CN102199543A - New Poria cocos strain and liquid fermentation method thereof - Google Patents
New Poria cocos strain and liquid fermentation method thereof Download PDFInfo
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Abstract
The invention provides a new Poria cocos strain and a liquid fermentation method thereof and relates to the field of Poria cocos liquid fermentation. The invention provides the Poria cocos strain conserved in the China center for type culture collection, wherein the CCTCC NO is M 2010361. The invention also provides a Poria cocos liquid fermentation method and the method comprises the following steps: a) preparing a fermentation seed: inoculating the Poria cocos strain of which CCTCC NO is M 2010361 in seed culture medium to obtain the Poria cocos seed; and b) fermenting: inoculating the seed obtained in the step a) in Poria cocos liquid fermentation culture medium to ferment and obtain fermentation liquor, namely the Poria cocos fermentation product. By adopting the fermentation method, the mycelium yield or extracellular polysaccharide yield of Poria cocos liquid fermentation can be increased, the demand of the market on Poria cocos can be met and a foundation is laid for the developments of the health food and medicine which are related to Poria cocos.
Description
Technical field
The present invention relates to medicinal fungi liquid fermenting field, particularly relate to the Poria cocos liquid fermenting.
Background technology
Poria cocos Poria cocos is a kind of perennial saprophytic fungus, belongs to Mycophyta, Basidiomycetes, Holobasidiomycetidae, Aphyllophorales, polyporaceae, Polyporus.The Poria cocos beginning is stated from Shennong's Herbal, is listed in top grade.Just there is the record of using Poria cocos in China as far back as latter stage in spring and autumn, and the history of artificial culture Poria cocos also had more than 1500 year.Traditional Chinese medicine is thought Poria cocos " Li Shui and just not hindering, mend and do not help heresy ", can promoting diuresis to eliminate damp pathogen, spleen-benefiting mind-tranquilizing; Modern medicine studies show that, the pharmacological action of Poria cocos mainly shows aspects such as diuresis, calmness, antitumor, enhancing immunity.Poria cocos is used as medicine with dry sclerotia, and different sites becomes medicinal materials such as white tuckahoe, Poria, fushen, pine among the Tuckahoe and Tuckahoe peel respectively through processing.The Poria cocos property of medicine is gentle, and effectiveness is extensive, is described as " panacea of dehumidifying ", " product on the elixir ", and for the key medicine of the multiple compatibility of medicines in a prescription and the important source material of Chinese patent medicine, in the conventional Chinese medicine prescription, the compatibility rate of Poria cocos reaches 70%.
At present to the existing more research of Poria cocos liquid fermenting.Replace traditional solid fermentation with new technologies such as liquid fermentation and culture and extraction separation and produce Poria cocos, carry out suitability for industrialized production serially, on a large scale, shorten the production time, save a large amount of timber." research of Poria cocos liquid fermentation condition " (Chengdu University of Traditional Chinese Medicine's journal of Li Yi etc., 2005 the 28th the 2nd phases of volume) reported a kind of method of Poria cocos liquid fermenting in, its fermentation strain is a Sichuan institute of agricultural sciences Poria cocos bacterial classification, the suitableeest fermention medium is: glucose 30g, yeast extract 3.9g, peptone 5.1g, K
2HPO
41g, MgSO
4.7H
2O 0.5g, water 1L; The optimum pH 5.5 of Poria cocos liquid fermenting, the cell age of liquid spawn are 2d, liquid-spawn inoculation amount 6%, and shake-flask culture time 7d, mycelium production is up to 11.88g/L.
" Poria cocos shake bottle fed-batch fermentation and fermentor tank feed supplement liquid fermenting " (medicine biotechnology of Li Yi etc., 2007 the 14th the 1st phases of volume) reported a kind of method of Poria cocos liquid fermenting in, the Poria cocos bacterial classification that its fermentation strain provides for the Sichuan institute of agricultural sciences: the mode that adds carbon source by batch feeding can improve the level of Poria cocos liquid fermenting, and mycelium production is up to 11.95g/L.
In sum, the fermentation level of existing Poria cocos liquid fermentation process is still not high, can not satisfy the demand of market to Poria cocos.
Contain materials such as abundant Mierocrystalline cellulose, starch, protein, lipid in the Chinese medicine, and medicinal fungi has the powerful enzyme system of materials such as decomposition of cellulose, starch, protein, lipid, therefore, medicinal fungi can utilize Chinese medicine matrix to carry out submerged fermentation.Simultaneously, some composition in the Chinese medicine (alkaloid, saponin(e, flavones etc.) can promote or Depressant with the production of fungi growth and meta-bolites.
" Chinese medicine is to the influence of medicinal fungi submerged fermentation " (food and fermentation industries of Yang Hailong etc., 2009 35 1 phases of volume) reported the influence of Chinese medicine in to glossy ganoderma, Grifola frondosa, black fungus, Cordyceps sinensis and coprinus comatus submerged fermentation level, different Chinese medicines have not same-action to the fermentation of different fungies, the Chinese medicine that has has promoter action, the Chinese medicine that has is inhibited, and the Chinese medicine that has is with promoter action and restraining effect.
At present also there is not bibliographical information in substratum, to add the method that Chinese medicine improves Poria cocos liquid fermenting level.
Summary of the invention
In order further to improve the Poria cocos fermentation level, the invention provides the method for a kind of new Poria cocos bacterial strain and a kind of new Poria cocos liquid fermenting.
At first, the invention provides a kind of Poria cocos bacterial strain, strain name is Poria cocos (Schw.) wolf.P6, and it is by China's typical culture collection center preservation, and preserving number is CCTCC NO:M2010361.This Poria cocos bacterial strain is preserved in Chinese typical culture collection center on December 21st, 2010, and it abbreviates CCTCC as, and deposit number is CCTCC NO:M 2010361.
The present invention provides a kind of Poria cocos liquid fermentation process again, and it comprises the steps:
A, preparation ferment-seeded: the Poria cocos bacterial strain of getting preserving number and be CCTCC NO:M 2010361 is inoculated in seed culture medium and cultivates, the Poria cocos seed;
B, fermentation: the seed that step a is obtained is inoculated in the Poria cocos liquid fermentation medium and ferments, fermented liquid, be the Poria cocos tunning.
The seed culture medium of step a is formed by following feedstock production: glucose 20g/L, yeast extract 4g/L, peptone 5g/L, K
2HPO
41g/L, MgSO
4.7H
2O 0.5g/L and distilled water; The culture temperature of step a is 25 ℃, and pH is 5.5;
The described Poria cocos liquid fermentation medium of step b is to be formed by following feedstock production: glucose 20g/L, yeast extract 3.5g/L, peptone 4.5g/L, K
2HPO
41g/L, MgSO
47H
2O 0.5g/L, distilled water and particle diameter are less than Chinese medicinal materials pressed powder 2.5g/L～20g/L of 250 μ m, and described Chinese medicinal materials is Semen Coicis, the Radix Astragali, Japanese Honeysuckle, Radix Glycyrrhizae, Herba Lophatheri, mulberry leaf, the red sage root, glossy ganoderma or wolfberry fruit.The amount of above-mentioned Semen Coicis pressed powder is preferably 2.5g/L～10g/L, more preferably 7.5g/L; The amount of the pressed powder of above-mentioned Radix Glycyrrhizae is preferably 2.5g/L～10g/L, more preferably 5g/L; The amount of the pressed powder of above-mentioned Herba Lophatheri is preferably 2.5g/L～15g/L, more preferably 7.5g/L; The amount of the pressed powder of above-mentioned mulberry leaf is preferably 2.5g/L～10g/L, more preferably 5g/L; The amount of the pressed powder of above-mentioned glossy ganoderma is preferably 2.5g/L～15g/L, more preferably 7.5g/L; The amount of the pressed powder of above-mentioned wolfberry fruit is preferably 2.5g/L～10g/L, more preferably 5g/L.
The fermentation process of step b is the liquid fed-batch fermentation, and concrete steps are as follows:
1. the Poria cocos seed is inoculated in the Poria cocos initial medium and ferments, described initial medium is formed by following feedstock production: glucose 100g, yeast extract 8.75g, peptone 11.25g, K
2HPO
45g, MgSO
4.7H
2O 2.5g, initial pH value 5.5, distilled water 4.05L, leavening temperature are 26 ℃, and the pH value is 5.5, and dissolved oxygen level is 60%～90%;
2. when Poria cocos liquid fermenting 48h, 60h, 72h and 84h, respectively 100mL supplemented medium or 200mL supplemented medium are mended in the fermentor tank, described supplemented medium is formed by following feedstock production: glucose 100g, yeast extract 8.75g, peptone 11.25g, distilled water 600mL;
3. ferment to fermentation time be 117～129h, collect fermented liquid, promptly get tunning.
The supplemented medium that 2. step adds when 48h, 60h, 72h and 84h is followed successively by 100mL, 200mL, 200mL and 100mL.
The present invention also provides a kind of Poria cocos tunning according to method for preparing.
Poria cocos bacterial strain provided by the invention has production performance and stability preferably, Poria cocos liquid fermentation process provided by the invention, can improve the level of Poria cocos liquid fermenting, mycelium dry weight, the exocellular polysaccharide content of Poria cocos are significantly improved, satisfy the demand of market, for the exploitation of relevant protective foods of Poria cocos and medicine lays the first stone to Poria cocos.
The embodiment of form is described in further detail foregoing of the present invention by the following examples.But this should be interpreted as that the scope of the above-mentioned theme of the present invention only limits to following embodiment.All technology that realizes based on foregoing of the present invention all belong to scope of the present invention.
Embodiment
1 experiment material
1.1 bacterial classification Poria cocos bacterial strain is bought the institute of agricultural sciences in Sichuan Province, preserving number is P0, and is identical with the Poria cocos bacterial strain that uses in " research of Poria cocos liquid fermentation condition " (Chengdu University of Traditional Chinese Medicine's journal, 2005 the 28th the 2nd phases of volume) of Li Yi etc.
1.2 instrument and reagent Bechtop (safe and sound company of Su Jing group); Full temperature vibrator (east, Harbin connection electronic technology development corporation, Ltd.); NBS Bioflo III type fermentor tank; Whizzer Beckman J2-HS; Electronic balance Sartorius BP121S.
Yeast extract, peptone are the Oxoid product, glucose, sucrose, K
2HPO
4, MgSO
47H
2O, NaNO
3, KCl and FeSO
4All adopt homemade analytical pure.
1.3 Chinese medicinal materials Semen Coicis (Coicis Semen), the Radix Astragali (Astragali Radix), Japanese Honeysuckle (Lonicerae Flos), Radix Glycyrrhizae (Glycyrrhizae Radix), Herba Lophatheri (Lophatheri Folum), mulberry leaf (Mori Folum), the red sage root (Salviae Miltirrrhizae Radix et Rhizoma), glossy ganoderma (Ganoderma) and wolfberry fruit (Lycii Rructus) are all purchased the big pharmacy of Yu Xinglin, pulverized 60 mesh sieves, dry to constant weight for 60 ℃ and be incorporated with in the plug bottle, labelled, it is standby to put into moisture eliminator.
The seed selection of embodiment 1 Poria cocos strain excellent
1, substratum
Perfect medium (TYEG substratum): Tryptones 10g, yeast extract 5g, K
2HPO
43g, glucose 1g, agar 15g, distilled water 1L, pH 7.0.1.05kg/cm
2, 121.3 ℃ of sterilization 20min.
Liquid fermenting seed culture medium: glucose 20g, yeast extract 4g, peptone 5g, K
2HPO
41g, MgSO
4.7H
2O 0.5g, initial pH 5.5, distilled water 1L.1.05kg/cm
2, 121.3 ℃ of sterilization 20min.
Liquid fermentation medium: glucose 20g, yeast extract 3.5g, peptone 4.5g, K
2HPO
41g, MgSO
4.7H
2O 0.5g, initial pH 5.5, distilled water 1L.1.05kg/cm
2, 121.3 ℃ of sterilization 20min.
2, selection by mutation
1) ultraviolet mutagenesis
A, the Poria cocos typical strain that preserving number is P0 is got in producing of spore suspension, places stroke-physiological saline solution, be prepared into spore suspension, pour in the little triangular flask that fills granulated glass sphere, fully shake 10min, make the spore homodisperse, filter, be spore suspension with sterilization absorbent cotton.Get above-mentioned spore suspension counting, adjusting spore suspension concentration is 10
6Individual spore/mL;
B, uviolizing is met hot 20min with the ultraviolet lamp switch opens.6cm aseptic plate 5 covers in cut-off footpath add above-mentioned spore suspension 3mL respectively, and aseptic stirring rod are put in the plate.The plate that fills bacteria suspension is put on the magnetic stirring apparatus, is 30cm in distance, and power is to shine 77s respectively under the ultraviolet lamp of 15W;
C is diluted under the red light, and above-mentioned processing bacteria suspension is diluted to 10 with 10 times of dilution methods
-2, 10
-3, 10
-4Four different extent of dilution;
D under red light, gets 10
-1, 10
-2, 10
-3Three dilution 0.1mL spore suspensions are coated on the concentration gradient perfect medium flat board that contains 2mg/mL Poria cocos exocellular polysaccharide, smoothen with aseptic glass scraper;
E wraps the above-mentioned flat board that smoothens with black cloth, put 28 ℃ of constant temperature culture carton upside downs and cultivate, and obtains Poria cocos list bacterium colony;
2) screening of strain excellent
A, step 1) is obtained Poria cocos list bacterium colony chooses on the perfect medium flat board that contains 4mg/mL Poria cocos exocellular polysaccharide with aseptic toothpick, pick out and grow preferably that bacterium colony is inoculated on the perfect medium flat board that contains 8mg/mL Poria cocos exocellular polysaccharide with aseptic toothpick, pick out and grow preferably that bacterium colony is inoculated on the perfect medium that contains 16mg/mL Poria cocos exocellular polysaccharide with aseptic toothpick, select the bacterial strain preferably of growing;
With preserving number is that typical strain and the above-mentioned bacterial strain that filters out of P0 is inoculated into respectively in the 250mL triangular flask that fills the 50mL liquid seed culture medium, and at 25 ℃, it is standby as liquid spawn that 150r/min shakes bottle shaking culture 5d; Draw the seed liquor 3.5mL that has prepared respectively, be inoculated in (ratio inoculation) in the 250mL triangular flask that fills 50mL liquid shaking bottle fermention medium in 7%, 26 ℃ of culture temperature, initial pH value of medium 5.5 is shaken and is cultivated 7d under bottle culture condition of rotating speed 150r/min.Get fermented liquid 50mL,, collect sedimentary mycelium pellet, dry to constant weight, accurately weigh for 60 ℃ with electronic balance through the centrifugal 15min of 6000r/min.
Through shake-flask culture, preserving number is that the mycelium dry weight of the typical strain of P0 is 11.08g/L, and in the bacterial strain that screening obtains, the mycelium dry weight that has a strain Poria cocos strain fermentation to obtain is 11.92g/L, has improved 7.58% than starting strain.Therefore, be strain excellent with this bacterial strain, be preserved in Chinese typical culture collection center, preserving number is CCTCC NO:M 2010361.
3), Detection of Stability
Picking step 2) screening the preserving number that obtains is the strain excellent of CCTCC NO:M 2010361, is inoculated in the 250mL triangular flask that fills the 50mL liquid seed culture medium.At 25 ℃, it is standby as liquid spawn that 150r/min shakes bottle shaking culture 5d, is called first-generation seed liquor.
Draw 250 μ L first-generation seed liquor with aseptic pipettor, be inoculated into (ratio inoculation) in the 250mL triangular flask that fills the 50mL liquid seed culture medium in 0.5%.25 ℃ of culture temperature, 150r/min shakes the seed liquor that bottle shaking culture 2d obtains s-generation strain excellent.S-generation seed is inoculated in the liquid seed culture medium, 25 ℃ of culture temperature, 150r/min shakes a bottle shaking culture 2d and obtains third generation strain excellent seed liquor again.The rest may be inferred, can obtain the tenth generation the strain excellent seed liquor.
Draw the seed liquor 3.5mL in ten generations of the strain excellent first-generation to the prepared respectively, be inoculated in (ratio by 7% is inoculated) in the 250mL triangular flask that fills the 50mL liquid fermentation medium.26 ℃ of culture temperature, initial pH value of medium 5.5 is shaken under bottle culture condition of rotating speed 150r/min, shakes a bottle shaking culture 7d.Get fermented liquid 50mL,, collect sedimentary mycelium pellet, dry to constant weight, accurately weigh for 60 ℃ with electronic balance through the centrifugal 15min of 6000r/min.
The result is as shown in table 1:
The stability of table 1 Poria cocos strain excellent
As known from Table 1, preserving number is that the mycelia dry weight in ten generations of the strain excellent first-generation to the of CCTCC NO:M 2010361 fluctuates between 11.70～11.97g/L, and fluctuating range is very little, shows that it has good genetic stability.
Present embodiment has proved that the preserving number that ultraviolet mutagenesis breeding of the present invention obtains is that CCTCC NO:M2010361 Poria cocos bacterial strain not only has good tolerance to polysaccharide, can produce more polysaccharide, and mycelial output also has raising.
Embodiment 2 Chinese medicines shake the influence of bottle liquid fermenting to Poria cocos
1, substratum
Seed culture medium: glucose 20g, yeast extract 4g, peptone 5g, K
2HPO
41g, MgSO
47H
2O 0.5g, distilled water 1L, initial pH 5.5.
Fermentation basic medium: glucose 20g, yeast extract 3.5g, peptone 4.5g, K
2HPO
41g, MgSO
47H
2O 0.5g, distilled water 1L, initial pH 5.5.
2, fermentation process
1), Poria cocos shakes bottle preparation of liquid fermenting bacterial classification
With the 50mL seed culture medium substratum 250mL triangular flask of packing into, the preserving number that the ratio inoculation embodiment 1 in 0.5% obtains is the Poria cocos bacterial strain of CCTCC NO:M 2010361, and 25 ℃ to shake bottle shaking culture 2d standby as the liquid fermenting bacterial classification.
2), inoculation culture
In shaking bottle liquid fermenting basic medium, 9 flavor Chinese medicine pressed powders (particle diameter of pressed powder is less than 250 μ m) such as Semen Coicis are added respectively and shake in bottle liquid fermenting basic medium, contrast and do not add Chinese medicine, in 1.05kg/cm with the concentration of 10g/L
2, 121.3 ℃ of sterilization 20min.Shake a bottle liquid fermentation and culture condition: the Poria cocos liquid spawn is inserted in 6% ratio in the cooling back, and 26 ℃ of culture temperature are shaken bottled amount 60mL (250mL triangular flask), and 150r/min shakes a bottle shaking culture 7d, promptly gets fermented liquid.
3), mycelium is weighed
Get fermented liquid in the centrifugal 15min of 6000r/min, collect sedimentary mycelium pellet and dry to constant weight for 60 ℃, weigh with precision balance.
4), the purifying of exocellular polysaccharide and weighing
Get the centrifugal 15min of fermented liquid 6000r/min, collect supernatant liquor; Remove albumen with the Sevag method after the supernatant concentration; The centrifugal 20min of 6000r/min, supernatant liquor adds 95% ethanol of 3 times of amounts, refrigeration 12h.The centrifugal 20min of 6000r/min, collecting precipitation, cryodrying gets the Poria cocos crude extracellular polysaccharide; Successively with dehydrated alcohol, acetone, ether washing, cryodrying gets the Poria cocos exocellular polysaccharide, accurately weighs with electronic balance with the outer polysaccharide just of Poria cocos born of the same parents.
3, result
Different Chinese medicines are as shown in table 2 to the influence that Poria cocos shakes bottle liquid fermenting mycelium production and exopolysaccharides:
The different Chinese medicines of table 2 are to the influence of Poria cocos submerged fermentation
| Sample | Contrast | Semen Coicis | The Radix Astragali | Japanese Honeysuckle | Wolfberry fruit |
| Dry weight (g/L) | 11.86 | 13.92 | 9.04 | 9.93 | 13.17 |
| Exocellular polysaccharide (g/L) | 5.34 | 8.41 | 6.14 | 6.23 | 9.72 |
| Sample | Glossy ganoderma | Mulberry leaf | Herba Lophatheri | Radix Glycyrrhizae | The red sage root |
| Dry weight (g/L) | 13.36 | 12.63 | 12.47 | 12.29 | 6.74 |
| Exocellular polysaccharide (g/L) | 8.26 | 7.35 | 7.12 | 7.82 | 5.93 |
Wherein contrast is the fermentation basic medium that does not add Chinese medicine, and other conditions are identical.Aspect Poria mycelium output, the Radix Astragali, Japanese Honeysuckle and the red sage root have the obvious suppression effect to the mycelial growth of Poria cocos, and remaining 6 flavor Chinese medicine has obvious facilitation to the mycelial growth of Poria cocos, and wherein remarkable with Semen Coicis, biomass has increased by 17.37%.Aspect the Poria cocos exopolysaccharides, 9 flavor Chinese medicines all help the raising of exopolysaccharides, and comparison is according to improving 11.05%～82.02% respectively.
Present embodiment has proved its their extract of Semen Coicis, the Radix Astragali, Japanese Honeysuckle, Radix Glycyrrhizae, Herba Lophatheri, mulberry leaf, the red sage root, glossy ganoderma or wolfberry fruit Poria cocos fermentation is all had promoter action, particularly Semen Coicis, Radix Glycyrrhizae, Herba Lophatheri, mulberry leaf, glossy ganoderma or wolfberry fruit and their extract fermentation has good promoter action to Poria cocos.
Dose shakes the influence of bottle liquid fermenting in embodiment 3 differences to Poria cocos
1, substratum
Seed culture medium: glucose 20g, yeast extract 4g, peptone 5g, K
2HPO
41g, MgSO
47H
2O 0.5g, distilled water 1L, initial pH 5.5.
Fermentation basic medium: glucose 20g, yeast extract 3.5g, peptone 4.5g, K
2HPO
41g, MgSO
47H
2O 0.5g, distilled water 1L, initial pH 5.5.
2, fermentation process
1), Poria cocos shakes bottle preparation of liquid fermenting bacterial classification
With the 50mL seed culture medium 250mL triangular flask of packing into, the preserving number that the ratio inoculation embodiment 1 in 0.5% obtains is the Poria cocos bacterial strain of CCTCC NO:M 2010361, and 25 ℃ to shake bottle shaking culture 2d standby as the liquid fermenting bacterial classification.
2), inoculation culture
In shaking bottle liquid fermenting basic medium, all Semen Coicis, Radix Glycyrrhizae, Herba Lophatheri, mulberry leaf, glossy ganoderma and wolfberry fruit pressed powder (particle diameter of pressed powder is less than 250 μ m) are added respectively and shake in bottle liquid fermenting basic medium with the concentration of 2.5g/L, 5g, 7.5g/L, 10g/L, 15g/L, 20g/L, contrast does not add Chinese medicine, in 1.05kg/cm
2, 121.3 ℃ of sterilization 20min.Shake a bottle liquid fermentation and culture condition: the Poria cocos liquid spawn is inserted in 6% ratio in the cooling back, and 26 ℃ of culture temperature are shaken bottled amount 60mL (250mL triangular flask), and 150r/min shakes a bottle shaking culture 7d, promptly gets fermented liquid.
3), mycelium is weighed
Get fermented liquid in the centrifugal 15min of 6000r/min, collect sedimentary mycelium pellet and dry to constant weight for 60 ℃, weigh with precision balance.
4), the purifying of exocellular polysaccharide and weighing
Get the centrifugal 15min of fermented liquid 6000r/min, collect supernatant liquor; Remove albumen with the Sevag method after the supernatant concentration; The centrifugal 20min of 6000r/min, supernatant liquor adds 95% ethanol of 3 times of amounts, refrigeration 12h.The centrifugal 20min of 6000r/min, collecting precipitation, cryodrying gets the Poria cocos crude extracellular polysaccharide; Successively with dehydrated alcohol, acetone, ether washing, cryodrying gets the Poria cocos exocellular polysaccharide, accurately weighs with electronic balance with the outer polysaccharide just of Poria cocos born of the same parents.
3, result
Poria mycelium output and exopolysaccharides result are as follows:
(1) that Poria cocos is shaken bottle liquid fermenting result is as shown in table 3 for different Semen Coicis additions, and mycelium dry weight is up to 14.41g/L when the Semen Coicis addition is 7.5g/L, and exopolysaccharides is up to 8.79g/L when the Semen Coicis addition is 15g/L.
Table 3 different concns Semen Coicis is to the influence of Poria cocos submerged fermentation
| Add concentration | Contrast | 2.5g/L | 5g/L | 7.5g/L | 10g/L | 15g/L | 20g/L |
| Dry weight (g/L) | 11.86 | 12.24 | 13.15 | 14.41 | 13.83 | 11.42 | 10.73 |
| Exocellular polysaccharide (g/L) | 5.34 | 5.53 | 6.12 | 7.23 | 8.37 | 8.79 | 8.46 |
(2) that Poria cocos is shaken bottle liquid fermenting result is as shown in table 4 for different Radix Glycyrrhizae additions, and mycelium dry weight is up to 12.66g/L when the Radix Glycyrrhizae addition is 5g/L, and exopolysaccharides is up to 7.71g/L when the Radix Glycyrrhizae addition is 10g/L.
Table 4 different concns Radix Glycyrrhizae is to the influence of Poria cocos submerged fermentation
| Add concentration | Contrast | 2.5g/L | 5g/L | 7.5g/L | 10g/L | 15g/L | 20g/L |
| Dry weight (g/L) | 11.86 | 12.03 | 12.66 | 12.51 | 12.23 | 10.81 | 8.34 |
| Exocellular polysaccharide (g/L) | 5.34 | 5.23 | 5.77 | 7.06 | 7.71 | 7.34 | 6.77 |
(3) that Poria cocos is shaken bottle liquid fermenting result is as shown in table 5 for different Herba Lophatheri additions, and mycelium dry weight is up to 13.55g/L when the Herba Lophatheri addition is 7.5g/L, and exopolysaccharides is up to 7.48g/L when the Herba Lophatheri addition is 15g/L.
Table 5 different concns Herba Lophatheri is to the influence of Poria cocos submerged fermentation
| Add concentration | Contrast | 2.5g/L | 5g/L | 7.5g/L | 10g/L | 15g/L | 20g/L |
| Dry weight (g/L) | 11.86 | 11.98 | 12.86 | 13.55 | 12.42 | 12.05 | 11.42 |
| Exocellular polysaccharide (g/L) | 5.34 | 5.24 | 5.62 | 6.97 | 7.21 | 7.48 | 6.97 |
(4) that Poria cocos is shaken bottle liquid fermenting result is as shown in table 6 for different mulberry leaf additions, and mycelium dry weight is up to 12.91g/L when the mulberry leaf addition is 7.5g/L, and exopolysaccharides is up to 7.81g/L when the mulberry leaf addition is 20g/L.
Table 6 different concns mulberry leaf are to the influence of Poria cocos submerged fermentation
| Add concentration | Contrast | 2.5g/L | 5g/L | 7.5g/L | 10g/L | 15g/L | 20g/L |
| Dry weight (g/L) | 11.86 | 12.22 | 12.91 | 12.72 | 12.51 | 11.02 | 9.36 |
| Exocellular polysaccharide (g/L) | 5.34 | 5.41 | 6.12 | 6.97 | 7.41 | 7.72 | 7.81 |
(5) that Poria cocos is shaken bottle liquid fermenting result is as shown in table 7 for different glossy ganoderma additions, and mycelium dry weight is up to 13.71g/L when the glossy ganoderma addition is 7.5g/L, and exopolysaccharides is up to 9.06g/L when the glossy ganoderma addition is 20g/L.
Table 7 different concns glossy ganoderma is to the influence of Poria cocos submerged fermentation
| Add concentration | Contrast | 2.5g/L | 5g/L | 7.5g/L | 10g/L | 15g/L | 20g/L |
| Dry weight (g/L) | 11.86 | 11.94 | 12.54 | 13.71 | 13.39 | 12.43 | 10.71 |
| Exocellular polysaccharide (g/L) | 5.34 | 5.51 | 6.43 | 7.39 | 8.43 | 8.87 | 9.06 |
(6) that Poria cocos is shaken bottle liquid fermenting result is as shown in table 8 for different wolfberry fruit dosages, and mycelium dry weight is up to 13.86g/L when the wolfberry fruit addition is 5g/L, and exopolysaccharides is up to 11.23g/L when wolfberry fruit addition 20g/L.
Table 8 different concns wolfberry fruit is to the influence of Poria cocos submerged fermentation
| Add concentration | Contrast | 2.5g/L | 5g/L | 7.5g/L | 10g/L | 15g/L | 20g/L |
| Dry weight (g/L) | 11.86 | 12.64 | 13.86 | 13.52 | 13.22 | 11.41 | 10.19 |
| Exocellular polysaccharide (g/L) | 5.34 | 5.91 | 7.12 | 8.33 | 9.77 | 10.51 | 11.23 |
The difference of 6 flavor Chinese medicine pressed powder additions such as Semen Coicis has bigger difference to the influence that Poria cocos shakes bottle liquid fermenting, the Chinese medicine addition is too high or too low all can to influence the growth of Poria cocos and the secretion of exocellular polysaccharide, and common every flavor Chinese medicine mycelium dry weight reaches the most moderate medicine addition when the highest and is different from exopolysaccharides and reaches the most moderate medicine addition when the highest.6 flavor Chinese medicines have bigger difference to the mycelium production and the exopolysaccharides of Poria cocos liquid fermenting, studies show that, the Poria mycelium dry weight is up to 14.41g/L when the Semen Coicis addition is 7.5g/L, and the Poria cocos exopolysaccharides is up to 11.23g/L when the wolfberry fruit addition is 20g/L.
Present embodiment has proved:
The Semen Coicis pressed powder that adds 2.5g/L～20g/L in the substratum can promote the Poria cocos liquid fermenting, fermentation has promoter action preferably to the Semen Coicis pressed powder of preferred 2.5g/L～10g/L to Poria cocos, and the mycelium production of the Poria cocos tunning that obtains when adding the Semen Coicis pressed powder of most preferred 7.5g/L in the substratum is the highest;
The Radix Glycyrrhizae pressed powder that adds 2.5g/L～20g/L in the substratum can promote the Poria cocos liquid fermenting, fermentation has better promoter action to the Radix Glycyrrhizae pressed powder of preferred 2.5g/L～10g/L to Poria cocos, and the mycelium production of the Poria cocos tunning that obtains when adding the Radix Glycyrrhizae pressed powder of most preferred 5g/L in the substratum is the highest;
The Herba Lophatheri pressed powder that adds 2.5g/L～20g/L in the substratum can promote the Poria cocos liquid fermenting, fermentation has promoter action preferably to the Herba Lophatheri pressed powder of preferred 2.5g/L～15g/L to Poria cocos, and the mycelium production of the Poria cocos tunning that obtains when adding the Herba Lophatheri pressed powder of most preferred 7.5g/L in the substratum is the highest;
The mulberry leaf pressed powder that adds 2.5g/L～20g/L in the substratum can promote the Poria cocos liquid fermenting, fermentation has promoter action preferably to the mulberry leaf pressed powder of preferred 2.5g/L～10g/L to Poria cocos, and the mycelium production of the Poria cocos tunning that obtains when adding the mulberry leaf pressed powder of most preferred 5g/L in the substratum is the highest;
The glossy ganoderma pressed powder that adds 2.5g/L～20g/L in the substratum can promote the Poria cocos liquid fermenting, fermentation has promoter action preferably to the glossy ganoderma pressed powder of preferred 2.5g/L～15g/L to Poria cocos, and the mycelium production of the Poria cocos tunning that obtains when adding the glossy ganoderma pressed powder of most preferred 7.5g/L in the substratum is the highest;
The wolfberry fruit pressed powder that adds 2.5g/L～20g/L in the substratum can promote the Poria cocos liquid fermenting, fermentation has promoter action preferably to the pressed powder of the wolfberry fruit of preferred 2.5g/L～10g/L to Poria cocos, and the mycelium production of the Poria cocos tunning that obtains when adding the wolfberry fruit pressed powder of most preferred 5g/L in the substratum is the highest.
Embodiment 4 liquid fed-batch fermentations
1, substratum
Seed culture medium: glucose 20g, yeast extract 4g, peptone 5g, K
2HPO
41g, MgSO
4.7H
2O0.5g, initial pH value 5.5, distilled water 1L.1.05Kg/cm
2, 121.3 ℃ of sterilization 20min.
Fermentor tank initial medium: glucose 100g, yeast extract 8.75g, peptone 11.25g, K
2HP
045g, MgSO
4.7H
2O 2.5g, initial pH value 5.5, distilled water 4.05L.1.05kg/cm
2, 121.3 ℃ of sterilization 20min.
Fermentor tank supplemented medium: glucose 100g, yeast extract 8.75g, peptone 11.25g, distilled water 600mL.0.56kg/cm
2, 112.6 ℃ of sterilization 30min.
2, fermentation process
1), Poria cocos shakes bottle preparation of liquid fermenting bacterial classification
With the 50mL seed culture medium 350mL triangular flask of packing into, the preserving number that the ratio inoculation embodiment 1 in 0.5% obtains is the Poria cocos bacterial strain of CCTCC NO:M 2010361, and 25 ℃ to shake bottle shaking culture 2d standby as the liquid fermenting bacterial classification.
2), inoculation culture
Under the flame protection; 350mL fermentor tank seed liquor is inoculated in the fermentor tank that 4.05L fermentor tank initial medium is housed ferments; when Poria cocos liquid fermenting 48h, 60h, 72h and 84h, divide by peristaltic pump to add the 100mL supplemented medium for four times or the 200mL supplemented medium is mended in the fermentor tank.Fermentation condition: leavening temperature is set at 26 ℃, and the pH value is set at 5.5, and mixing speed and air flow are adjusted according to the height of dissolved oxygen level, and dissolved oxygen level is controlled at 60%～90%.Simultaneously, investigated the best fermentation termination of Poria cocos fermentor tank liquid fermenting under the different feed supplement conditions respectively, definite step of fermentation termination: 1. behind Poria cocos liquid fermenting 110h, every 1h sampling 20mL; 2. the biomass of Poria mycelium and exocellular polysaccharide content in the working sample; 3. according to the biomass of the Poria mycelium that records and the size that exocellular polysaccharide contains numerical quantity, and combining with fermentation time and fermentation costs, determine fermentation termination.Stop fermentation during to fermentation termination, the fermented liquid that obtains is tunning.
3), mycelium is weighed
Get fermented liquid in the centrifugal 15min of 6000r/min, collect sedimentary mycelium pellet and dry to constant weight for 60 ℃, weigh with precision balance.
4), the purifying of exocellular polysaccharide and weighing
Get the centrifugal 15min of fermented liquid 6000r/min, collect supernatant liquor; Remove albumen with the Sevag method after the supernatant concentration; The centrifugal 20min of 6000r/min, supernatant liquor adds 95% ethanol of 3 times of amounts, refrigeration 12h.The centrifugal 20min of 6000r/min, collecting precipitation, cryodrying gets the Poria cocos crude extracellular polysaccharide; Successively with dehydrated alcohol, acetone, ether washing, cryodrying gets the Poria cocos exocellular polysaccharide, accurately weighs with electronic balance with the outer polysaccharide just of Poria cocos born of the same parents.
3, result
Different time and the influence of different feed supplement modes to Poria cocos fermentor tank liquid fermenting the results are shown in Table 9:
Table 9 different time and of the influence of different feed supplement modes to Poria cocos fermentor tank liquid fermenting
Different time and the influence of different glucose concn feed supplements to fermentation termination, experimental result sees Table 10:
Table 10 different time and of the influence of different feed supplement modes to Poria cocos fermentor tank liquid fermenting terminal point
Take all factors into consideration different time and of the influence of different feed supplement modes to Poria cocos fermentor tank liquid fermenting mycelium production, exopolysaccharides and fermentation termination, best zymotechnique is: when fermentation culture 48h, 60h, 72h and 84h, add the supplemented medium of 100mL, 200mL, 200mL and 100mL respectively.Under best zymotechnique, mycelium production reaches 12.56g/L, and exopolysaccharides reaches 5.51g/L, and fermentation time is 117h.
Fermentation has promoter action to the mode that present embodiment has proved the feed supplement liquid fermenting to Poria cocos, particularly when fermentation culture 48h, 60h, 72h and 84h, the supplemented medium that adds 100mL, 200mL, 200mL and 100mL respectively is particularly remarkable to the promoter action of the output of Poria mycelium output and exocellular polysaccharide.
Embodiment 5 Semen Coiciss are to the influence of Poria cocos fermentor tank liquid fed-batch fermentation
1, substratum
Seed culture medium: glucose 20g, yeast extract 4g, peptone 5g, K
2HPO
41g, MgSO
4.7H
2O 0.5g, initial pH value 5.5, distilled water 1L.1.05Kg/cm
2, 121.3 ℃ of sterilization 20min.
Ferment tank substratum: glucose 100g, yeast extract 8.75g, peptone 11.25g, K
2HPO
45g, MgSO
4.7H
2O 2.5g, initial pH value 5.5, distilled water 4.05L.1.05Kg/cm
2, 121.3 ℃ of sterilization 20min.
Fermentor tank supplemented medium: glucose 100g, yeast extract 8.75g, peptone 11.25g, distilled water 600mL.0.56Kg/cm
2, 112.6 ℃ of sterilization 30min.
2, fermentation step
1), Poria cocos shakes bottle preparation of liquid fermenting bacterial classification
The 350mL liquid seed culture medium of packing in the 1L triangular flask is inoculated freezing glycerol stock in 0.5% ratio, 25 ℃ of culture temperature, and it is standby as liquid spawn that 150r/min shakes bottle shaking culture 2d.
2), inoculation culture
Concentration with 2.5g/L in 4.05L fermentor tank initial medium adds Semen Coicis, and contrast does not add Chinese medicine, 1.05kg/cm
2, 121.3 ℃ of sterilization 20min.
Under the flame protection; 350mL fermentor tank seed liquor is inoculated in the fermentor tank that above-mentioned substratum is housed ferments; when Poria cocos liquid fermenting 48h, 60h, 72h and 84h, successively the supplemented medium of 100mL, 200mL, 200mL and 100mL is mended in the fermentor tank by the peristaltic pump branch.Fermentation condition: leavening temperature is set at 26 ℃, and the pH value is set at 5.5, and mixing speed and air flow are adjusted according to the height of dissolved oxygen level, and dissolved oxygen level is controlled at 60%～90%, and fermentation culture 117h promptly gets fermented liquid.
3), mycelium is weighed
Get fermented liquid in the centrifugal 15min of 6000r/min, collect sedimentary mycelium pellet and dry to constant weight for 60 ℃, weigh with precision balance.
4), the purifying of liquid fermenting Poria cocos exocellular polysaccharide and weighing
The centrifugal 15min of fermented liquid 6000r/min collects supernatant liquor.Remove albumen with the Sevag method after the supernatant concentration.The centrifugal 20min of 6000r/min, supernatant liquor adds 95% ethanol of 3 times of amounts, refrigeration 12h.The centrifugal 20min of 6000r/min, collecting precipitation, cryodrying gets the Poria cocos crude extracellular polysaccharide.Successively with dehydrated alcohol, acetone, ether washing, cryodrying gets the Poria cocos exocellular polysaccharide with the outer polysaccharide just of Poria cocos born of the same parents.With the accurate weighing of electronic balance.
3, result
Poria cocos fermentor tank liquid fermenting mycelium production and exopolysaccharides result are as shown in table 11
Table 11 Semen Coicis is to the influence of Poria cocos submerged fermentation
| Sample | Contrast | Semen Coicis |
| Dry weight (g/L) | 12.56 | 14.92 |
| Exocellular polysaccharide (g/L) | 5.51 | 7.76 |
Concentration is that the Semen Coicis pressed powder of 7.5g/L has promoter action to Poria cocos fermentor tank feed supplement liquid fermenting, and mycelium production improves 24.85%, and exopolysaccharides improves 44.17%.
The improvement that present embodiment has proved substratum has obvious facilitation in conjunction with the method for liquid fed-batch fermentation to the output and the exocellular polysaccharide content of Poria mycelium.
To sum up, Poria cocos bacterial strain provided by the invention has production performance and stability preferably, liquid fermentation process and the liquid fed-batch fermentation method that in substratum, adds the Chinese medicine pressed powder provided by the invention, can improve the level of Poria cocos liquid fermenting, and significantly improve the mycelium dry weight and the exopolysaccharides of Poria cocos, satisfy the demand of market, for the exploitation of relevant protective foods of Poria cocos and medicine lays the first stone to Poria cocos.
Claims (10)
1. a Poria cocos bacterial strain (Poria cocos), it is characterized in that: it is the bacterial strain by China's typical culture collection center preservation, and preserving number is CCTCC NO:M 2010361.
2. the method for a Poria cocos liquid fermenting, it is characterized in that: it comprises the steps:
A, preparation ferment-seeded: the Poria cocos bacterial strain of getting preserving number and be CCTCC NO:M 2010361 is inoculated in seed culture medium and cultivates, the Poria cocos seed;
B, fermentation: the seed that step a is obtained is inoculated in the Poria cocos liquid fermentation medium and ferments, fermented liquid, be the Poria cocos tunning.
3. method according to claim 2 is characterized in that: the seed culture medium of step a is formed by following feedstock production: glucose 20g/L, yeast extract 4g/L, peptone 5g/L, K
2HPO
41g/L, MgSO
4.7H
2O 0.5g/L and distilled water.
4. method according to claim 2 is characterized in that: the culture temperature of step a is 25 ℃, and pH is 5.5.
5. method according to claim 2 is characterized in that: the described Poria cocos liquid fermentation medium of step b is formed by following feedstock production: glucose 20g/L, yeast extract 3.5g/L, peptone 4.5g/L, K
2HPO
41g/L, MgSO
47H
2O 0.5g/L, distilled water and particle diameter are less than Chinese medicinal materials pressed powder 2.5g/L～20g/L of 250 μ m, and described Chinese medicinal materials is Semen Coicis, the Radix Astragali, Japanese Honeysuckle, Radix Glycyrrhizae, Herba Lophatheri, mulberry leaf, the red sage root, glossy ganoderma or wolfberry fruit.
6. method according to claim 5 is characterized in that: described Chinese medicinal materials is:
Semen Coicis, its concentration are 2.5g/L～10g/L;
Perhaps Radix Glycyrrhizae, its concentration is 2.5g/L～10g/L;
Perhaps Herba Lophatheri, its concentration is 2.5g/L～15g/L;
Perhaps mulberry leaf, its concentration is 2.5g/L～10g/L;
Perhaps glossy ganoderma, its concentration is 2.5g/L～15g/L;
Perhaps wolfberry fruit, its concentration 2.5g/L～10g/L.
7. method according to claim 6 is characterized in that: described Chinese medicinal materials is:
Semen Coicis, its concentration are 7.5g/L;
Perhaps Radix Glycyrrhizae, its concentration is 5g/L;
Perhaps Herba Lophatheri, its concentration is 7.5g/L;
Perhaps mulberry leaf, its concentration is 5g/L;
Perhaps glossy ganoderma, its concentration is 7.5g/L;
Perhaps wolfberry fruit, its concentration is 5g/L.
8. method according to claim 2 is characterized in that: the fermentation process of described step b is the liquid fed-batch fermentation, and concrete steps are as follows:
1. the Poria cocos seed is inoculated in the Poria cocos initial medium and ferments, described initial medium is formed by following feedstock production: glucose 100g, yeast extract 8.75g, peptone 11.25g, K
2HPO
45g, MgSO
4.7H
2O 2.5g, initial pH value 5.5, distilled water 4.05L, leavening temperature are 26 ℃, and the pH value is 5.5, and dissolved oxygen level is 60%～90%;
2. when Poria cocos liquid fermenting 48h, 60h, 72h and 84h, respectively 100mL supplemented medium or 200mL supplemented medium are mended in the fermentor tank, described supplemented medium is formed by following feedstock production: glucose 100g, yeast extract 8.75g, peptone 11.25g, distilled water 600mL;
3. ferment to fermentation time be 117～129h, collect fermented liquid, promptly get tunning.
9. method according to claim 8 is characterized in that, the supplemented medium that 2. described step adds when 48h, 60h, 72h and 84h is followed successively by 100mL, 200mL, 200mL and 100mL.
10. the Poria cocos tunning for preparing of any described method of claim 2～9.
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