CN104920069A - High-density liquid strain and fermenting method thereof - Google Patents
High-density liquid strain and fermenting method thereof Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G18/00—Cultivation of mushrooms
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- C05—FERTILISERS; MANUFACTURE THEREOF
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- C05G—MIXTURES OF FERTILISERS COVERED INDIVIDUALLY BY DIFFERENT SUBCLASSES OF CLASS C05; MIXTURES OF ONE OR MORE FERTILISERS WITH MATERIALS NOT HAVING A SPECIFIC FERTILISING ACTIVITY, e.g. PESTICIDES, SOIL-CONDITIONERS, WETTING AGENTS; FERTILISERS CHARACTERISED BY THEIR FORM
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Abstract
The invention discloses a high-density liquid strain and a fermenting method thereof, and belongs to the technical field of culture of pleurotus eryngii. According to the preparation method, Chinese herbal medicine extracts which can improve the cold resistance, thermal stress, immunity and hybrid bacteria resistance of the strain are scientifically composited in the culture medium at each stage of liquid seeds, seeds in a shake flask, seeds in a seed tank, and liquid strain in a fermenting tank; meanwhile, sawdust which has the raw material adaptability and also has the function of a defoaming agent is scientifically composited; in addition, the mode of supplemental inoculating and sectional adjustable temperature fermenting is carried out, in order to reduce the cell age of the strain and the difference in the number of generation of proliferation; chitosan which is coated and has the special function is added by a flowing manner at a proper time so as to culture the liquid strain which is strong in fermenting hypha, high in activity, small in mycelium pellet diameter, large in density, and uniform to distribute; the density of the mycelium pellet is 4.5 to 5.5x105 units/ L; the diameter of the mycelium pellet is 0.5 to 0.7m; the dry weight of the mycelium pellet is 90 to 100g/L; the germination costs 4 to 5 hours; the exopolysaccharide is 3.0 to 3.5g/L.
Description
Technical field
The present invention relates to planting almond abalone mushroom technical field, be specifically related to a kind of high density liquid bacterial classification and fermentation process thereof.
Background technology
Xingbao mushroom (PleurotuseryngiiQuel.) has another name called pleurotus eryngii, there is bacterial context plumpness, the advantages such as quality is tender and crisp, particularly its stem dense structure, solid, milky white, tasty and refreshing, be called as " flat mushroom king ", " dried scallop mushroom ", there is the mouthfeel of almond flavor and abalone, be applicable to fresh-keeping, processing, firmly get liking of people, it is nutritious, be rich in protein, carbohydrate, vitamin and calcium, magnesium, copper, the mineral matters such as zinc, immune function of human body can be improved, have anticancer to human body, reducing blood lipid, the effects such as ease constipation stomach and beauty treatment, there is high cultivating value and commercial value.
Domestic and international planting almond abalone mushroom is based on solid spawn at present; and liquid spawn relative to solid spawn have with short production cycle, cell age is consistent, fruiting is neat, be convenient to management, bacterial classification cost is low, inoculation convenient and the advantage such as quick, is conducive to the scale of Edible Fungi, batch production.In recent years due to the fast development of industrial cultivation technique, for shortening bacterial classification manufacturing cycle, improve strain quality and production efficiency, domestic and international manufacturer research and development liquid spawn is used for substituting during current Xingbao mushroom is produced the solid spawn generally used, and in actual production, start application.
Pleurotus eryngii liquid strain produces great-hearted Xingbao mushroom mycelium by liquid fermentation process.At present, domesticly not yet promulgate quality and quality that unified national standard carrys out specification pleurotus eryngii liquid strain, but people sum up effect and have worked out some regional characters standards in actual application, as Liaoning Province's provincial standard " DB21/T 1693-2008 edible fungi liquid strain production technology regulation " etc., clear stipulaties in these standards, qualified edible fungi liquid strain should " visible distinctive hypha form, spherical and plexi mycelium distributes in a large number, mycelia is sturdy, in mycelia, protoplasm is evenly distributed " " bacterium liquid slightly thickness, there is a large amount of sheet or spherical mycelium suspended, be evenly distributed ", that is excellent liquid spawn mycelium pellet density is high, diameter is little, be evenly distributed, on the other hand, in production application, pleurotus eryngii liquid strain carries out mechanical inoculation operation by production line inoculation device, because inoculation device spout is narrow, for preventing bacterium liquid blocking pipe, also need the pretreating process before increasing liquid-spawn inoculation (the general method of mechanical crushing that adopts makes the mycelium pellet diameter in bacterium liquid diminish, and bacterium liquid is become evenly).Therefore, the physical characteristic such as size, density, the uniformity of Xingbao mushroom liquid fermentation mycelium pellet is the crucial Con trolling index of high-quality liquid spawn.
At present, about the research report of Xingbao mushroom liquid culture and patented technology major part concentrate on shaking flask level, scale fermentation research less, substantially the optimization aspect of zymotechnique is concentrated on, especially more in the improvement of fermentation medium: application number be 201410568173.3 Chinese patent disclose a kind of Xingbao mushroom liquid fermentation medium of improvement and utilize it to cultivate the method for pleurotus eryngii liquid strain, add the gelatin of 0.1-0.3g/100ml in the fermentation medium, the mixture of the one or both in agar, adopt and stir, shaking table concussion is cultivated, in above-mentioned disclosed Patent media, agar addition solidifies more than 0.4g/100ml, liquid culture cannot be carried out, wayward, there is the failed hidden danger of fermentation, and it is few that the liquid spawn amount obtained cultivated by shaking table, be not suitable for scale, mechanization production, strain quality is not fine simultaneously.Application number be 201410244283.4 Chinese patent disclose a kind of production method of edible fungi liquid strain, cultivate at solid culture medium after activated for the edible mushroom strains preserved, then pulverize, continue sealing on solid culture medium and cultivate 2-3 days, contact mixing with the agar solution of 0.15-0.2% and obtain liquid spawn, in fact still, there is the defect of solid fermentation in above-mentioned disclosed patent, and pulverizing is wherein culture technology and the easy pollution microbes of allocating technology again, high-speed stirred making beating and the high shear force pulverized injure quite large to bacterial classification.North gardening. an edible mushroom section discloses the preparation research [2009 (11) 230-232] of the pleurotus eryngii liquid strain that Liu Guanhui etc. delivers, fermentation medium and fermentation condition are optimized, but still it is not comprehensive, well can not be applicable to growth, breeding, the physical characteristic such as size, density, the uniformity of the zymophyte pompon of the liquid spawn obtained is not bery desirable, and same exist stirring shearing force having a strong impact on mycelium pellet quality.
Air-blowing fermentation is fermentation mode oxygen or filtrated air passed into from airlift fermentation pot bottom, possesses the effect of logical oxygen and soft stirring simultaneously, logical oxygen efficiency is high, dissolved oxygen is effective, effectively can prevent stirring-type from fermenting shearing force to the impact of pleurotus eryngii liquid strain quality, the liquid spawn mycelia obtained is sturdy, modest viscosity, mycelium pellet diameter is little, density is high, be evenly distributed.Application number be 201310479951.7 Chinese patent disclose a kind of cultural method of pleurotus eryngii liquid strain, traditional potato carbon source and wheat bran nitrogenous source are replaced with bean cake powder, pour medium into fermentation tank, 121 DEG C of sterilizing 60-120 minute, after sterilizing, water drenches and cools and pass into filtrated air; The liquid mother that inoculated and cultured is good plants, inoculum concentration is 1-2 ‰, fermentation tank culture temperature is 21-26 DEG C, venting pressure is 0.5-1.5 MPa, cultivate and obtain pleurotus eryngii liquid strain in 7-10 days, but above-mentioned disclosed Patent media not comprehensive nutrition, zymotechnique is delayed, fermentation period is long, and the liquid spawn quality obtained is still undesirable.
To sum up, according to the strain properties of Xingbao mushroom, by Optimal Medium and condition of culture, air-blowing fermentation is adopted to be still the unremitting pursue of those skilled in the art for high-quality, high yield, pleurotus eryngii liquid strain that cost is low.
Summary of the invention
Technical problem solved by the invention overcomes the defect of existing air-blowing fermentation for pleurotus eryngii liquid strain, in Spawn incubation various stages medium, the composite bacterial classification that improves of science resists cold, the Chinese herbal medicine extract of heat stress and immunity and adaptability raw material wood dust, segmentation is adopted to add the mode of inoculation and temperature-variable fermentation, shorten bacterial classification cell age, propagation algebraic step distance, in good time stream add bag by after shitosan adjustment fermentation medium viscosity, turn out a kind of fermented hypha sturdy, active high, mycelium pellet diameter is little, density is large, be evenly distributed, the pleurotus eryngii liquid strain that fermentation period is short.
In order to achieve the above object, the present invention is by the following technical solutions:
A fermentation process for high density liquid bacterial classification, comprises the steps:
1) go bail for the Xingbao mushroom slant strains 0.2cm kept
2bacterial classification block 2 pieces be inoculated in plating medium and activate, 25 DEG C of constant temperature culture 10 days, so activation 2 times, obtains dull and stereotyped seed;
2) by dull and stereotyped seed diameter be 0.5cm card punch intercept 0.2cm
2bacterial classification block 3 pieces access 250mL triangular flask in, liquid seed culture medium liquid amount is 100mL, in 25 DEG C of constant temperature culture 2 days, then puts into constant-temperature table, 25 DEG C, 100r/min shaken cultivation 3 days liquid seeds;
3) by liquid seeds with 10% inoculum concentration be inoculated in 500mL shaking flask, Shake flask medium liquid amount 250mL, in 25 DEG C, 150r/min vibrate constant temperature culture 4 days shake-flask seed;
4) by shake-flask seed with 10% inoculum concentration be inoculated in 5L seeding tank, seed tank culture base liquid amount 3L, in 25 DEG C, under throughput 4L/min condition constant temperature culture 4 days seeding tank seed;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 30-32 DEG C, throughput 3-5L/min ferment at constant temperature 12-24h, then 18-22 DEG C is cooled to the speed of 0.3-0.5 DEG C/min, 8-10g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 10-16h under throughput 6-8L/min condition, last with the ramp of 0.2-0.4 DEG C/min to 24-26 DEG C, under throughput 5-7L/min condition, namely ferment at constant temperature 45-52h obtains high density liquid bacterial classification;
Described Xingbao mushroom slant strains is commercial Xingbao mushroom purifying bacterial strain;
Described plating medium consists of: potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1.5g/L, agar 20g/L, VB
10.02g/L, surplus is water, pH value 6.0;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.3-0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1-0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.5-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2-0.4g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8-1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3-0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.5-2g/L, wood dust 0.6-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Preferably, the preparation method of described Chinese herbal medicine extract, comprises the steps: to count by weight, take the Radix Astragali 30 parts, rhizoma zingiberis 30 parts, dried orange peel 30 parts, cow-bezoar 25 parts, the root bark of tree peony 25 parts, 20 parts, Radix Glycyrrhizae, the wind-weed 18 parts, honeysuckle 18 parts, cordate houttuynia 17 parts, the radix paeoniae rubrathe 15 parts, the root of large-flowered skullcap 15 parts, Ligusticum wallichii 10 parts, Radix Angelicae Sinensis 8 parts; Put in the supersonic wave cleaning machine filling 0.2% sodium bicarbonate solution and clean 12min in 200W, 30KHz, drain, said herbal medicine being crushed to particle diameter is less than 2 millimeters, puts Homogeneous phase mixing in container and adds the water of 5 times of weight, obtaining mixed material, control temperature 80 DEG C keeps 3h, then being cooled to 45 DEG C, is 4 by lactic acid adjust ph, under power 200W condition, carry out Microwave Extraction 12min, under power 250W, frequency 35KHz condition, carry out ultrasonic assistant extraction simultaneously; Then the mixed enzyme adding mixed material gross weight 1.0% carries out enzymolysis, be 6.2 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixed material 2 times of w ethanol and propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 1:2, control temperature to 70 DEG C keeps 3.5h, filters, obtains the first filtrate; Add the water of filter residue 2 times of weight, control temperature 90 DEG C keeps 2h, is then cooled to 30 DEG C, filters, obtains the second filtrate; First filtrate and the second filtrate are merged according to mass ratio 3:2, filter vacuum concentrates postlyophilization, pulverizes and obtains Chinese herbal medicine extract;
Described mixed enzyme is dextranase, zytase, pentosanase, pectase 3:2:2:1 Homogeneous phase mixing in mass ratio.
Preferably, the preparation method of described wood dust comprises the steps: without mouldy, anosis worm sawmilling end or waste wood removing foreign material, be placed in the supersonic wave cleaning machine of the sodium bicarbonate solution that mass percent concentration 0.5% is housed in 200W, 30KHz cleans 10min, rinse, drain, put into steam still in 0.3-0.4MPa boiling 20-30min, take out, drain, with vegetable oil 100:1 Homogeneous phase mixing in mass ratio, then mixture is put microwave dryer in 2000W, 120 DEG C of microwave irradiation 5min, make it moisture and reach 8-10%, last ultramicro grinding is to particle diameter 0.3-0.5mm, pack and obtain wood dust,
More preferably, described drying process is: microwave irradiation 10s, stops 10s.
Preferably, the preparation method of described fermentation medium is: according to each raw material of culture medium prescription precise, first soybean cake powder quality 4-6 water is doubly got, be 5-6 by lactic acid adjust ph, add the thermostableα-amylase of 5u/g soybean cake powder, add soybean cake powder while stirring, be warming up to 90-95 DEG C, insulation, liquefaction 10-15min, then 50-60 DEG C is cooled to, insulation, add the carbohydrase of 40u/g soybean cake powder and the protease hydrolytic 20-30min of 30u/g soybean cake powder while stirring, add residue water and other raw material while stirring, Homogeneous phase mixing, adjusted to ph is 6.0, namely 121-123 DEG C of sterilizing 30-40min obtain fermentation medium,
Preferably, described bag is by the preparation method of shitosan, comprising the steps: to get the mass percent concentration that shitosan adds its quality 30-50% is in the cyclohexaamylose aqueous solution of 10-12%, make softwood, granulate with 10-20 mesh sieve, obtain wet granular, put in power 2000W, 60-80 DEG C of dry 4-6min in microwave dryer, namely obtain bag by shitosan with the quick whole grain of 10-20 mesh sieve.
The detection method of liquid spawn primary quality measure of the present invention:
The mensuration of mycelium pellet dry weight
Get 50ml zymotic fluid 80 object screen clothes to filter, collect bacterium ball, repeatedly rinse with clear water, and to be placed in constant temp. drying box 80 DEG C and to dry after 4 hours and weigh, and weigh once every half an hour, until twice weighing difference is no more than 2mg, be constant weight.Finally calculate the mean value of dry mycelial weight.
The mensuration of mycelium pellet density
Get zymotic fluid 1ml in culture dish with pipette, bacterium ball quantity is counted, repeat numeration three times, finally calculate the mean value of Peloton density.
The mensuration of mycelium pellet diameter
Get zymotic fluid 1ml with pipette, get 10 bacterium balls with tweezers and line up a queue of, measure total length, then average, duplicate measurements three times, calculate the mean value of bacterium bulb diameter.
The mensuration of the dull and stereotyped sprout time of mycelium pellet tieback
From the culture fluid of fermentation medium, get a bacterium ball be placed in PDA culture medium flat plate central authorities, each fermentation medium does three repetitions, the PDA flat board being placed with bacterium ball is placed in incubator 25 DEG C cultivate, observed once every 1 hour, by the time after having observed one group of bacterium ball sprouting, change observation in every 0.5 hour into once, and record the sprout time of each experimental group, finally calculate the mean value of sprout time after bacterium ball tieback flat board in each fermentation medium.
The mensuration of exocellular polysaccharide content
Get zymotic fluid 50ml, suction filtration, get supernatant, add the industrial alcohol of two volumes, precipitate polysaccharides spends the night, then centrifugal 10 minutes of 3600r/min, collects polysaccharide, dries at being placed in 40 DEG C, weigh once every half an hour after weighing after 2 hours, until twice weighing difference is no more than 2mg, be constant weight.
Another object of the present invention is to provide high density liquid bacterial classification prepared by said method.
Described high density liquid bacterial classification is through ferment tank 67-92h, and mycelium pellet density is 4.5-5.5 X 10
5individual/L, mycelium pellet diameter is 0.5-0.7mm, and mycelium pellet dry weight is 90-100g/L, and sprout time is 4-5h, and exocellular polysaccharide is 3.0-3.5g/L.
A further object of the invention is to provide the application of above-specified high density liquid spawn in planting almond abalone mushroom.
Beneficial effect:
The present invention is according to the characteristic of the former bacterial strain of Xingbao mushroom, at liquid seeds, shake-flask seed, resist cold from few composite bacterial classification that improves of science at the most in seeding tank seed and fermentor liquid bacterial classification each stage medium, heat stress, the Chinese herbal medicine extract of immunity and antibiotic property, science is composite simultaneously both had adaptability to raw material, there is again the wood dust of defoamer function, by taming step by step, rejuvenation expands cultivates, enhance pleurotus eryngii quel strains high temperature resistance and Cold stress ability, improve the ability of producing multiple digestive enzyme, excite its multiplication capacity and propagation density, shorten sprout time, improve the output of mycelia and exocellular polysaccharide, improve the adaptability of bacterial strain to yeasting and follow-up planting environment, adopt the mode adding inoculation and segmentation temperature-variable fermentation simultaneously, shorten bacterial classification cell age and propagation algebraic step distance, in good time stream add bag by after there is thickening property, intermiscibility, stability, the viscosity of the shitosan adjustment fermentation medium of the specific function such as adsorptivity and biocidal property and stability, improve homogeneity and dissolved oxygen concentration that mycelium pellet distributes in zymotic fluid, and break the preparation method of traditional zymotic medium, to the further biological enzymolysis of soybean cake powder wherein, shorten fermentation period, turn out a kind of fermented hypha sturdy, active high, mycelium pellet diameter is little, density is large, the liquid spawn be evenly distributed, its mycelium pellet density is 4.5-5.5 X 10
5individual/L, mycelium pellet diameter is 0.5-0.7mm, and mycelium pellet dry weight is 90-100g/L, and sprout time is 4-5h, and exocellular polysaccharide is 3.0-3.5g/L.Improve the Quality and yield of liquid spawn comprehensively, reduce production cost, for the high-quality of Xingbao mushroom, high yield and large-scale planting have established solid foundation, show through experiment in cultivation: liquid spawn prepared by the present invention is healthy and strong, vigor is high, fast growth, mycelial yield and quality high, anti-miscellaneous bacteria ability is strong, long shelf-life, bacterium time shorten 22.22% is sent out compared with commercial liquid bacterial classification, miscellaneous bacteria infection rate reduces by 96.67%, one-level mushroom rate improves 5%, production capacity is commercial liquid bacterial classification more than 2 times, embody higher economic worth, more be applicable to batch production, large-scale planting.
Concrete know-why is:
1. the composite bacterial classification that significantly improves of Chinese herbal medicine extract science of the present invention resists cold, heat stress, the Chinese herbal medicine of immunity and disease resistance is raw material, through ultrasonic cleaning, low temperature enzymolysis, prepared by water extraction alcohol extracting and ultrasonic assistant Microwave Extraction, extract active constituent content is high, cost is low, can effectively by taming step by step, rejuvenation expands cultivates, enhance pleurotus eryngii quel strains high temperature resistance and Cold stress ability, improve cellulase-producing, protease, the ability of the digestive enzymes such as amylase, excite its multiplication capacity and propagation density, shorten sprout time, effectively improve the density of liquid spawn, the output of dry mycelial weight and exocellular polysaccharide, reduce mycelium pellet diameter, enhance the adaptability of bacterial strain to yeasting and follow-up planting environment, enhance the resisting stress of high density liquid bacterial classification comprehensively, disease resistance and immunocompetence, adopt ultrasonic cleaning effectively can kill worm's ovum in raw material and miscellaneous bacteria simultaneously, reduce heavy metal ion content and persticide residue, the raw material reducing Xingbao mushroom enriching heavy metal ion may, improve the foodsafety of edible mushroom and the shelf-life of liquid spawn.
2. wood dust of the present invention with the wood chip of high cellulose content and high lignin content and vegetable oil for raw material, adopt ultrasonic cleaning, autoclaving and microwave drying process preparation, its quality is fine and soft soft, trophic fiber cellulose content is high, be very beneficial for pleurotus eryngii quel strains fermentation, network is enriched simultaneously, evenly, absorption affinity is strong, vegetable oil adhesion amount is large, both can be the nutrition that Xingbao mushroom provides abundant, also can solve air-blowing under high ventilation to ferment the problem of foam easy to foaming, also for the cultivation in later stage provides environmental suitability ability, and adopt ultrasonic cleaning effectively can kill worm's ovum in raw material and miscellaneous bacteria, reduce heavy metal ion content and persticide residue, the raw material reducing Xingbao mushroom enriching heavy metal ion may, improve the foodsafety of edible mushroom.
3. instant invention overcomes the method that tradition prepares fermentation medium, soybean cake powder is wherein liquefied and enzymolysis further, starch wherein and protein macromolecule nutriment are degraded to Small molecular, are convenient to Xingbao mushroom and play ferment and fermentation, shorten Xingbao mushroom fermentation period.
4. bag of the present invention is by shitosan, there is the multi-functional of shitosan and cyclohexaamylose simultaneously, appropriateness have adjusted the viscosity of Xingbao mushroom zymotic fluid, intermiscibility, stability, adsorptivity and biocidal property, improves homogeneity and dissolved oxygen concentration that mycelium pellet distributes in zymotic fluid.
5. the fermentation process of high density liquid bacterial classification of the present invention adopts air-blowing zymotechnique, technique is simple, easy to operate, fermentation period short (fermentation tank 67-92h), can scale and mechanization production, spread cultivation and fermentation process medium and zymotechnique by optimizing bacterial classification, segmentation is adopted to add the mode of inoculation and temperature-variable fermentation, shorten cell age and propagation algebraic step distance, effectively improve biologically active and the fermenting power of liquid spawn, significantly improve the density of mycelium pellet, the output of dry mycelial weight and exocellular polysaccharide, reduce mycelium pellet diameter, improve the Quality and yield of liquid spawn comprehensively, reduce production cost.
Embodiment
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, but not limits the scope of the invention, and the spirit and scope of the invention only limited by claims.To those skilled in the art, under the prerequisite not deviating from essence of the present invention and scope, the various change carry out the material component in these embodiments and consumption or change also belong to protection scope of the present invention.
Prepared by embodiment 1 raw material
1. the preparation of Chinese herbal medicine extract:
The preparation method of described Chinese herbal medicine extract, comprises the steps: to count by weight, takes the Radix Astragali 30 parts, rhizoma zingiberis 30 parts, dried orange peel 30 parts, cow-bezoar 25 parts, the root bark of tree peony 25 parts, 20 parts, Radix Glycyrrhizae, the wind-weed 18 parts, honeysuckle 18 parts, cordate houttuynia 17 parts, the radix paeoniae rubrathe 15 parts, the root of large-flowered skullcap 15 parts, Ligusticum wallichii 10 parts, Radix Angelicae Sinensis 8 parts; Put in the supersonic wave cleaning machine filling 0.2% sodium bicarbonate solution and clean 12min in 200W, 30KHz, drain, said herbal medicine being crushed to particle diameter is less than 2 millimeters, puts Homogeneous phase mixing in container and adds the water of 5 times of weight, obtaining mixed material, control temperature 80 DEG C keeps 3h, then being cooled to 45 DEG C, is 4 by lactic acid adjust ph, under power 200W condition, carry out Microwave Extraction 12min, under power 250W, frequency 35KHz condition, carry out ultrasonic assistant extraction simultaneously; Then the mixed enzyme adding mixed material gross weight 1.0% carries out enzymolysis, be 6.2 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixed material 2 times of w ethanol and propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 1:2, control temperature to 70 DEG C keeps 3.5h, filters, obtains the first filtrate; Add the water of filter residue 2 times of weight, control temperature 90 DEG C keeps 2h, is then cooled to 30 DEG C, filters, obtains the second filtrate; First filtrate and the second filtrate are merged according to mass ratio 3:2, filter vacuum concentrates postlyophilization, pulverizes and obtains Chinese herbal medicine extract;
Described mixed enzyme is dextranase, zytase, pentosanase, pectase 3:2:2:1 Homogeneous phase mixing in mass ratio.
2. the preparation of wood dust:
The preparation method of described wood dust, comprising the steps: will without mouldy, anosis worm sawmilling end or waste wood removing foreign material, be placed in the supersonic wave cleaning machine of the sodium bicarbonate solution that mass percent concentration 0.5% is housed in 200W, 30KHz cleans 10min, rinse, drain, put into steam still in 0.35MPa boiling 25min, take out, drain, with vegetable oil 100:1 Homogeneous phase mixing in mass ratio, then mixture is put microwave dryer in 2000W, 120 DEG C of microwave irradiation 5min, wherein microwave irradiation 10s, stop 10s, last ultramicro grinding is to particle diameter 0.4mm, pack and obtain wood dust.
3. bag is by the preparation of shitosan:
Described bag is by the preparation method of shitosan, comprising the steps: to get the mass percent concentration that shitosan adds its quality 40% is in the cyclohexaamylose aqueous solution of 11%, make softwood, granulate with 20 mesh sieves, obtain wet granular, put in power 2000W, 70 DEG C of dry 5min in microwave dryer, namely obtain bag by shitosan with the quick whole grain of 20 mesh sieve.
The Chinese herbal medicine extract used in following embodiment 2-7, wood dust and bag are embodiment 1 by shitosan to be prepared, and " No. 3, Longhai City " that pleurotus eryngii quel strains provides for edible mushroom research institute of Longhai City of Zhangzhou City of Fujian Province, other raw material is commercial.
Embodiment 2
A fermentation process for high density liquid bacterial classification, comprises the steps:
1) go bail for the Xingbao mushroom slant strains 0.2cm kept
2bacterial classification block 2 pieces be inoculated in plating medium and activate, 25 DEG C of constant temperature culture 10 days, so activation 2 times, obtains dull and stereotyped seed;
2) by dull and stereotyped seed diameter be 0.5cm card punch intercept 0.2cm
2bacterial classification block 3 pieces access 250mL triangular flask in, liquid seed culture medium liquid amount is 100mL, in 25 DEG C of constant temperature culture 2 days, then puts into constant-temperature table, 25 DEG C, 100r/min shaken cultivation 3 days liquid seeds;
3) by liquid seeds with 10% inoculum concentration be inoculated in 500mL shaking flask, Shake flask medium liquid amount 250mL, in 25 DEG C, 150r/min vibrate constant temperature culture 4 days shake-flask seed;
4) by shake-flask seed with 10% inoculum concentration be inoculated in 5L seeding tank, seed tank culture base liquid amount 3L, in 25 DEG C, under throughput 4L/min condition constant temperature culture 4 days seeding tank seed;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 31 DEG C, throughput 4L/min ferment at constant temperature 18h, then 20 DEG C are cooled to 0.4 DEG C/min speed, 9g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 13h under throughput 7L/min condition, finally with 0.3 DEG C/min ramp to 25 DEG C, under throughput 6L/min condition, namely ferment at constant temperature 48h obtains high density liquid bacterial classification;
Described plating medium consists of: potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1.5g/L, agar 20g/L, VB
10.02g/L, surplus is water, pH value 6.0;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.4g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.8g/L, wood dust 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
The preparation method of described fermentation medium is: according to each raw material of culture medium prescription precise, first the water of soybean cake powder quality 5 times is got, be 5.5 by lactic acid adjust ph, add the thermostableα-amylase of 5u/g soybean cake powder, add soybean cake powder while stirring, be warming up to 93 DEG C, insulation, liquefaction 12min, then 55 DEG C are cooled to, insulation, add the carbohydrase of 40u/g soybean cake powder and the protease hydrolytic 25min of 30u/g soybean cake powder while stirring, add residue water and other raw material while stirring, Homogeneous phase mixing, adjusted to ph is 6.0, namely 121 DEG C of sterilizing 40min obtain fermentation medium.
The high density liquid bacterial classification mycelium pellet density prepared through said method is 5.5 X 10
5individual/L, mycelium pellet diameter is 0.5mm, and mycelium pellet dry weight is 100g/L, and exocellular polysaccharide is 3.5g/L.
Embodiment 3
A fermentation process for high density liquid bacterial classification, comprises the steps:
Step 1) to 4) with embodiment 2;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 30 DEG C, throughput 3L/min ferment at constant temperature 12h, then 18 DEG C are cooled to the speed of 0.3 DEG C/min, 8g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 10h under throughput 6L/min condition, finally with the ramp to 24 DEG C of 0.2 DEG C/min, under throughput 5L/min condition, namely ferment at constant temperature 45h obtains high density liquid bacterial classification;
Described plating medium composition is with embodiment 2;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.3g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.5g/L, wood dust 0.6g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
The preparation method of described fermentation medium is: according to each raw material of culture medium prescription precise, first the water of soybean cake powder quality 4 times is got, be 5 by lactic acid adjust ph, add the thermostableα-amylase of 5u/g soybean cake powder, add soybean cake powder while stirring, be warming up to 90 DEG C, insulation, liquefaction 10min, then 50 DEG C are cooled to, insulation, add the carbohydrase of 40u/g soybean cake powder and the protease hydrolytic 20min of 30u/g soybean cake powder while stirring, add residue water and other raw material while stirring, Homogeneous phase mixing, adjusted to ph is 6.0, namely 123 DEG C of sterilizing 30min obtain fermentation medium.
The high density liquid bacterial classification mycelium pellet density prepared through said method is 5.0 X 10
5individual/L, mycelium pellet diameter is 0.6mm, and mycelium pellet dry weight is 96g/L, and exocellular polysaccharide is 3.2g/L.
Embodiment 4
A fermentation process for high density liquid bacterial classification, comprises the steps:
Step 1) to 4) with embodiment 2;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 32 DEG C, throughput 5L/min ferment at constant temperature 24h, then 22 DEG C are cooled to the speed of 0.5 DEG C/min, 10g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 16h under throughput 8L/min condition, finally with the ramp to 26 DEG C of 0.4 DEG C/min, under throughput 7L/min condition, namely ferment at constant temperature 52h obtains high density liquid bacterial classification;
Described plating medium composition is with embodiment 2;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 2g/L, wood dust 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
The preparation method of described fermentation medium is: according to each raw material of culture medium prescription precise, first the water of soybean cake powder quality 6 times is got, be 6 by lactic acid adjust ph, add the thermostableα-amylase of 5u/g soybean cake powder, add soybean cake powder while stirring, be warming up to 95 DEG C, insulation, liquefaction 15min, then 60 DEG C are cooled to, insulation, add the carbohydrase of 40u/g soybean cake powder and the protease hydrolytic 30min of 30u/g soybean cake powder while stirring, add residue water and other raw material while stirring, Homogeneous phase mixing, adjusted to ph is 6.0, namely 121 DEG C of sterilizing 30min obtain fermentation medium.
The high density liquid bacterial classification mycelium pellet density prepared through said method is 4.8 X 10
5individual/L, mycelium pellet diameter is 0.5mm, and mycelium pellet dry weight is 92g/L, and exocellular polysaccharide is 3.1g/L.
Embodiment 5
A fermentation process for high density liquid bacterial classification, comprises the steps:
Step 1) to 4) with embodiment 2;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 32 DEG C, throughput 3L/min ferment at constant temperature 24h, then 22 DEG C are cooled to the speed of 0.3 DEG C/min, 8g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 10h under throughput 8L/min condition, finally with the ramp to 24 DEG C of 0.4 DEG C/min, under throughput 7L/min condition, namely ferment at constant temperature 45h obtains high density liquid bacterial classification;
Described plating medium composition is with embodiment 2;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.3g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.5g/L, wood dust 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
The preparation method of described fermentation medium is: according to each raw material of culture medium prescription precise, first the water of soybean cake powder quality 4 times is got, be 6 by lactic acid adjust ph, add the thermostableα-amylase of 5u/g soybean cake powder, add soybean cake powder while stirring, be warming up to 90 DEG C, insulation, liquefaction 15min, then 50 DEG C are cooled to, insulation, add the carbohydrase of 40u/g soybean cake powder and the protease hydrolytic 30min of 30u/g soybean cake powder while stirring, add residue water and other raw material while stirring, Homogeneous phase mixing, adjusted to ph is 6.0, namely 121 DEG C of sterilizing 40min obtain fermentation medium.
The high density liquid bacterial classification mycelium pellet density prepared through said method is 4.6 X 10
5individual/L, mycelium pellet diameter is 0.7mm, and mycelium pellet dry weight is 90g/L, and exocellular polysaccharide is 3.1g/L.
Embodiment 6
A fermentation process for high density liquid bacterial classification, comprises the steps:
Step 1) to 4) with embodiment 2;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 30 DEG C, throughput 5L/min ferment at constant temperature 12h, then 18 DEG C are cooled to the speed of 0.5 DEG C/min, 10g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 16h under throughput 6L/min condition, finally with the ramp to 26 DEG C of 0.2 DEG C/min, under throughput 5L/min condition, namely ferment at constant temperature 52h obtains high density liquid bacterial classification;
Described plating medium composition is with embodiment 2;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 2g/L, wood dust 0.6g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0;
The preparation method of described fermentation medium is: according to each raw material of culture medium prescription precise, first the water of soybean cake powder quality 6 times is got, be 5 by lactic acid adjust ph, add the thermostableα-amylase of 5u/g soybean cake powder, add soybean cake powder while stirring, be warming up to 95 DEG C, insulation, liquefaction 10min, then 60 DEG C are cooled to, insulation, add the carbohydrase of 40u/g soybean cake powder and the protease hydrolytic 20min of 30u/g soybean cake powder while stirring, add residue water and other raw material while stirring, Homogeneous phase mixing, adjusted to ph is 6.0, namely 123 DEG C of sterilizing 30min obtain fermentation medium.
The high density liquid bacterial classification mycelium pellet density prepared through said method is 5.2 X 10
5individual/L, mycelium pellet diameter is 0.6mm, and mycelium pellet dry weight is 98g/L, and exocellular polysaccharide is 3.4g/L.
Embodiment 7
A fermentation process for high density liquid bacterial classification, comprises the steps:
Step 1) to 4) with embodiment 2;
5) first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 31 DEG C, throughput 4L/min ferment at constant temperature 18h, then 20 DEG C are cooled to 0.4 DEG C/min speed, 9g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 13h under throughput 7L/min condition, finally with 0.3 DEG C/min ramp to 25 DEG C, under throughput 6L/min condition, namely ferment at constant temperature 48h obtains high density liquid bacterial classification;
Described plating medium consists of: potato 200g/L, glucose 20g/L, peptone 5g/L, potassium dihydrogen phosphate 3g/L, magnesium sulfate 1.5g/L, agar 20g/L, VB
10.02g/L, surplus is water, pH value 6.0;
Described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.4g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.4g/L, VB
10.01g/L, surplus is water, pH value 6.0;
Consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.8g/L, wood dust 0.8g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0.
The high density liquid bacterial classification mycelium pellet density prepared through said method is 4.5 X 10
5individual/L, mycelium pellet diameter is 0.7mm, and mycelium pellet dry weight is 90g/L, and exocellular polysaccharide is 3.0g/L.
The experiment in cultivation of embodiment 8 high density liquid bacterial classification of the present invention
1. test strain: the liquid spawn of preparing with the embodiment of the present invention 2 and commercially available " No. 3, Longhai City " liquid spawn, for test strain, are divided into experimental group and control group;
2. inoculation method: experimental group adopts conventional method inoculation with control group with identical inoculum concentration and inoculum density, inoculates 2000 bottles respectively;
3. production management: experimental group and control group adopt same management method
3.1 hair's tube reasons
After inoculation, bacterium bottle is placed on constant temperature culture indoor, lucifuge is cultivated.Temperature controls at 23 DEG C, relative moisture 70% ~ 75%.The nutrient that this stage is used in planting material decomposes completely and accumulates.The key of bacteria developing period management is insulation, moisturizing, if early stage, temperature was too low, the duration is longer, can reduce spawn activity, and mycelia speed of production slows down.The dense Bai Houzai of all bacterium bottle mycelia continues to cultivate 15d, and nutrient is thoroughly decomposed, and mycelia reaches physiological ripening.
3.2 uncorks, mycelium stimulation
After bacterium bottle latter stage of ripening, carry out uncork, mycelium stimulation process according to a conventional method.In mycelium stimulation process, during liquid spawn mycelium stimulation, remove surperficial thin layer.After mycelium stimulation completes, bacterium bottle is moved into mushroom room, bacterium bottle is inverted, and makes mycelia restoration ecosystem.After mycelia recovers, temperature controls at 14 DEG C ~ 16 DEG C, and relative moisture is 90 ~ 95%, illumination 500 ~ 800Lx, CO
2concentration 600 ~ 1500ppm, fruiting to be buddingged.
3.3 sporophore growth management
Temperature controls at 15 ~ 18 DEG C, and air humidity remains on 85% ~ 90%, CO
2concentration is between 1500 ~ 1800ppm, and sporophore growth is rapid.When mushroom lid launches substantially, a damp mushroom of gathering when spore not yet launches.
4. quality and yield index measure
The mycelial concentration of observed and recorded experimental group and control group culture bottle, mycelia color, a bacterium time, miscellaneous bacteria infection state, observation and comparison fruit body proterties, measure fruit body stem length, cap size and stem diameter, single bottle of output, and statistical computation miscellaneous bacteria infection rate, one-level mushroom rate and biological transformation ratio, result is as table 1-3:
Biological conversion rate (%)=fruit body fresh goods output (g)/composts or fertilisers of cultivating dry weight (g) × 100%
Table 1
| Project | Send out the bacterium time | Mycelia color | Mycelial concentration | Miscellaneous bacteria infection rate |
| Experimental group | 14 days | White | Dense | 0.5％ |
| Control group | 18 days | White partially yellow | Lighter | 15％ |
Above result shows: liquid spawn of the present invention is healthy and strong, vigor is high, fast growth, mycelial yield and quality high, anti-miscellaneous bacteria ability is strong, sends out bacterium time shorten 22.22% compared with control group, and miscellaneous bacteria infection rate reduces by 96.67%.
Table 2
| Project | Stem average length (cm) | Cap average diameter (cm) | Stem average diameter (cm) | One-level mushroom rate |
| Experimental group | 15.17 | 7.34 | 6.28 | 97％ |
| Control group | 10.36 | 7.79 | 3.42 | 92％ |
Above result shows: it is suitable that the fruit body cap after liquid strain cultivation of the present invention and stem grow ratio, attractive appearance, and credit rating is high, and one-level mushroom rate improves 5% compared with control group.
Table 3
| Project | Single bottle of average weight (g) | Planting material dry weight (g) | Biological transformation ratio (%) |
| Experimental group | 123.41 | 180 | 68.56 |
| Control group | 91.73 | 180 | 50.96 |
Above result shows: the fruit body after liquid strain cultivation of the present invention is high only according to output, and biological transformation ratio is high, has good economic benefit, is more applicable to batch production, large-scale planting.Compared with control group, biological transformation ratio improves 34.79%.
Meanwhile, liquid spawn mycelium pellet density of the present invention is high is 5.5 X 10
5individual/L, commercial liquid bacterial classification mycelium pellet density is up to 2.7 X 10
5individual/L, compared with control group, its production capacity is commercial liquid bacterial classification more than 2 times, embodies higher economic worth.
It should be noted that: abalone mushroom liquid culture prepared by embodiment of the present invention 3-7 has above-mentioned experiment effect equally, between each embodiment and little with above-mentioned experiment effect otherness.
Claims (10)
1. the fermentation process of a high density liquid bacterial classification, it is characterized in that, comprise the steps: by preserve Xingbao mushroom slant strains through plating medium activation after successively through liquid seed culture medium, shake-flask seed medium and seed tank culture base expand cultivation step by step and obtain seeding tank seed, first seeding tank seed is inoculated in 30L fermentation tank with 5% inoculum concentration, fermentation medium liquid amount 15L, in 30-32 DEG C, throughput 3-5L/min ferment at constant temperature 12-24h, then 18-22 DEG C is cooled to the speed of 0.3-0.5 DEG C/min, 8-10g bag is added by shitosan and 900mL seeding tank seed in fermentation tank, ferment at constant temperature 10-16h under throughput 6-8L/min condition, last with the ramp of 0.2-0.4 DEG C/min to 24-26 DEG C, under throughput 5-7L/min condition, namely ferment at constant temperature 45-52h obtains high density liquid bacterial classification,
It is in the cyclohexaamylose aqueous solution of 10-12% that described bag is comprised the steps: to get by the preparation method of shitosan the mass percent concentration that shitosan adds its quality 30-50%, make softwood, granulate with 10-20 mesh sieve, obtain wet granular, put in power 2000W, 60-80 DEG C of dry 4-6min in microwave dryer, namely obtain bag by shitosan with the quick whole grain of 10-20 mesh sieve.
2. the fermentation process of a kind of high density liquid bacterial classification as claimed in claim 1, it is characterized in that, described liquid seed culture medium consists of: glucose 30g/L, Yeast diffusion juice 5g/L, peptone 2g/L, agar 2g/L, Chinese herbal medicine extract 0.3-0.5g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.1-0.3g/L, VB
10.01g/L, surplus is water, pH value 6.0.
3. the fermentation process of a kind of high density liquid bacterial classification as claimed in claim 1, it is characterized in that, described Shake flask medium consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.5-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.2-0.4g/L, VB
10.01g/L, surplus is water, pH value 6.0.
4. the fermentation process of a kind of high density liquid bacterial classification as claimed in claim 1, it is characterized in that, described seed tank culture base consists of: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 0.8-1.2g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, wood dust 0.3-0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0.
5. the fermentation process of a kind of high density liquid bacterial classification as claimed in claim 1, it is characterized in that, consisting of of described fermentation medium: soybean cake powder 60g/L, glucose 10g/L, peptone 5g/L, Chinese herbal medicine extract 1.5-2g/L, wood dust 0.6-1.0g/L, potassium dihydrogen phosphate 0.5g/L, magnesium sulfate 0.5g/L, VB
10.01g/L, surplus is water, pH value 6.0.
6. the fermentation process of a kind of high density liquid bacterial classification as described in as arbitrary in claim 2-5, is characterized in that, the preparation method of described Chinese herbal medicine extract, comprise the steps: to count by weight, take the Radix Astragali 30 parts, rhizoma zingiberis 30 parts, dried orange peel 30 parts, cow-bezoar 25 parts, the root bark of tree peony 25 parts, 20 parts, Radix Glycyrrhizae, the wind-weed 18 parts, honeysuckle 18 parts, cordate houttuynia 17 parts, the radix paeoniae rubrathe 15 parts, the root of large-flowered skullcap 15 parts, Ligusticum wallichii 10 parts, Radix Angelicae Sinensis 8 parts; Put in the supersonic wave cleaning machine filling 0.2% sodium bicarbonate solution and clean 12min in 200W, 30KHz, drain, said herbal medicine being crushed to particle diameter is less than 2 millimeters, puts Homogeneous phase mixing in container and adds the water of 5 times of weight, obtaining mixed material, control temperature 80 DEG C keeps 3h, then being cooled to 45 DEG C, is 4 by lactic acid adjust ph, under power 200W condition, carry out Microwave Extraction 12min, under power 250W, frequency 35KHz condition, carry out ultrasonic assistant extraction simultaneously; Then the mixed enzyme adding mixed material gross weight 1.0% carries out enzymolysis, be 6.2 by lactic acid adjust ph, enzymolysis 3h, finally add the mixture of mixed material 2 times of w ethanol and propyl alcohol, the mass ratio of ethanol and propyl alcohol mixing is 1:2, control temperature to 70 DEG C keeps 3.5h, filters, obtains the first filtrate; Add the water of filter residue 2 times of weight, control temperature 90 DEG C keeps 2h, is then cooled to 30 DEG C, filters, obtains the second filtrate; First filtrate and the second filtrate are merged according to mass ratio 3:2, filter vacuum concentrates postlyophilization, pulverizes and obtains Chinese herbal medicine extract;
Described mixed enzyme is dextranase, zytase, pentosanase, pectase 3:2:2:1 Homogeneous phase mixing in mass ratio.
7. the fermentation process of a kind of high density liquid bacterial classification as described in as arbitrary in claim 2-5, it is characterized in that, the preparation method of described wood dust comprises the steps: without mouldy, anosis worm sawmilling end or waste wood removing foreign material, be placed in the supersonic wave cleaning machine of the sodium bicarbonate solution that mass percent concentration 0.5% is housed in 200W, 30KHz cleans 10min, rinse, drain, put into steam still in 0.3-0.4MPa boiling 20-30min, take out, drain, with vegetable oil 100:1 Homogeneous phase mixing in mass ratio, then mixture is put microwave dryer in 2000W, 120 DEG C of microwave irradiation 5min, last ultramicro grinding is to particle diameter 0.3-0.5mm, pack and obtain wood dust.
8. the fermentation process of a kind of high density liquid bacterial classification as claimed in claim 7, is characterized in that, microwave irradiation 10s during microwave drying, stops 10s.
9. the high density liquid bacterial classification that the fermentation process as described in as arbitrary in claim 1-8 is obtained.
10. the application of high density liquid bacterial classification in planting almond abalone mushroom as claimed in claim 9.
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| CN111771621A (en) * | 2020-07-20 | 2020-10-16 | 长治学院 | Method for producing oyster mushroom liquid strain by using compound sophora flavescens alcohol sediment |
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| CN110819563B (en) * | 2019-11-18 | 2022-03-01 | 曲阜师范大学 | Brevibacillus brevis 3C and application thereof in promoting growth of pleurotus eryngii |
| CN111615996B (en) * | 2020-05-19 | 2021-09-21 | 江西省农业科学院农业应用微生物研究所（江西省农村能源研究中心） | Stropharia rugosoannulata strain breeding method |
| CN115873718B (en) * | 2022-10-25 | 2024-08-16 | 江苏神华药业有限公司 | Liquid fermentation medium for armillaria mellea mycelium and preparation method and application thereof |
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| CN111771621A (en) * | 2020-07-20 | 2020-10-16 | 长治学院 | Method for producing oyster mushroom liquid strain by using compound sophora flavescens alcohol sediment |
Also Published As
| Publication number | Publication date |
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| CN108251313A (en) | 2018-07-06 |
| CN108271620A (en) | 2018-07-13 |
| CN104920069B (en) | 2018-03-30 |
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