CN105009941A - Cordyceps sinensis continuous submerged fermentation liquor and fermentation powder production method - Google Patents
Cordyceps sinensis continuous submerged fermentation liquor and fermentation powder production method Download PDFInfo
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Abstract
The present invention relates to the cordyceps sinensis technology field, in particular to a cordyceps sinensis continuous submerged fermentation liquor and fermentation powder production method. The method is carried out as the following steps of carrying out the mother-specie purification of the companion fungus and the cordyceps sinensis; b carrying out the fermentation tank strain culture of the companion fungus and the cordyceps sinensis; c mixing the continuous submerged fermentation liquor; d canning and sterilizing the fermentation liquor; e filtering, freeze-drying and powdering the solid matter. By separating and purifying the wild cordyceps sinensis bacterial strain of the Qinghai-Tibet Plateau, and extracting the artificial fermented cordyceps sinensis liquor and cordyceps sinensis powder by a continuous submerged fermentation technology, multiple key indexes of the artificial fermented cordyceps sinensis liquor and cordyceps sinensis powder all exceed the natural cordyceps sinensis, and have the medicinal effect of stable quality. The method of the present invention is mainly applied to the human body health care treatment.
Description
Technical field
The present invention relates to Cordyceps sinensis technical field, more specifically, relate to the production method of the continuous submerged fermentation liquid of Cordyceps sinensis and yeast powder.
Background technology
Cordyceps sinensis is the organism of a kind of special worm and mycosymbiosis, its mycelium infects the larva of bat moth (Lepidoptera Hepialidae bat Hepialus insect) by various mode, using the organic substance in its body as nutrient energy, parasitic life is carried out in source, after constantly growing and breaking up, final mycelium is twisted together and is formed stroma and stretches out host's shell, thus the organism of a kind of special entomogenous fungi symbiosis formed.Medicinal part is the complex of sclerotium and stroma.Use recorded first by Cordyceps sinensis is Qing Dynasty Wu Yi Lip river " this thick grass is new ", thinks that Cordyceps sinensis nature and flavor are sweet in book, temperature.Function is invigorated the lung and the kidney, preventing phlegm from forming and stopping coughing.Can be used for that chronic cough void is breathed heavily, the illness that puerperal asthenia, impotence are sombre etc. " void ".According to the study: Cordyceps sinensis is mainly containing compositions such as Cordycepin, Cordyceps sinensis acid, adenosine and polysaccharide; Cordycepin can suppress the growth of the germ such as streptococcus, Actinobacillus mallei bacillus anthracis, is again anticancer active substance, to the regulating action that endocrine system and the nervous system of human body have had; Cordycepic acid can change microcirculation in human body, has obvious reducing blood lipid and Antitussive and Expectorant Effect; Cordyceps sinensis polysaccharide is immunomodulator, can strengthen body to virus and parasitic resistance.Because high-quality natural cs output is extremely limited, cannot satisfy the demands, the source of goods is in great shortage, expensive, for meeting the demand of consumers in general, is necessary to improve prior art.
Summary of the invention
In order to overcome deficiency existing in prior art, provide the production method of the continuous submerged fermentation liquid of a kind of Cordyceps sinensis and yeast powder.
In order to solve the problems of the technologies described above, the technical solution used in the present invention is:
The production method of the continuous submerged fermentation liquid of Cordyceps sinensis and yeast powder, carry out according to following steps:
The mother of a, concomitance bacterium and Cordyceps sinensis plants purifying;
The fermentation tank Spawn incubation of b, concomitance bacterium and Cordyceps sinensis;
C, mix continuous submerged fermentation;
The preparation of d, zymotic fluid;
The preparation of e, yeast powder.
In described a step, association starter kind purifying carries out in such a way: 1) preparation raw material; 2) medium is made; 3) packing test tube; 4) Sai Pisai; 5) test tube wrapping; 6) medium sterilization; 7) inclined-plane is put; 8) sterilization effect is checked; 9) female kind is inoculated; 10) bacterial classification is cultivated;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L, inorganic ions (initial pH is 5.6-6.0): 0.3g CaCl22H2O, 1g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2; Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, agar 18-20 gram, PH nature;
Above-mentioned making medium is specially: the potato selecting high-quality, peeling, cut out eye, ream dark green part, be cut into the thin slice of long 0.5 millimeter, take 200 grams, add water 1000 milliliters, boil rear low baking temperature and keep 30 minutes, potato is boiled and not crisp, i.e. 8 ripe left and right, then by 2 layers of adhesional wetting filtered through gauze, add agar afterwards and continue heating, and continuous glass bar is stirred to agar dissolves completely, finally add sucrose successively, glucose, tryptone, yeast extract, inorganic ions, continue to heat and be stirred to all to dissolve, finally complement to 1000 milliliters with clear water, packing can be carried out.
In described a step, Cordyceps sinensis mother kind purifying carries out in such a way: 1) preparation raw material; 2) medium is made; 3) inoculate; 4) cultivate;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, 50 grams, wheat bran, potato 200 grams, glucose 20 grams, peptone 5 grams, 18-20 grams, agar, potassium dihydrogen phosphate l.5 gram, 0.75 gram, magnesium sulfate, PH nature;
Above-mentioned making medium is specially: added by potato in the water of 1000ml, boil rear low baking temperature and keep 30 minutes, then by double-deck adhesional wetting filtered through gauze, add agar afterwards and continue heating, and continuous glass bar is stirred to agar dissolves completely, then add wheat bran, glucose, potassium dihydrogen phosphate, peptone, magnesium sulfate successively, continue to heat and be stirred to all to dissolve, finally complement to 1000 milliliters with clear water, complete medium and make.
In described b step, concomitance bacterium fermentation tank Spawn incubation carries out in such a way: 1) preparation raw material; 2) make; 3) inoculate; 4) cultivate;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L, inorganic ions (initial pH is 5.6-6.0): 0.3 g CaCl22H2O, 1 g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01 g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2; Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, PH nature;
Above-mentionedly to be made as: formula is above made liquid nutrient medium, loads 1000ml shaking flask, put into stirrer, be stoppered bottleneck, wrap moistureproof paper, put into autoclave, seal pot
mouthful, open vent valve, close vent valve when there being a large amount of steam to discharge, temperature rises to 121 DEG C-123 DEG C, and time remaining is 30 minutes, then allows it naturally cool, and during pressure back to zero, opens sterilizing pot cover and takes out liquid bottles.
In described b step, Cordyceps sinensis fermentation tank Spawn incubation carries out in such a way: 1) preparation raw material; 2) make; 3) inoculate; 4) cultivate;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, 50 grams, wheat bran, potato 200 grams, glucose 20 grams, peptone 5 grams, potassium dihydrogen phosphate l.5 gram, 0.75 gram, magnesium sulfate, PH nature;
Above-mentionedly to be made as: formula is above made liquid nutrient medium, loads 1000ml shaking flask, put into stirrer, be stoppered bottleneck, wrap moistureproof paper, put into autoclave, seal pot
mouthful, open vent valve, close vent valve when there being a large amount of steam to discharge, temperature rises to 121 DEG C-123 DEG C, and time remaining is 30 minutes, then allows it naturally cool, and during pressure back to zero, opens sterilizing pot cover and takes out liquid bottles.
Described step c is carried out in such a way: 1) fermentation tank sterilization treatment; 2) continuous submerged fermentation liquid nutrient medium is mixed; 3) charging sterilizing; 4) inoculate; 5) cultivation of mixed culture fermentation liquid;
The continuous submerged fermentation liquid nutrient medium of above-mentioned mixing is configured according to following component, sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L; Inorganic ions (initial pH is 5.6-6.0): 0.3g CaCl22H2O, 1g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2, inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, PH nature;
Above-mentioned inoculation is that cultured concomitance bacterium fermented bacterium and Cordyceps sinensis fermented bacterium are carried out combined inoculation.
Described Step d is carried out in such a way: use filter to start to filter temperature 25 ~ 28 DEG C, reflux and filter liquid 10 minutes, collects the filtrate of clarification, obtain Cordyceps fermented liquid; Filter the liquid of gained through super high temperature instantaneous sterilizing machine sterilization, sterilization temperature (110 ± 5) DEG C, 10 seconds time, sterilized liquid is put and is cooled to 50 ~ 55 DEG C rapidly to temporary storage tank, concentrates; Concentrated under vacuum 0.096 ~ 0.099 MPa, fluid temperature 50 ~ 55 DEG C, steam pressure 0.196 ~ 0.294 MPa, be concentrated into after soluble solid reaches 42% ~ 45% and carry out re-pasteurization; Liquid is loaded in special-purpose bottle (bag) by the liquid liquid-filling machine concentrated, then 90 DEG C of sterilizations 30 minutes; By finished product constant refrigeration in-18 ~-25 DEG C of freezers.
Described step e is carried out in such a way: after the whole drying of fermentation solid content low-temperature freeze-dry equipment after filtration, then wear into 120 object fine powders with low temperature milling equipment, obtain Cordyceps sinensis yeast powder, then carry out vacuum packaging preservation.
The beneficial effect that compared with prior art the present invention has is:
The present invention is passing through the wild cordyceps strain separating to Qinghai-Tibet Platean, purification, and by continuous submerged fermentation technology, extracts Chinese caterpillar fungus liquid and the Chinese caterpillar fungus powder of artificial fermentation.Multinomial key index all exceedes natural Cordyceps sinensis, and has the characteristic of steady quality drug effect.Contained Chinese caterpillar fungus selenium be anticancer, oxidize-resistant physiology active in one of extremely important trace element, in cordycepin, reach 0.21gm, far above the 0.0049gm of natural cs.Nucleosides is one of principle active component in Chinese caterpillar fungus, and have the effects such as anticancer, antiviral, antibacterial, excellent natural cs can reach 0.1% order of magnitude, and cordycepin even can reach 0.30%.The com-parison and analysis result of natural cs and cordycepin mycelia amino acid content, cordycepin mycelia amino acid content exceeds one times than natural cs.Cordyceps fermented liquid and Cordyceps sinensis yeast powder are breathed heavily chronic cough void after part population is taken, and labor is coughed spitting of blood, positive fistula seminal emission, soreness of waist and knee joint, insomnia, and constipation, anaemia, ventilate, hyperglycaemia etc. have good curative effect.
Embodiment
Below by specific embodiment, the present invention is described in further detail.
Technological process of the present invention is carried out in the following manner: 1) mother of concomitance bacterium and Cordyceps sinensis plants purifying; 2) the fermentation tank Spawn incubation of concomitance bacterium and Cordyceps sinensis; 3) continuous submerged fermentation is mixed; 4) preparation of zymotic fluid; 5) preparation of yeast powder.
Aweto strain is a special bacterial classification, for concomitance bacterium and Cordyceps sinensis symbiosis mushroom, first first to cultivate association starter kind and concomitance bacterium liquid fermentation tank bacterial classification before manufacture respectively and Cordyceps sinensis mother plants and Cordyceps sinensis liquid fermentation tank bacterial classification, finally by its two kinds of zymotic fluid bacterial classifications combined inoculation more further continuously submerged fermentation can obtain Cordyceps fermented liquid and yeast powder.
One, female kind purifying
(1), concomitance bacterium Mother culture
1, fill a prescription: 1000ml is example
Sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L; Inorganic ions (initial pH is 5.6-6.0): 0.3g CaCl22H2O, 1g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2.Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, 18-20 grams, agar, PH nature.
2, medium makes.Select the potato of high-quality, peeling, cuts out eye, reams dark green part, is cut into the thin slice of long 0.5 millimeter, takes 200 grams, add water 1000 milliliters, boil rear low baking temperature and keep 30 minutes, and potato is boiled and not crisp, i.e. 8 ripe left and right.Then by 2 layers of adhesional wetting filtered through gauze, add agar afterwards and continue heating, and continuous glass bar is stirred to agar dissolves completely, finally adds sucrose, glucose, tryptone, yeast extract, inorganic ions successively, continuation is heated and is stirred to and all dissolves.Finally complement to 1000 milliliters with clear water, can packing be carried out.
3, packing test tube.Left hand holds 5 test tube mouths of pipe upward, and lower end is neat, above moves test tube to funnel flexible pipe, inserts in vitro about 3 centimetres.The right hand clutches flexible pipe flow-stopping clip, makes medium liquid flow into test tube, and often prop up that test tube loading amount is about test tube length 1/5.Now unclamp flow-stopping clip liquid to stop flowing into.Flexible pipe is moved away by under test tube, then packing the 2nd test tube, other test tubes of Using such method packing, until all install.Packing test tube requires that speed wants fast, especially at low temperature season, to prevent culture medium solidifying.Loading amount is wanted accurately, because the loading amount inclined-plane be put on the high side shortens, waste medium again, charge weight is on the low side, and make the inclined-plane that is put into too thin, nutrition is few, and mycelia is difficult to normal growth.Note avoiding medium to be attached to the test tube mouth of pipe, in order to avoid cause the phenomenon of adhesion skin plug, also easily cause skin to multiply miscellaneous bacteria beyond the Great Wall, leave hidden danger to strain quality.
4, Sai Pisai, fills in test tube with hand special skin plug, and skin plug will be close to the wrinkle resistant line of tube wall, and the skin plug part filled in pipe is about 2.5 centimetres, and exposed parts accounts for 1/3 of Pi Sai overall length.
5, test tube wrapping.Test tube is stoppered Pi Saihou, gets the test tube of 10 same specifications, is encased by tampon, and extends downwardly in the middle part of test tube, be bundled into one with bonnet cotton with brown paper or double-deck newspaper.
6, medium sterilization.Mother culture Quito adopts portable high-pressure sterilizing pot to carry out sterilizing, and sterilization process is: flexibly whether the manometer of inspection high-pressure steam sterilizing pan and each position, reliably; Especially note checking whether safety valve blocks or become rusty dead.In pot, add about 3 kilograms of running water, add to support lower edge.The medium tampon tied upwards vertically is put into bacterium basket (bucket), and inserted in the wireway of sleeve lining by the blast pipe that pot cover hangs down, then both hands are exerted oneself simultaneously, respectively by two of diagonal screw tightenings.Close air bleeding valve after capping and start heating, when being heated to pressure and reaching 0.05 MPa, open air bleeding valve, drain cold air, after Pressure Drop to 0, can air bleeding valve be closed.Preferably arrange 2 times according to upper method.Continue heating, start timing when pressure increase to 0.11 MPa, keep 30 minutes under 0.11 ~ 0.14 MPa, stop heating, make it cool voluntarily and be down to 0 DEG C.Open air bleeding valve, then pot door is opened the gap heat radiation of 10 cm.
7, inclined-plane is put.After observing newspaper drying, open pot cover, take out in time when medium temperature drops to about 60 DEG C and put inclined-plane.Notice that medium will cover bottom test tube, chamfer length should with 3/5ths of test tube length.When in vitro medium is packed up for subsequent use after solidifying completely.The medium prepared should be placed on aeration-drying place and preserve.
If just put inclined-plane when temperature is very high, culture medium solidifying rear surface there will be more condensed water, unfavorable mycelial growth.After being well placed inclined-plane, cover medium with abundant cotton, medium slow cooling is cooled, also can reduce condensed water and occur.
8, sterilization effect inspection.Get part test tube slant at random, carry out blank at being placed on 28 DEG C and cultivate, after 3 days, check on inclined-plane with or without bacterium and mold colony.If find that there is miscellaneous bacteria, illustrate that sterilizing is not thorough, sterilizing again.
9, female kind is inoculated
Before inoculation, medium is put into inoculating hood to carry out disinfection, sterilization requires to be undertaken by aforementioned.During inoculation, hand and inoculation hook with 75% cotton ball soaked in alcohol wipe, light alcolhol burner, flame periphery space is made to form aseptic area, left hand is parallel picks up female kind test tube and the slant tube for inoculation side by side, and upwards, the mouth of pipe will flush in two test tube slants, right hand thumb and forefinger hold inoculation hook, sterilizing that the flame of alcolhol burner burns.The part of test tube that inoculation hook is allowed for access all will be carried out calcination.Left hand test tube is moved to by flame, right-handed little finger of toe, the third finger and palm, under the other folder respectively of flame, the tampon of two test tubes, bakes on flame a little by test tube mouth, to kill the miscellaneous bacteria on the mouth of pipe, subsequently the mouth of pipe is moved to apart from flame 1 ~ 2 centimeters, the inoculation of calcination rake is stretched in strain tube, first contacts inboard wall of test tube, make it cool, the aerial hyphae hook of being climbed wall again falls, and is cut off by medium front end thinnest part, goes out outside pipe together with aerial hyphae in the lump hook.With inoculation hook (shovel, rake), mother's kind is slit into many blockages, then picking one fritter in length and breadth again, moves into rapidly the middle front part of the test tube slant of inoculation, extract inoculation hook gently out, roasting test tube mouth once, crosses rapidly the tampon of flame beyond the Great Wall again, and a mother plants generally can expand and connects 30 ~ 40, test tube.
Technical essential: whole process must carry out sterile working on flame side, the inoculation block in female kind and aging will the removing of periphery, the mycelia block selecting cell age short is produced.Before test tube takes out from inoculating hood, should be labelled by the top of a tube facing side, write bacterium numbering, inoculation date exactly.
10, Spawn incubation
By connecting the slant tube of planting, putting into biochemical cultivation case, cultivating 90 days under temperature being set to 16--18 DEG C of temperature, in incubation, all wanting every day careful backlight to observe, find that there is any doubtful Chang Xianxiang and all will eliminate.
(2), Cordyceps sinensis mother plants purifying
1, fill a prescription: 1000ml is example
50 grams, wheat bran, potato 200 grams, glucose 20 grams, peptone 5 grams, 18-20 grams, agar, potassium dihydrogen phosphate l.5 gram, 0.75 gram, magnesium sulfate, PH nature.
2, make
The same concomitance bacterium of preparation method of Cordyceps sinensis mother culture media.
3, inoculate
The slant tube of mother's kind used and brand-new is put into inoculating hood (or clean bench), sterilize after 20-30 minute by hand with 75% medicinal alcohol clean (sterilization), stretch into and after inoculating hood (or clean bench) waits for 5 minutes, to start inoculation (before inoculation all inoculating tools all will calcination sterilizing on alcolhol burner flame) again, 0.5cm2 mono-fritter mycelia is cut above alcolhol burner flame, put on the slant tube medium of brand-new, be stoppered test tube mouth.
4, cultivate
By connecting the slant tube of planting, putting into biochemical cultivation case, cultivating 90 days under temperature being set to 16--18 DEG C of temperature, in incubation, all wanting every day careful backlight to observe, find that there is any doubtful Chang Xianxiang and all will eliminate.
Two, fermentation tank Spawn incubation
(1) concomitance bacterium fermented bacterium is cultivated
Sucrose 50 g/L, tryptone 10 g/L, yeast extract 3 g/L; Inorganic ions (initial pH is 5.6-6.0): 0.3 g CaCl22H2O, 1 g KH2PO4,0.5 g MgSO47H2O, 1 g NaCl, 0.01 g CuSO45H2O, 0.01 g FeSO47H2O, 0.01 g MnCl24H2O, 0.01 g ZnCl2.Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, PH nature.
2, make
Making liquid nutrient medium by filling a prescription above, loading 1000ml shaking flask, putting into stirrer.Be stoppered bottleneck, wrap moistureproof paper, put into autoclave, seal pot
mouthful, open vent valve, close vent valve when there being a large amount of steam to discharge, after temperature rises to 121 DEG C-123 DEG C, timing 30 minutes, allows it naturally cool, and during pressure back to zero, opens sterilizing pot cover and takes out liquid bottles.
3, inoculate
Liquid bottles bottle and the good mother of purifying are planted after putting into inoculating hood (or clean bench) sterilization together, remove moistureproof paper, back-out cotton stockaded village, above alcolhol burner flame, the mycelia block of picking 3 square centimeters, left hand is picked up shaking flask and is sealed cotton stockaded village, and mycelia is accessed rapidly by the right hand, then covers tampon at once.
4, cultivate
The shaking flask to connect kind is put into biochemical cultivation case, and temperature to be set at the temperature of the most applicable growth of this bacterium static gas wave refrigerator 4 days, backlight is observed, and without any miscellaneous bacteria, to be then placed on electromagnetic constant-temperature shaking table 18 DEG C of stir culture 60 days.Concomitance bacterium fermented bacterium cultivate after will with Cordyceps sinensis fermented bacterium more further combined inoculation carry out submerged fermentation.
(2), Cordyceps sinensis fermented bacterium is cultivated
1, fill a prescription
50 grams, wheat bran, potato 200 grams, glucose 20 grams, peptone 5 grams, potassium dihydrogen phosphate l.5 gram, 0.75 gram, magnesium sulfate, PH nature.
2, make
Making liquid nutrient medium by filling a prescription above, loading 1000 ㏕ shaking flasks, putting into stirrer.Be stoppered bottleneck, wrap moistureproof paper, put into autoclave, seal pot
mouthful, open vent valve, close vent valve when there being a large amount of steam to discharge, after temperature rises to I21 DEG C-123 DEG C, timing 30 minutes, allows it naturally cool, and during pressure back to zero, opens sterilizing pot cover and takes out liquid bottles.
3. inoculate
Liquid bottles bottle and the good mother of purifying are planted after putting into inoculating hood (or clean bench) sterilization together, remove moistureproof paper, back-out cotton stockaded village, above alcolhol burner flame, the mycelia block of picking 3 square centimeters, left hand is picked up shaking flask and is sealed cotton stockaded village, and bacterial classification accesses rapidly by the right hand, then covers tampon at once.
4, cultivate
The shaking flask to connect kind is put into biochemical cultivation case, and temperature to be set at the temperature of the most applicable growth of this bacterium static gas wave refrigerator 4 days, backlight is observed, and without any miscellaneous bacteria, to be then placed on electromagnetic constant-temperature shaking table 16-18 DEG C of stir culture 60 days.Cordyceps sinensis fermented bacterium cultivate after will with concomitance bacterium fermented bacterium more further combined inoculation carry out submerged fermentation.
Three, continuous submerged fermentation is mixed
1, fermentation tank sterilizing place is buried
Sterilizing feed supplement tank is added water to 70% of cumulative volume.Open feed supplement tank power switch, temperature is set to 121 DEG C-125 DEG C, after getting to this temperature, close air header road valve, open steam valve, allow steam enter three grades, level Four successively by secondary filter, finally arrive fermentation tank, after fermentation jar temperature reaches ll8 DEG C-120 DEG C, valve, sample valve will be inoculated, air bleeding valve is opened a bit, steam is allowed slowly to discharge a bit, sterilizing (will arrange the condensed water of a filter bottom generation for every 3-5 minutes in whole process) is carried out to it, keep after 30 minutes, first open fermentation tank drain tap below, then sterilizing feed supplement tank discharging opening valve is slowly opened a bit, high-temperature high pressure water is allowed to flow out a little, after its threeway preheating, open the steam valve entering punishment in advance pipeline, after drain tap after fermentation tank has a large amount of steam to discharge, close fermentation tank drain tap below, open fermentation tank charging aperture valve below, then sample valve is closed, the rapid metal tube head that will sample inserts in formaldehyde bottle, close inoculation and drain tap on fermentation tank deck, first open inoculation and rob controlled valve, again fermentation tank is gone out kind of a mouth valve slowly to open a bit, high-temperature high pressure water is allowed to flow out a little, after its threeway preheating, open the steam valve entering seed output pipe road, when inoculation snatch away kind of a mouth have a large amount of steam discharge after, gas output is turned down, have a small amount of steam to discharge, sterilizing is closed inoculating gun switch and is gone out bacterial classification mouth valve after 30 minutes, for subsequent use with aseptic pouch seal.Temperature be set to lower than room temperature, open air pump, close steam sub valve, open air total valve, dry up filter core, allow filtrated air enter in tank, adjustment tank pressure, at 0.03-0.05MPa, closes the drain tap below filter, allows whole system keep malleation.With 75% medicinal alcohol cotton balls, the inoculation mouth valve of fermentation tank top center is sealed after terminating.
2, continuous submerged fermentation liquid nutrient medium is mixed
Sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L; Inorganic ions (initial pH is 5.6-6.0): 0.3g CaCl22H2O, 1g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2.Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, PH nature.
3, charging sterilizing
First sterilizing feed supplement tank steam total valve and discharging opening valve is closed, the water (if the too poor need of water quality carry out purified treatment) of required loading amount 70% will be added in feed supplement tank, open sterilizing feed supplement tank power switch, temperature is set to 95 DEG C, all raw materials with hot water dissolving together, when in feed supplement tank, temperature is raised to 95 DEG C, open discharging opening valve and pass into a small amount of air, first defoamer is added under the stirring of air, when feed temperature is close to 95 DEG C, close discharging opening valve, after more than 95 DEG C, close all valves on feed supplement tank; Temperature is set to 121 DEG C, opens timer (sterilization time sets 30 minutes); Connected by heat exchanger circulating water pipeline, after reaching sterilising temp, by the charge door valve at sterilization tank top, drain tap is all opened a bit, allows steam discharge a little, to its sterilizing, after reaching sterilization time, closes heating power supply and all valves in sterilization tank top.First discharge 500mL-1000mL feed liquid from the sewage draining exit below sterilizing feed supplement tank, again down cycles water is led to, then open feed supplement tank discharging opening valve, by the composts or fertilisers of cultivating of bacterium of having gone out, enter fermentation tank after being lowered the temperature by heat exchanger, sterilizing temperature controller temperature is controlled below 25 DEG C.
4, inoculate
Within 2 hours, with ozone, all for culturing room environment are carried out disinfection in advance, then cultured shake-flask seed is taken out, from inoculation valve, bacterial classification is accessed under Alcohol Flame protection, general two people's cooperations are carried out, be enclosed within inoculation mouth with the fire ring soaking alcohol, close air intake valve, open drain tap, allow pressure zero in fermentation tank, the firstly light fire ring, the cotton ball soaked in alcohol of inoculation valve port is taken away with tweezers, open inoculation valve, the hand-held shaking flask of second people, metal pipe mouth is put into flame, the first tweezers pull out the tampon in tube head, pipe inserts in inoculation valve by the second people, tiltedly put forward shaking flask in aweto strain and concomitance bacterium mixing access fermentation tank, pipe is extracted after having connect, valve-off, remove fire ring, inoculate salty new use 75% medical cotton ball to seal, open air intake valve, pass into filtrated air fermented and cultured.
5, the cultivation of mixed culture fermentation liquid
With the impeller speed stir culture 72 hours of 180 revs/min after inoculation, middlely detect sugar, nitrogen, pH value, liquid asepsis degree every sampling in 12 hours.Putting tank standard is that zymotic fluid pH value drops to 5, and residual sugar content is down to about 2.5, and ammoniacal nitrogen is no more than 30 mg/litre, and bacterium ball concentration reaches 1000 ~ 1500/milliliter, or through 3000 revs/min centrifugal 10 points, solid content weight in wet base is at 20 ~ 25 grams/100 milliliters.Check that zymotic fluid qualitative items comprises: purity test, microscopy or slat chain conveyor are without bacterium, mould; Vigor checks, microscopy bacterium ball edge mycelia branch is fine and closely woven, and color depth with during violet staining, hyphal cell protoplasm not yet occurs agglutination phenomenon, static 5 points of mash, and solid content does not sink; Bacterium ball size, 80% bacterium bulb diameter is less than 1 millimeter; Temperature controller temperature is set to 23-25 DEG C, and in tank, pressure keeps 0.03-0.04MPa, throughput: material: gas is in 1:(0.2-0.5) v/v ferments 10 days.
Four, aweto fluid separation applications filters and packages (preparation of zymotic fluid)
1, use special filtering machine to start to filter temperature 25 ~ 28 DEG C, reflux and filter liquid 10 minutes, collects the filtrate of clarification.Obtain Cordyceps fermented liquid.
2, a sterilization: filter the liquid of gained through super high temperature instantaneous sterilizing machine sterilization, sterilization temperature (110 ± 5) DEG C, 10 seconds time.Sterilized liquid is put and is cooled to 50 ~ 55 DEG C rapidly to temporary storage tank, concentrates.
3, Vacuum Concentration: concentrated under vacuum 0.096 ~ 0.099 MPa, fluid temperature 50 ~ 55 DEG C, steam pressure 0.196 ~ 0.294 MPa, is concentrated into after soluble solid reaches 42% ~ 45% and carries out re-pasteurization.
4, re-pasteurization: liquid is loaded in special-purpose bottle (bag) by the liquid liquid-filling machine concentrated, then 90 DEG C of sterilizations 30 minutes.
5, refrigerate: by finished product constant refrigeration in-18 ~-25 DEG C of freezers.
6, product quality indicator
(1). organoleptic indicator
Color and luster: pitchy liquid is translucent; Flavour and smell: there is the distinctive fragrance of Inonotus obliquus and smell, without being charred taste when being diluted to Normal juice concentration, free from extraneous odour; Tissue morphology: be organized as translucent uniform liquid, five layerings, deposited phenomenon; Impurity: without exogenous impurity.
(2). physical and chemical index
Soluble solid 42% ± 1%; PH value 5.0 ~ 7.0.
(3). microbiological indicator
Total number of bacteria≤3000/milliliter; Coliform, pathogenic bacteria must not detect.
Five, solid content freeze-drying abrasive dust (preparation of yeast powder) is filtered
1, be laid in lyophilized plate by worm winter, filtering fermentation liquor summer solid content solid content, should keep its high uniformity when thing is laid in lyophilized plate in filter, to reduce batch interpolation of water content after freeze-drying, height can not be greater than 2cm, in order to avoid affect effect;
2, treat in lyophilized plate that lyophilized products height can not lower than 1.5cm;
3, open freeze dryer, ruuning situation is shown in
following table:
4, after the whole drying of fermentation solid content low-temperature freeze-dry equipment after filtration, then wear into 120 object fine powders with low temperature milling equipment, then carry out vacuum packaging preservation.
Claims (8)
1. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis and yeast powder, is characterized in that, carry out according to following steps:
The mother of a, concomitance bacterium and Cordyceps sinensis plants purifying;
The fermentation tank Spawn incubation of b, concomitance bacterium and Cordyceps sinensis;
C, mix continuous submerged fermentation;
The preparation of d, zymotic fluid;
The preparation of e, yeast powder.
2. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, is characterized in that, in described a step, association starter kind purifying carries out in such a way: 1) preparation raw material; 2) medium is made; 3) packing test tube; 4) Sai Pisai; 5) test tube wrapping; 6) medium sterilization; 7) inclined-plane is put; 8) sterilization effect is checked; 9) female kind is inoculated; 10) bacterial classification is cultivated;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L, inorganic ions (initial pH is 5.6-6.0): 0.3g CaCl22H2O, 1g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2; Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, agar 18-20 gram, PH nature;
Above-mentioned making medium is specially: the potato selecting high-quality, peeling, cut out eye, ream dark green part, be cut into the thin slice of long 0.5 millimeter, take 200 grams, add water 1000 milliliters, boil rear low baking temperature and keep 30 minutes, potato is boiled and not crisp, i.e. 8 ripe left and right, then by 2 layers of adhesional wetting filtered through gauze, add agar afterwards and continue heating, and continuous glass bar is stirred to agar dissolves completely, finally add sucrose successively, glucose, tryptone, yeast extract, inorganic ions, continue to heat and be stirred to all to dissolve, finally complement to 1000 milliliters with clear water, packing can be carried out.
3. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, is characterized in that, in described a step, Cordyceps sinensis mother kind purifying carries out in such a way: 1) preparation raw material; 2) medium is made; 3) inoculate; 4) cultivate;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, 50 grams, wheat bran, potato 200 grams, glucose 20 grams, peptone 5 grams, 18-20 grams, agar, potassium dihydrogen phosphate l.5 gram, 0.75 gram, magnesium sulfate, PH nature;
Above-mentioned making medium is specially: added by potato in the water of 1000ml, boil rear low baking temperature and keep 30 minutes, then by double-deck adhesional wetting filtered through gauze, add agar afterwards and continue heating, and continuous glass bar is stirred to agar dissolves completely, then add wheat bran, glucose, potassium dihydrogen phosphate, peptone, magnesium sulfate successively, continue to heat and be stirred to all to dissolve, finally complement to 1000 milliliters with clear water, complete medium and make.
4. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, is characterized in that, in described b step, concomitance bacterium fermentation tank Spawn incubation carries out in such a way: 1) preparation raw material; 2) make; 3) inoculate; 4) cultivate;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L, inorganic ions (initial pH is 5.6-6.0): 0.3 g CaCl22H2O, 1 g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01 g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2; Inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, PH nature;
Above-mentionedly to be made as: formula is above made liquid nutrient medium, loads 1000ml shaking flask, put into stirrer, be stoppered bottleneck, wrap moistureproof paper, put into autoclave, seal pot mouth, open vent valve, close vent valve when there being a large amount of steam to discharge, temperature rises to 121 DEG C-123 DEG C, time remaining is 30 minutes, then allow it naturally cool, during pressure back to zero, open sterilizing pot cover and take out liquid bottles.
5. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, is characterized in that, in described b step, Cordyceps sinensis fermentation tank Spawn incubation carries out in such a way: 1) preparation raw material; 2) make; 3) inoculate; 4) cultivate;
Above-mentioned preparation raw material is specially: be configured respectively according to following component, 50 grams, wheat bran, potato 200 grams, glucose 20 grams, peptone 5 grams, potassium dihydrogen phosphate l.5 gram, 0.75 gram, magnesium sulfate, PH nature;
Above-mentionedly to be made as: formula is above made liquid nutrient medium, loads 1000ml shaking flask, put into stirrer, be stoppered bottleneck, wrap moistureproof paper, put into autoclave, seal pot mouth, open vent valve, close vent valve when there being a large amount of steam to discharge, temperature rises to 121 DEG C-123 DEG C, time remaining is 30 minutes, then allow it naturally cool, during pressure back to zero, open sterilizing pot cover and take out liquid bottles.
6. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, it is characterized in that, described step c is carried out in such a way: 1) fermentation tank sterilization treatment; 2) continuous submerged fermentation liquid nutrient medium is mixed; 3) charging sterilizing; 4) inoculate; 5) cultivation of mixed culture fermentation liquid;
The continuous submerged fermentation liquid nutrient medium of above-mentioned mixing is configured according to following component, sucrose 50g/L, tryptone 10g/L, yeast extract 3g/L; Inorganic ions (initial pH is 5.6-6.0): 0.3g CaCl22H2O, 1g KH2PO4,0.5g MgSO47H2O, 1g NaCl, 0.01g CuSO45H2O, 0.01g FeSO47H2O, 0.01g MnCl24H2O, 0.01g ZnCl2, inorganic ions can be made into 10 × mother liquor 4 DEG C of preservations, potato 200 grams, glucose 20 grams, PH nature;
Above-mentioned inoculation is that cultured concomitance bacterium fermented bacterium and Cordyceps sinensis fermented bacterium are carried out combined inoculation.
7. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, it is characterized in that, described Step d is carried out in such a way: use filter to start to filter temperature 25 ~ 28 DEG C, reflux and filter liquid 10 minutes, collect the filtrate of clarification, obtain Cordyceps fermented liquid; Filter the liquid of gained through super high temperature instantaneous sterilizing machine sterilization, sterilization temperature (110 ± 5) DEG C, 10 seconds time, sterilized liquid is put and is cooled to 50 ~ 55 DEG C rapidly to temporary storage tank, concentrates; Concentrated under vacuum 0.096 ~ 0.099 MPa, fluid temperature 50 ~ 55 DEG C, steam pressure 0.196 ~ 0.294 MPa, be concentrated into after soluble solid reaches 42% ~ 45% and carry out re-pasteurization; Liquid is loaded in special-purpose bottle (bag) by the liquid liquid-filling machine concentrated, then 90 DEG C of sterilizations 30 minutes; By finished product constant refrigeration in-18 ~-25 DEG C of freezers.
8. the production method of the continuous submerged fermentation liquid of Cordyceps sinensis according to claim 1 and yeast powder, it is characterized in that, described step e is carried out in such a way: after the whole drying of fermentation solid content low-temperature freeze-dry equipment after filtration, 120 object fine powders are worn into again with low temperature milling equipment, obtain Cordyceps sinensis yeast powder, then carry out vacuum packaging preservation.
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