of immunoglobulin.Selenium has apparent inhibition to kinds cancers such as colon cancer, cutaneum carcinoma, liver cancer, breast cancer and prevents The effect of shield has stronger anticancer activity in the intracorporal mesostate methyl selenol of machine.Selenium and vitamin E, garlic The nutrients such as element, linoleic acid, germanium, zinc have effects that collaboration is oxidation resistant, increase antioxidant activity.Meanwhile selenium have mitigate and Alleviate the effect of heavy metal toxicity.Therefore in recent years, selenium-enriched food, Selenium-enriched health food are very burning hot.Edible mushroom is as Enriching Selenium The carrier of element, because its cultivated area is wide, easy to operate, concentration effect is significant, is just being widely used in China.Traditional hedgehog hydnum Mushroom selenium-rich method is selenium element to be added in cultivation matrix, and then obtain selenium-enriched hericium erinaceus fructification.And with edible fungus liquid The utilization of fermentation technique adds selenium element in Hericium erinaceus fermentation process, so that it may selenium-enriched hericium erinaceus mycelium and fermentation liquid are obtained, It is used directly for extracting active constituent and as nutraceutical additive, can also be used as strain production selenium-enriched hericium erinaceus, And such method mycelium has a extensive future to the absorptivity and conversion ratio highest of selenium, has great market latent Power.
Summary of the invention
Good quality and high output selenium-enriched hericium erinaceus strain is produced using liquid fermentation technology the purpose of the present invention is to provide a kind of Method to solve the problems, such as Hericium erinaceus production cycle length, low efficiency, low output, at high cost in the prior art, while improving monkey The content of organic selenium in head mushroom, conducive to meeting the needs of hedgehog hydnum mushroom industrialized cultivation and in the market selenium-rich product.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of production method of good quality and high output selenium-enriched hericium erinaceus strain, which is characterized in that the process flow of this method is as follows:
Step 1, production parent species
Bacterial strain inoculation shovel is aseptically taken 1 by the screening Hericium erinaceus strain excellent that mycelial growth rate is fast, growing way is strong Block size is about 0.5cm2Fungus block, be inoculated on PDA enriched medium inclined-plane, 25 DEG C of culture 5-7d obtain slant strains, then Slant strains are transferred on PDA enriched medium plate, 25 DEG C of cultures cover with plate until mycelia, obtain parent species；
Step 2, production shaking flask strain
The Hericium erinaceus parent species of plate culture are taken into bacterium piece 3-4 piece with diameter 5mm punch, access is equipped with the level-one of fluid nutrient medium In shaking flask, level-one shaking flask strain is cultivated to obtain；Again by level-one shaking flask strain according to 7-9%(v/v) inoculum concentration be transferred to equipped with liquid In the second-level shake flask of culture medium, second-level shake flask strain is cultivated to obtain；Finally by second-level shake flask strain according to 7-9%(v/v) inoculum concentration It is transferred in the three-level shaking flask equipped with fluid nutrient medium, cultivates to obtain three-level shaking flask strain；
Step 3, shaking flask strain electric field magnetic field are dealt with altogether
The shaking flask strain made first is placed in the high-voltage electrostatic field of 100KV/m and handles 1min, is then placed into 50mT's 5min is handled in uniform magnetic field；
Step 4, production Selenium-enriched fermentation strain
1. fermentative medium formula: corn flour 30-36g/L, casein 5-9g/L, soybean stalk powder 15-20g/L, vinegar grain 6- 10g/L, maize cob meal 7-10g/L, black fungus rich in selenium mushroom bran 5-6g/L, citric acid 2.4-3.0g/L, selenium-rich alfalfa meal 6.8- 7.4g/L, MgSO4·7H2O0.9-1.1g/L, KH2PO41.8-2.2g/L, Na2SeO337 ~ 43mg/L, astragalus root dregs 1.4- 2.0g/L, lichens powder 9-10g/L, fly maggot powder 3.6-4.8g/L, oyster shell powder 2.6-3.0g/L, VB650-70mg/L, glycerol 1.7-2.3mL/L, edible defoaming agent 0.25-0.35g/L, surplus are magnetized water, and initial pH is adjusted to 5.3-5.7；According to above Formula prepares fermentation medium；
2. fermentor cleaning and sky go out: sterilizing at 121-125 DEG C of temperature, pressure 0.11-0.13Mpa after fermentor is cleaned 30min；
3. charging sterilizing: prepared fermentation medium being added in fermentor from feeding port, the loading amount of fermentation medium is hair The 50-60% of fermentation tank capacity opens air pump and stirs 2-3min, sterilizes at 121-125 DEG C of temperature, pressure 0.11-0.13Mpa 40min；
4. inoculation: after sterilizing, when fermentation medium is cooled to 30 DEG C or less, general's treated shaking flask strain is according to sterile Operation accesses fermentor, inoculum concentration 11-13% from inoculation mouth；
5. fermented and cultured: starting fermentor enters cultivation conditions, and cultivation temperature is set as 25-27 DEG C, and blender revolving speed is set as 140-160r/min, ventilatory capacity are set as the 1-3 days 0.8 v/v/min, then 1.2 v/v/min to fermentation ends, when culture Between 6-8d.
In the step 1, the ingredient of PDA enriched medium are as follows: potato 180-220g/L, trehalose 17-23g/L, fine jade Rouge 18-22g/L, peptone 3-4g/L, lemon juice 3-5mL/L, brewer's wort 10-14mL/L, VB120-30mg/L, VB2 20- 30mg/L, MgSO4·7H2O0.4-0.6g/L, KH2PO40.8-1.2g/L, surplus are tap water, and pH is adjusted to 4.8-5.2.
In the step 2, the ingredient of fluid nutrient medium are as follows: soybean starch 4-6g/L, protein hydrolysate 0.6-1.0g/L, grape Sugared 20-26g/L, sweet potato powder 14-18g/L, brewer's wort 10-14mL/L, succus liquiritiae 8-9mL/L, astragalus root dregs 1.4-2.0g/L, MnSO4 0.22-0.26g/L, MgSO4·7H2O0.4-0.6g/L, KH2PO40.8-1.2g/L, choline chloride 0.5-0.7mg/L, IAA 1.3-1.5mg/L, surplus are tap water, and pH is adjusted to 4.8-5.2.
In the step 2, the liquid amount of shaking flask strains at different levels is that every 500mL shaking flask fills 150mL fluid nutrient medium.
In the step 2, the condition of culture of shaking flask strains at different levels is equal are as follows: 25-27 DEG C of shaking table temperature, shaking speed 140- 160r/min, incubation time 4-5d.
In the step 4, sterile working when inoculation refers to 75% ethanol of inoculation mouth, and with 95% ethyl alcohol calcination, Sebific duct in shaking flask is inserted in inoculation mouth by flame snare in inoculation mouth, and quickly.
Magnetized water refers to by Management of magnetic water device treated tap water, the type of Management of magnetic water device used in the step 4 Number be CFG/L-40, flow 7.0-9.0t/h, vertical centre magnetic field strength be 160mT.
The beneficial effects are mainly reflected as follows the following aspects:
1) of the invention to produce hedgehog fungus bacterial using liquid fermentation technology, with short production cycle, mycelia grows fast, yield height, simultaneously Selenium element is added in the fermentation medium, so that sorption enhanced is organic selenium to mycelium during the fermentation, has obtained selenium-rich monkey Head mushroom strains, the strain is for that can obtain selenium-enriched hericium erinaceus after producing.Compared with traditional Hericium erinaceus selenium-rich method, the present invention Method selenium element utilization rate and high conversion rate, the content of organic selenium is high in strain, so that in the hericium erinaceus fruiting body produced The content of organic selenium is also improved.
2) present invention is directed to the growth characteristics of Hericium erinaceus, optimizes liquid fermentation and culture process；By to Hericium erinaceus shaking flask Strain carries out electric field magnetic field and deals with altogether, and magnetized water is used during Selenium-enriched fermentation, can promote hericium mycelium The accumulation of biomass improves its enrichment and conversion capability to selenium.
3) selenium-enriched hericium erinaceus cultivating bacterial spawn Hericium erinaceus of the invention is used, organic selenium content is 87.84mg/ in fructification Kg is 3 times of traditional selenium-enriched hericium erinaceus, selenium-rich significant effect.
Specific embodiment
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, combined with specific embodiments below to this Invention is described further.
The measuring method of some indexs is as follows in embodiment:
1) hypha biomass measures: taking 100mL strain liquid that mycelium is collected by filtration, the 5000r/min after distillation water washing 3 ~ 4 times It is centrifuged 10min, precipitating is separated with supernatant.Precipitating is taken to be put into baking oven, drying to constant weight at 80 DEG C, it is dry for electronic balance weighing Weight.
2) bacterium bulb diameter measures: taking 1mL strain liquid to be diluted with water 10 times, takes 20 bacterium balls in culture dish along fixation at random Straight line be in line, with slide calliper rule measure total length, be repeated 3 times, take its average value.
3) Peloton density measures: taking 1mL fermentation liquid to be diluted with water 10 times, sets black box paper in culture dish underlay, counts Quantity.
4) total Determination of Selenium: the mycelia that drying to constant weight is worn into uniform powder and carries out digestion process.Accurately weigh this Powder 1g is added 10mL nitric acid and places more than for 24 hours, and then low-temperature heat dissolves it all, and 3mL perchloric acid is added after slightly cooling down Continue to heat, it is cooling after constant volume to 50mL.Sample after taking 10mL digestion process, is diluted with water 40mL, and pH is adjusted to 2- 3, the EDTA solution 2mL that mass fraction is 5% is added, 3, the 3- diaminobenzidine solution that mass fraction is 0.5% is then added 2mL shakes up.It is taken out after being protected from light 30min, pH is adjusted to 7,10mL toluene oscillation extraction 2min, stratification, first is added Benzene layer is put into cuvette, and absorbance is measured at 430nm, obtains corresponding concentration according to selenium standard curve, calculates in mycelia Total Se content.
5) organic selenium content measures: accurately weighing above-mentioned mycelia powder 1g, without digestion process, distilled water constant volume is added to arrive 50mL measures inorganic Se content by the method for the total Se content of said determination.Organic selenium content=inorganic the Se content of total Se content-.
Embodiment 1
It is tested according to the production procedure of good quality and high output selenium-enriched hericium erinaceus strain provided by the invention, steps are as follows:
Step 1, production parent species
The screening Hericium erinaceus strain excellent " Changshan hedgehog hydnum " that mycelial growth rate is fast, growing way is strong, aseptically, bacterial strain is used It is about 0.5cm that inoculation shovel, which takes 1 block size,2Fungus block, be inoculated on PDA enriched medium inclined-plane, 25 DEG C of culture 6d obtain inclined-plane Strain, then slant strains are transferred on PDA enriched medium plate, 25 DEG C of cultures cover with plate until mycelia, obtain parent species. The ingredient of PDA enriched medium are as follows: potato 200g/L, trehalose 20g/L, agar 20g/L, peptone 3.5g/L, lemon juice 4mL/L, brewer's wort 12mL/L, VB125mg/L, VB225mg/L, MgSO4·7H2O0.5g/L, KH2PO41.0g/L, surplus are Water, pH are adjusted to 5.0.
Step 2, production shaking flask strain
The Hericium erinaceus parent species of plate culture are taken into bacterium piece 3-4 piece with diameter 5mm punch, access is equipped with fluid nutrient medium (ingredient Are as follows: soybean starch 5g/L, protein hydrolysate 0.8g/L, glucose 23g/L, sweet potato powder 16g/L, brewer's wort 12mL/L, succus liquiritiae 8.5mL/L, astragalus root dregs 1.7g/L, MnSO4 0.24g/L, MgSO4·7H2O0.5g/L, KH2PO41.0g/L, choline chloride 0.6mg/L, IAA 1.4mg/L, surplus are water, and pH is adjusted in level-one shaking flask 5.0), cultivate to obtain level-one shaking flask strain；Again will Level-one shaking flask strain is according to 8%(v/v) inoculum concentration be transferred in the second-level shake flask equipped with fluid nutrient medium, cultivate second level is shaken Bottle strain；Finally by second-level shake flask strain according to 8%(v/v) inoculum concentration be transferred in the three-level shaking flask equipped with fluid nutrient medium, Cultivate to obtain three-level shaking flask strain；The liquid amount of shaking flasks at different levels is that every 500mL shaking flask fills 150mL fluid nutrient medium, shaking flask bacterium at different levels The condition of culture of kind is equal are as follows: and 26 DEG C of shaking table temperature, shaking speed 150r/min, incubation time 5d.To shaking flask bacterium after culture Kind carries out the measurement of hypha biomass, bacterium bulb diameter and Peloton density.
Step 3, shaking flask strain electric field magnetic field are dealt with altogether
The shaking flask strain made first is placed in the high-voltage electrostatic field of 100KV/m and handles 1min, is then placed into 50mT's 5min is handled in uniform magnetic field.
Step 4, production Selenium-enriched fermentation strain
1. fermentative medium formula: corn flour 33g/L, casein 7g/L, soybean stalk powder 17.5g/L, vinegar grain 8g/L, corncob Powder 8.5g/L, black fungus rich in selenium mushroom bran 5.5g/L, citric acid 2.7g/L, selenium-rich alfalfa meal 7.1g/L, MgSO4·7H2O1.0g/L, KH2PO42.0g/L, Na2SeO340mg/L, astragalus root dregs 1.7g/L, lichens powder 9.5g/L, fly maggot powder 4.0g/L, oyster shell powder 2.8g/L, VB660mg/L, glycerol 2.0mL/L, edible defoaming agent 0.3g/L, surplus are magnetized water, and initial pH is adjusted to 5.5； Fermentation medium is prepared according to the above formula；Magnetized water refers to by Management of magnetic water device treated tap water, at magnetic water used The model CFG/L-40 of device, flow 7.0-9.0t/h are managed, vertical centre magnetic field strength is 160mT；
2. fermentor cleaning and sky go out: sterilizing at 121-125 DEG C of temperature, pressure 0.11-0.13Mpa after fermentor is cleaned 30min；
3. charging sterilizing: prepared fermentation medium being added in fermentor from feeding port, the loading amount of fermentation medium is hair The 50-60% of fermentation tank capacity opens air pump and stirs 2-3min, sterilizes at 121-125 DEG C of temperature, pressure 0.11-0.13Mpa 40min；
4. inoculation: after sterilizing, when fermentation medium is cooled to 30 DEG C or less, general's treated shaking flask strain is according to sterile (75% ethanol of inoculation mouth, and with 95% ethyl alcohol calcination, flame snare, and quickly will be in shaking flasks in inoculation mouth for operation Sebific duct is inserted in inoculation mouth) fermentor, inoculum concentration 12% are accessed from inoculation mouth；
5. fermented and cultured: starting fermentor enters cultivation conditions, and cultivation temperature is set as 26 DEG C, and blender revolving speed is set as 150r/min, ventilatory capacity are set as the 1-3 days 0.8 v/v/min, then 1.2 v/v/min to fermentation ends, incubation time 7d. The measurement of hypha biomass, total Se content and organic selenium content is carried out to fermenting microbe after fermentation.
Embodiment 2
For fluid nutrient medium more of the invention and influence of the conventional liq culture medium to Hericium erinaceus shaking flask growth, this reality It applies example and the fluid nutrient medium used in 1 step 2 of embodiment is replaced with into following conventional medium: culture medium A (glucose 30g/L, Yeast extract 10g/L, KH2PO4 1g/L、MgSO4·7H2O0.5g/L), culture medium B(soluble starch 25g/L, yeast extract 25g/ L、KH2PO4 1g/L、MgSO4·7H2O1g/L), culture medium C (maltose 20g/L, wheat bran 15g/L, peptone 20g/L, yeast Powder 2g/L, KH2PO4 1.5g/L、MgSO4·7H2O0.75g/L), other conditions are constant, measure the mycelia biology of shaking flask strain Amount, bacterium bulb diameter and Peloton density, statistics such as the following table 1 together with the result of embodiment 1.
When carrying out shaking flask culture using fluid nutrient medium of the invention it can be seen from 1 result of table, Hericium erinaceus shaking flask strain Hypha biomass and Peloton density all reach maximum value, and bacterium bulb diameter is of moderate size.As liquid spawn, hypha biomass Bigger, bacterium bulb diameter is smaller (1-2mm), and mycelium pellet quantity is more, and in inoculation, mycelium germination point is more, mycelial growth rate Also faster, ferment effect is with regard to more preferable.Compared with conventional liq culture medium, fluid nutrient medium of the invention optimizes carbon source, nitrogen source Ingredient and ratio, be also additionally added to traditional Chinese medicine ingredients and growth promoter, therefore the promotion to Hericium erinaceus shaking flask growth Effect is more preferable.
Embodiment 3
1) in order to prove electric field magnetic field deal with altogether on Selenium-enriched fermentation strain generate it is positive influence, the present embodiment is to making Shaking flask strain carries out the following processing: 1) independent electric field treatment；2) individually magnetic field processing；3) it does not handle, measures the bacterium of fermenting microbe Silk biomass, total Se content and organic selenium content, statistics such as the following table 2 together with the result of embodiment 1.
It can be seen from 2 result of table compared with not handling, electric field magnetic field deals with altogether, independent electric field treatment and independent magnetic When the processing of field, the mycelial biomass of Hericium erinaceus fermenting microbe, total Se content and organic selenium content are improved.With it is independent It is compared using a certain processing mode, electric field magnetic field deals with significant effect altogether, illustrates to handle shaking flask strain with the processing mode, Mycelia vigor can be more excited, mycelium growth vigor is strong after inoculation, and metabolism is vigorous, and then improves the ability of mycelia Enriching Selenium.
2) in order to prove to prepare influence of the fermentation medium to Hericium erinaceus fermenting microbe growth and selenium-rich using magnetized water, this Embodiment prepares fermentation medium using tap water, and other conditions are constant, measures the hypha biomass of fermenting microbe, total Se content And organic selenium content, statistics such as the following table 3 together with the result of embodiment 1.
It can be seen from 3 result of table compared with preparing fermentation medium using tap water, after magnetized water, Hericium erinaceus The mycelial biomass of fermenting microbe, total Se content and organic selenium content are improved.It is similar to the effect of magnetic field processing, magnetization Water can also play the role of exciting mycelia vigor, promote mycelia growth, improve mycelia Enriching Selenium ability.
Embodiment 4
In order to which fermentation medium and normal fermentation culture medium more of the invention are to the shadow of Hericium erinaceus fermenting microbe growth and selenium-rich It rings, the fermentation medium used in 1 step 4 of embodiment is replaced with following conventional medium: culture medium A (sucrose by the present embodiment 30g, yeast extract 10g, corn flour 10g, KH2PO4 1g/L、MgSO4·7H2O0.5g/L), culture medium B(glucose 30g/L, beans Cake powder 7g/L, wheat bran 2 g/L, KH2PO4 1g/L、MgSO4·7H2O0.5g/L), culture medium C (brewer's wort 30mL/L, peptone 20g/L、KH2PO4 1.5g/L、MgSO4·7H2O0.75g/L), Na is equally added2SeO3 40mg/L, other conditions are constant, survey Hypha biomass, total Se content and the organic selenium content for determining Hericium erinaceus fermenting microbe count as follows together with the result of embodiment 1 Table 4.
Selenium-enriched fermentation culture medium of the invention is used it can be seen from 4 result of table, hypha biomass, total Se content and is had Machine Se content reaches peak, illustrates that Selenium-enriched fermentation culture medium of the invention not only contributes to the accumulation of hypha biomass, also Be conducive to the enrichment of selenium.Selenium-enriched fermentation culture medium of the invention is full of nutrition, and carbon source, nitrogen source proportion rationally, are not only added to Na2SeO3As inorganic selenium source, black fungus rich in selenium mushroom bran, selenium-rich alfalfa meal, astragalus root dregs, fly maggot powder, oyster shell powder also added The equal higher raw material of Se contents is used in compounding as other nutritional ingredients in organic selenium source, with culture medium, can promote hedgehog hydnum Absorption and conversion of the mushroom silk to selenium, moreover it is possible to which the growth for promoting mycelia reaches higher effect.
Embodiment 5
The present embodiment is the Optimum Experiment of some important factor in order in the production process to Selenium-enriched fermentation strain.
1) influence of the cultivation temperature to Hericium erinaceus fermenting microbe growth and selenium-rich: this test is provided with 20 DEG C, 23 DEG C, 26 DEG C, the fermented and cultured temperature of 29 DEG C of several gradients, measure the hypha biomass of Hericium erinaceus fermenting microbe, total Se content respectively and have As a result machine Se content counts such as the following table 5.
Hericium erinaceus fermenting microbe can be grown within the temperature range of setting it can be seen from 5 result of table, when temperature is lower When, mycelial growth breeding is slower, and biomass is lower, when the temperature is excessively high, understands shadow mycelium to the concentration effect of selenium, at 26 DEG C When, hypha biomass, total Se content and organic selenium content all reach maximum, therefore select 26 DEG C for optimum culturing temperature.
2) influence of the fermentation medium initial pH value to Hericium erinaceus liquid Selenium-enriched fermentation: this test be provided with 4.5,5.5, 6.5, the initial pH value of 7.5 several gradients measures hypha biomass, total Se content and the organic selenium of Hericium erinaceus fermenting microbe respectively As a result content counts such as the following table 6.
The height of fermentation medium initial pH value directly affects mycelial growth, and it is quick that suitable pH value is conducive to mycelium Growth and breeding.It can be seen from 6 result of table when initial pH value is 5.5, hypha biomass, total Se content and organic selenium contain Amount all reaches maximum, therefore selects 5.5 for optimal initial pH value.
3) influence of the inoculum concentration to Hericium erinaceus liquid Selenium-enriched fermentation: this test is provided with 8%, 12%, 16%, 20% several gradients Inoculum concentration, respectively measure Hericium erinaceus fermenting microbe hypha biomass, total Se content and organic selenium content, as a result count it is as follows Table 7.
The number of inoculum concentration directly affects yield, the cultivation cycle of target product, and inoculum concentration can be accelerated greatly mycelial Growth so as to shorten culture period, but will lead to that mycelial growth is excessively prosperous, nutriment consumption is too fast, and later stage fermentation lacks of staying power, The synthesis of the final accumulation for influencing biomass and metabolite.It can be seen from 7 result of table when inoculum concentration is 12%, Hericium erinaceus The hypha biomass of fermenting microbe, total Se content and organic selenium content reach maximum value, therefore select 12% for optimum inoculation amount.
4) Na in fermentation medium2SeO3Influence of the concentration to Hericium erinaceus liquid Selenium-enriched fermentation: this test is prepared for Na2SeO3Concentration is respectively the fermentation medium of 20,40,60,80mg/L, measures the mycelia biology of Hericium erinaceus fermenting microbe respectively As a result amount, total Se content and organic selenium content count such as the following table 8.
Se content is with Na in fermentation medium in mycelium it can be seen from 8 result of table2SeO3The rising of concentration and on It rises, works as Na2SeO3Concentration tends towards stability when reaching 40mg/L, and works as Na2SeO3Concentration reach 60mg/L and it is higher when, mycelia Biomass is reduced therewith, illustrates the Na of high concentration2SeO3The growth that can inhibit hericium mycelium, is unfavorable for mycelium pair instead The enrichment of selenium.Work as Na2SeO3When concentration is 40mg/L, the hypha biomass of Hericium erinaceus fermenting microbe reaches maximum value, therefore selects 40mg/L is optimum N a2SeO3Concentration.
Embodiment 6
Hericium erinaceus culture test is carried out using selenium-enriched hericium erinaceus strain of the invention, cultural method is according to following conventional method:
1) compost is prepared according to following quality proportioning: grass meal 50%, sawdust 26%, wheat skin 20%, land plaster 2%, sugar 1%, peroxophosphoric acid Calcium 1% slowly sprays first by major ingredient uniform mixing, then by other auxiliary materials after such as land plaster, sugar, calcium superphosphate are dissolved in water Enter in compost, material-water ratio is 1 to 1.2 to 1.5, and water content is made to reach 70% or so.The pH of compost is controlled between 4-5.
2) compost is fitted into polyethylene plastic bag, compost is compacted by when charging, prick it is suitable for reading after carry out normal-pressure sterilization, 100 DEG C of holdings thoroughly sterilizing in 12-14 hours, when material temperature is down to 30 DEG C or less, is aseptically inoculated with.After inoculation, Bacterium bag is moved in into culturing room's bacterium germination, the control of culture room temperature is at 23-27 DEG C, air humidity 65% or so, shading culture, in bacterium Silk growth animated period, temperature are reduced to 20 DEG C or so.Until the mycelia of bacterium bag is covered with substantially, should in time by bacterium bag move in mushroom shed into Row flower bud fruiting.
3) at 15-20 DEG C, air humidity controlled in 85-90%, sporophore growth stage the control of fruiting stage mushroom shed temperature Give the scattering light irradiation of 200-400lx.
4) when Hericium erinaceus grows 7-10 days, bacteria thorn about 0.5cm long, spore is harvested before largely launching.
The selenium-enriched hericium erinaceus of harvesting is subjected to total Se content together with conventional solid cultivation of selenium-rich Hericium erinaceus and organic selenium contains The measurement of amount, as a result such as the following table 9.
As seen from the results in Table 9, Hericium erinaceus culture test, the hedgehog hydnum of harvest are carried out using selenium-enriched hericium erinaceus strain of the invention Total Se content is 90.38mg/kg, organic selenium content 87.84mg/kg in massee fruiting bodies, is organic in traditional selenium-enriched hericium erinaceus Se content illustrates that the absorptivity of method selenium of the invention and conversion ratio are higher, selenium-rich effect is more preferable 3 times more.
The present invention can be summarized with others without prejudice to the concrete form of spirit or essential characteristics of the invention.Therefore, nothing By from the point of view of which point, the embodiment above of the invention can only all be considered the description of the invention and cannot limit invention, Claims indicate the scope of the present invention, and above-mentioned explanation does not point out the scope of the present invention, therefore, with the present invention The comparable meaning and scope of claims in any variation, be all considered as being included within the scope of the claims.

Claims (7)

1. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain, which is characterized in that the process flow of this method is as follows:
Step 1, production parent species
Bacterial strain inoculation shovel is aseptically taken 1 by the screening Hericium erinaceus strain excellent that mycelial growth rate is fast, growing way is strong Block size is about 0.5cm2Fungus block, be inoculated on PDA enriched medium inclined-plane, 25 DEG C of culture 5-7d obtain slant strains, then Slant strains are transferred on PDA enriched medium plate, 25 DEG C of cultures cover with plate until mycelia, obtain parent species；
Step 2, production shaking flask strain
The Hericium erinaceus parent species of plate culture are taken into bacterium piece 3-4 piece with diameter 5mm punch, access is equipped with the level-one of fluid nutrient medium In shaking flask, level-one shaking flask strain is cultivated to obtain；Again by level-one shaking flask strain according to 7-9%(v/v) inoculum concentration be transferred to equipped with liquid In the second-level shake flask of culture medium, second-level shake flask strain is cultivated to obtain；Finally by second-level shake flask strain according to 7-9%(v/v) inoculum concentration It is transferred in the three-level shaking flask equipped with fluid nutrient medium, cultivates to obtain three-level shaking flask strain；
Step 3, shaking flask strain electric field magnetic field are dealt with altogether
The shaking flask strain made first is placed in the high-voltage electrostatic field of 100KV/m and handles 1min, is then placed into 50mT's 5min is handled in uniform magnetic field；
Step 4, production Selenium-enriched fermentation strain
1. fermentative medium formula: corn flour 30-36g/L, casein 5-9g/L, soybean stalk powder 15-20g/L, vinegar grain 6- 10g/L, maize cob meal 7-10g/L, black fungus rich in selenium mushroom bran 5-6g/L, citric acid 2.4-3.0g/L, selenium-rich alfalfa meal 6.8- 7.4g/L, MgSO4·7H2O0.9-1.1g/L, KH2PO41.8-2.2g/L, Na2SeO337 ~ 43mg/L, astragalus root dregs 1.4- 2.0g/L, lichens powder 9-10g/L, fly maggot powder 3.6-4.8g/L, oyster shell powder 2.6-3.0g/L, VB650-70mg/L, glycerol 1.7-2.3mL/L, edible defoaming agent 0.25-0.35g/L, surplus are magnetized water, and initial pH is adjusted to 5.3-5.7；According to above Formula prepares fermentation medium；
2. fermentor cleaning and sky go out: sterilizing at 121-125 DEG C of temperature, pressure 0.11-0.13Mpa after fermentor is cleaned 30min；
3. charging sterilizing: prepared fermentation medium being added in fermentor from feeding port, the loading amount of fermentation medium is hair The 50-60% of fermentation tank capacity opens air pump and stirs 2-3min, sterilizes at 121-125 DEG C of temperature, pressure 0.11-0.13Mpa 40min；
4. inoculation: after sterilizing, when fermentation medium is cooled to 30 DEG C or less, general's treated shaking flask strain is according to sterile Operation accesses fermentor, inoculum concentration 11-13% from inoculation mouth；
5. fermented and cultured: starting fermentor enters cultivation conditions, and cultivation temperature is set as 25-27 DEG C, and blender revolving speed is set as 140-160r/min, ventilatory capacity are set as the 1-3 days 0.8 v/v/min, then 1.2 v/v/min to fermentation ends, when culture Between 6-8d.
2. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain described in accordance with the claim 1, which is characterized in that described In step 1, the ingredient of PDA enriched medium are as follows: potato 180-220g/L, trehalose 17-23g/L, agar 18-22g/L, egg White peptone 3-4g/L, lemon juice 3-5mL/L, brewer's wort 10-14mL/L, VB120-30mg/L, VB220-30mg/L, MgSO4· 7H2O0.4-0.6g/L, KH2PO40.8-1.2g/L, surplus are tap water, and pH is adjusted to 4.8-5.2.
3. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain described in accordance with the claim 1, which is characterized in that described In step 2, the ingredient of fluid nutrient medium are as follows: soybean starch 4-6g/L, protein hydrolysate 0.6-1.0g/L, glucose 20-26g/L, Sweet potato powder 14-18g/L, brewer's wort 10-14mL/L, succus liquiritiae 8-9mL/L, astragalus root dregs 1.4-2.0g/L, MnSO4 0.22- 0.26g/L, MgSO4·7H2O0.4-0.6g/L, KH2PO40.8-1.2g/L, choline chloride 0.5-0.7mg/L, IAA 1.3- 1.5mg/L, surplus are tap water, and pH is adjusted to 4.8-5.2.
4. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain described in accordance with the claim 1, which is characterized in that described In step 2, the liquid amount of shaking flask strains at different levels is that every 500mL shaking flask fills 150mL fluid nutrient medium.
5. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain described in accordance with the claim 1, which is characterized in that described In step 2, the condition of culture of shaking flask strains at different levels is equal are as follows: 25-27 DEG C of shaking table temperature, shaking speed 140-160r/min, culture Time 4-5d.
6. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain described in accordance with the claim 1, which is characterized in that described In step 4, sterile working when inoculation refers to 75% ethanol of inoculation mouth, and with 95% ethyl alcohol calcination, and flame snare is connecing On kind mouth, and quickly the sebific duct in shaking flask is inserted in inoculation mouth.
7. a kind of production method of good quality and high output selenium-enriched hericium erinaceus strain described in accordance with the claim 1, which is characterized in that described Magnetized water refers to by Management of magnetic water device treated tap water, the model CFG/L- of Management of magnetic water device used in step 4 40, flow 7.0-9.0t/h, vertical centre magnetic field strength are 160mT.
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