CN109112175A - A method of building microbial co culture system high yield Pleurotus tuber-regium exocellular polysaccharide - Google Patents
A method of building microbial co culture system high yield Pleurotus tuber-regium exocellular polysaccharide Download PDFInfo
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Abstract
The invention discloses a kind of methods for constructing microbial co culture system high yield Pleurotus tuber-regium exocellular polysaccharide, belong to technical field of bioengineering.The present invention is co-cultured by using Pleurotus tuber-regium mycelium and lactic acid bacteria, and the inoculum concentration of lactic acid bacteria and co-cultures the time when optimizing co-cultivation, improves polysaccharide yield, and improve its dissolubility.Strain used in the present invention is edible bacterial strain, and securely and reliably, used medium raw material is cheap and easy to get, pollution-free, it is easy to accomplish.The method of the present invention process simplicity, strong operability are, it can be achieved that scale, industrialized production.
Description
Technical field
The present invention relates to a kind of methods for constructing microbial co culture system high yield Pleurotus tuber-regium exocellular polysaccharide, belong to biological work
Journey technical field.
Background technique
Pleurotus tuber-regium (Pleurotus tuber-regium), scientific name sclerotium oyster mushroom also known as pleurotus tuberregium (Franch.) Singer, core ear mushroom, Poria cocos side
Ear, Nan Yang Poria cocos etc. belong to Basidiomycotina, and Hymenomycetes, Agaricales, Pleurotaceae, Pleurotus is that one kind is distributed mainly on the torrid zone
And the rare edible and medicinal fungi of subtropical zone.Pleurotus tuber-regium not only has high nutritive value, is also rich in multiple biological activities
Ingredient, polysaccharide are concerned in recent years as the wherein highest ingredient of content.Studies have shown that Pleurotus tuber-regium polysaccharide can significantly improve
Body's immunity has good application prospect in fields such as medicine, health care product, food, pharmaceutical carriers.Pleurotus tuber-regium mycelium warp
Liquid state fermentation can generate exocellular polysaccharide, though the polysaccharide basic structure and sclerotium and fruitbody polysaccharide are variant, all have height
The form of branching, therefore there is huge potential research and application value.The side of production Pleurotus tuber-regium mycelium cell exo polysaccharides at present
Method is only by the liquid state fermentation of Pleurotus tuber-regium mycelium mostly, but that there are yields is lower, dissolubility is poor for obtained exocellular polysaccharide
The defects of.These problems seriously limit its further fundamental and applied research.
Lactic acid bacteria is the common name of a kind of bacterium that a large amount of lactic acid can be generated using fermentable carbohydrate, is widely present in
In the enteron aisle of human body, the facilitation of adjustment effect and human health to human body intestinal canal Tiny ecosystem is of increasing concern,
As research hotspot in recent years.There is complicated interaction between different microorganisms, be often applied to food and agricultural
In production, corresponding co-culture system is also often constructed to volume increase or unknown metabolite for known bioactive substance
Increase.But do not find the application example that polysaccharide yield is improved using lactic acid bacteria and macro fungi mixed fermentation also at present.
Summary of the invention
It is to utilize lactic acid bacteria and tiger the first purpose of the invention is to provide a kind of method of high yield Pleurotus tuber-regium exocellular polysaccharide
Two kinds of microorganisms of milk mycelium construct co-culture system.Specifically includes the following steps:
(1) Pleurotus tuber-regium mycelium is added to preculture in seed culture fluid；Lactic acid bacteria is added in fluid nutrient medium
Carry out preculture；
(2) culture solution obtained after Pleurotus tuber-regium mycelium preculture is linked into liquid state fermentation 7d in fermentation medium；
(3) when the fermentation of step (2) is carried out to 3-6d, the culture solution obtained after lactic acid bacteria preculture is inoculated into tiger
In milk mycelium fermentation liquid, co-cultured；
(4) it is centrifuged, filters after fermentation, gained filtrate is fermentation liquid, and ethyl alcohol is added into fermentation liquid, is stirred, quiet
Collection precipitating, dissolution precipitating are postponed, freeze-drying obtains Pleurotus tuber-regium exocellular polysaccharide.
In one embodiment of the invention, the mycelial preculture of Pleurotus tuber-regium described in step (1) is by brave milk
Mushroom silk is added in seed culture fluid, the preculture 2-4d under the conditions of 160-200rpm, 26-32 DEG C.
In one embodiment of the invention, the preculture of lactic acid bacteria described in step (1) is that lactic acid bacteria is added
Into MRS culture medium, the preculture 18-36h under the conditions of 100-120rpm, 30-40 DEG C.
In one embodiment of the invention, liquid state fermentation described in step (2) refers to Pleurotus tuber-regium Mycelium culture
The inoculum concentration of liquid is 5-10%, accesses fermentation medium, ferment 7d at 150-200rpm, 25-32 DEG C.
In one embodiment of the invention, in step (1) in the mycelial seed culture fluid of Pleurotus tuber-regium and step (2)
Fermentative medium formula are as follows: glucose 20-40g/L, yeast extract 3-5g/L, potassium dihydrogen phosphate (KH2PO4) 0.5-1.5g/L, seven
Water magnesium sulfate (MgSO4·7H2O)0.4-1.0g/L。
In one embodiment of the invention, co-cultivation described in step (3) refers to when fermentation is to 3-6d, will
Lactobacillus inoculum is into Pleurotus tuber-regium mycelium fermentation broth, inoculum concentration 2.5-10%, under the conditions of 150-200rpm, 26-35 DEG C
Co-culture 1-4d.
In one embodiment of the invention, centrifugation refers to that 3000-5000g is centrifuged 5-10min in step (4).
In one embodiment of the invention, described in step (4) obtain exocellular polysaccharide method are as follows: to centrifugation,
95% ethyl alcohol of 3-5 times of volume of fermentation liquid is added in filtered ferment filtrate, stirring stands 5-8h, by sediment with water-soluble
It is freeze-dried after solution.
The present invention be directed to it is existing produce Pleurotus tuber-regium exocellular polysaccharide method low output, dissolubility is poor the problems such as, using tiger
The mode that milk mycelium and lactic acid bacteria co-culture improves polysaccharide yield, and improves its dissolubility.By adjusting cream when co-culturing
The inoculum concentration of sour bacterium and co-cultivation time, the yield of gained Pleurotus tuber-regium exocellular polysaccharide can achieve 522.60mg/L, compared with control
Group 1 (liquid state fermentation of Pleurotus tuber-regium mycelium is used only) output increased is to 3.7 times.Two kinds of microbial co cultures are able to achieve mutually beneficial total
Raw, gained yield of extracellular polysaccharide significantly improves.Method according to the present invention, process simplicity, strong operability are, it can be achieved that scale
Change, industrialized production.
Specific embodiment
(1) method of polyoses content is surveyed:
Measurement of the polysaccharide content uses phend-sulphuric acid.It is molten that the sulfuric acid that 1mL concentration is 12M is dispersed by 5mg polysaccharide sample
In liquid, in 40 DEG C of water-bath 10min.Then plus 5mL deionized water, sample solution after dilution is placed in boiling water bath continue it is anti-
60min is answered, is cooled to room temperature after reaction.It takes 1mL reaction solution to dilute 10 times with concentration for the sulfuric acid of 2M, then 1mL is taken to dilute
5% phenol solution of 1mL is added in liquid, and concussion mixes, and the 5mL concentrated sulfuric acid (18M) solution is added, after reacting 30min, in 490nm
Place's measurement absorbance value.Standard curve is prepared by glucose, and linear concentration is respectively 12.5 μ g/mL, 25 μ g/mL, 50 μ g/mL
100 μ g/mL of and, is dissolved with the sulfuric acid solution of 2M, and measuring method is consistent with sample.After measurement, by the absorbance value generation of sample
Enter in standard items curve, obtain polysaccharide concentration, calculates total sugar content.
(2) culture medium
Pleurotus tuber-regium seed culture fluid and liquid fermentation medium: glucose 20-40g/L, yeast extract 3-5g/L, di(2-ethylhexyl)phosphate
Hydrogen potassium (KH2PO4) 0.5-1.5g/L, epsom salt (MgSO4·7H2O)0.4-1.0g/L。
MRS culture medium: peptone 10.0g/mL；Beef extract 10.0g/mL；Yeast extract 5.0g/mL；Diammonium hydrogen citrate
[(NH4)2HC6H5O7]2.0g/mL；Glucose (C6H12O6·H2O)20.0g/mL；Tween 80 1.0mL/L；Sodium acetate
(CH3COONa·3H2O)5.0g/mL；Dipotassium hydrogen phosphate (K2HPO4·3H2O)2.0g/mL；Magnesium sulfate (MgSO4·7H2O)
0.58g/mL；Manganese sulfate (MnSO4·H2O)0.25g/mL；Agar 18.0g/mL；pH 6.2-6.6.
Embodiment 1
The PDA solid medium for covering with Pleurotus tuber-regium mycelia is cut into 1cm2Seed culture fluid after being added to sterilizing after fritter
In, 180rpm, 30 DEG C of culture 3d, the liquid state fermentation culture medium progress liquid state fermentation after access sterilizing, inoculum concentration 5.0%,
180rpm, 30 DEG C of temperature, fermentation time is 7 days.Wherein seed liquor liquid amount is 100mL/250mL triangular flask, and fermentation liquid fills liquid
Amount is 190mL/500mL.
Lactobacillus solution 100rpm in MRS culture medium, 37 DEG C, preculture 18h.Take Pleurotus tuber-regium fermentation liquid total volume
5.0% lactobacillus solution is inoculated in Pleurotus tuber-regium fermentation liquid, and inoculation time is the 6d of Pleurotus tuber-regium liquid state fermentation, co-cultures temperature
Degree is 30 DEG C, and co-cultivation revolving speed is 180rpm.Terminate to ferment in Pleurotus tuber-regium total fermentation time 7d, i.e. the co-cultivation time is 1d.
After fermentation, 15min is centrifuged using 3000g revolving speed, and uses quantitative filter paper filtering to remove suspended matter, obtained
Filtrate be that Pleurotus tuber-regium-lactic acid bacteria co-cultures fermentation liquid.
95% ethyl alcohol of 4 times of volumes is added in resulting fermentation liquid, stirring stands 8h, by sediment with the water of 2 times of volumes
Freeze-drying is after dissolution to get exocellular polysaccharide.
Embodiment 2
The PDA solid medium for covering with Pleurotus tuber-regium mycelia is cut into 1cm2Seed culture fluid after being added to sterilizing after fritter
In, 180rpm, 30 DEG C of culture 3d, the liquid state fermentation culture medium progress liquid state fermentation after access sterilizing, inoculum concentration 10%,
160rpm, 30 DEG C of temperature, fermentation time is 7 days.Wherein seed liquor liquid amount is 90mL/250mL triangular flask, zymotic fluid liquid amount
For 180mL/500mL.
For 24 hours, cultivation temperature is 37 DEG C to preculture to lactobacillus solution, shaking speed 100rpm in MRS culture medium.It takes
The lactobacillus solution of Pleurotus tuber-regium fermentation liquid total volume 2.5% is inoculated in Pleurotus tuber-regium fermentation liquid, and inoculation time is Pleurotus tuber-regium fermentation
The 5d of time, co-culturing temperature is 30 DEG C, and co-cultivation revolving speed is 180rpm.Terminate to send out in Pleurotus tuber-regium total fermentation time 7d
Ferment, i.e. co-cultivation time are 2 days.
After fermentation, 10min is centrifuged using 4000g revolving speed, and uses quantitative filter paper filtering to remove suspended matter, obtained
Filtrate be that Pleurotus tuber-regium-lactic acid bacteria co-cultures fermentation liquid.
95% ethyl alcohol of 4 times of volumes is added in resulting fermentation liquid, stirring stands 5h, by sediment with the water of 2 times of volumes
Freeze-drying is after dissolution to get exocellular polysaccharide.
Embodiment 3
The PDA solid medium for covering with Pleurotus tuber-regium mycelia is cut into 1cm2Seed culture fluid after being added to sterilizing after fritter
In, 180rpm, 30 DEG C of culture 3d, the liquid state fermentation culture medium progress liquid state fermentation after access sterilizing, inoculum concentration 5.0%,
180rpm, 30 DEG C of temperature, fermentation time is 7 days.Wherein seed liquor liquid amount is 100mL/250mL triangular flask, and fermentation liquid fills liquid
Amount is 190mL/500mL.
Lactobacillus solution 100rpm in MRS culture medium, 37 DEG C, preculture 18h.Take Pleurotus tuber-regium fermentation liquid total volume
5.0% lactobacillus solution is inoculated in Pleurotus tuber-regium fermentation liquid, and inoculation time is the 5d of Pleurotus tuber-regium liquid state fermentation, co-cultures temperature
Degree is 30 DEG C, and co-cultivation revolving speed is 180rpm.Terminate to ferment in Pleurotus tuber-regium total fermentation time 7d, i.e. the co-cultivation time is 2d.
After fermentation, 15min is centrifuged using 3000g revolving speed, and uses quantitative filter paper filtering to remove suspended matter, obtained
Filtrate be that Pleurotus tuber-regium-lactic acid bacteria co-cultures fermentation liquid.
95% ethyl alcohol of 4 times of volumes is added in resulting fermentation liquid, stirs, stirring stands 8h, by sediment with 2 times of bodies
Freeze-drying is after long-pending water dissolution to get exocellular polysaccharide.
Embodiment 4
The PDA solid medium for covering with Pleurotus tuber-regium mycelia is cut into 1cm2Seed culture fluid after being added to sterilizing after fritter
In, 180rpm, 30 DEG C of culture 3d, the liquid state fermentation culture medium progress liquid state fermentation after access sterilizing, inoculum concentration 10%,
160rpm, 30 DEG C of temperature, fermentation time is 7 days.Wherein seed liquor liquid amount is 90mL/250mL triangular flask, zymotic fluid liquid amount
For 180mL/500mL.
For 24 hours, cultivation temperature is 37 DEG C to preculture to lactobacillus solution, shaking speed 100rpm in MRS culture medium.It takes
The lactobacillus solution of Pleurotus tuber-regium fermentation liquid total volume 7.5% is inoculated in Pleurotus tuber-regium fermentation liquid, and inoculation time is Pleurotus tuber-regium fermentation
The 5d of time, co-culturing temperature is 30 DEG C, and co-cultivation revolving speed is 180rpm.Terminate to send out in Pleurotus tuber-regium total fermentation time 7d
Ferment, i.e. co-cultivation time are 2 days.
After fermentation, 10min is centrifuged using 4000g revolving speed, and uses quantitative filter paper filtering to remove suspended matter, obtained
Filtrate be that Pleurotus tuber-regium-lactic acid bacteria co-cultures fermentation liquid.
95% ethyl alcohol of 4 times of volumes is added in resulting fermentation liquid, stirring stands 5h, by sediment with the water of 2 times of volumes
Freeze-drying is after dissolution to get exocellular polysaccharide.
Embodiment 5
The PDA solid medium for covering with Pleurotus tuber-regium mycelia is cut into 1cm2Seed culture fluid after being added to sterilizing after fritter
In, 180rpm, 30 DEG C of culture 3d, the liquid state fermentation culture medium progress liquid state fermentation after access sterilizing, inoculum concentration 10%,
160rpm, 30 DEG C of temperature, fermentation time is 7 days.Wherein seed liquor liquid amount is 90mL/250mL triangular flask, zymotic fluid liquid amount
For 180mL/500mL.
For 24 hours, cultivation temperature is 37 DEG C to preculture to lactobacillus solution, shaking speed 100rpm in MRS culture medium.It takes
The lactobacillus solution of Pleurotus tuber-regium fermentation liquid total volume 10% is inoculated in Pleurotus tuber-regium fermentation liquid, when inoculation time is that Pleurotus tuber-regium ferments
Between 5d, co-culture temperature be 30 DEG C, co-cultivations revolving speed be 180rpm.Terminate to ferment in Pleurotus tuber-regium total fermentation time 7d,
Co-culturing the time is 2 days.
After fermentation, 10min is centrifuged using 4000g revolving speed, and uses quantitative filter paper filtering to remove suspended matter, obtained
Filtrate be that Pleurotus tuber-regium-lactic acid bacteria co-cultures fermentation liquid.
95% ethyl alcohol of 4 times of volumes is added in resulting fermentation liquid, stirring stands 5h, by sediment with the water of 2 times of volumes
Freeze-drying is after dissolution to get exocellular polysaccharide.
Comparative example
More preferably to verify influence of the co-culture system employed in the present invention to yield of extracellular polysaccharide, We conducted such as
Under comparative test.
Comparative example 1: the lactobacillus inoculum time is the same day of Pleurotus tuber-regium fermentation liquid inoculation, i.e. the co-cultivation time is 7d, remaining
Condition is the same as the embodiment of the present invention 1；
Comparative example 2: the lactobacillus inoculum time is the 1d of Pleurotus tuber-regium fermentation, i.e. the co-cultivation time is 6d, remaining condition is equal
With the embodiment of the present invention 1；
Comparative example 3: the lactobacillus inoculum time is the 2d of Pleurotus tuber-regium fermentation, i.e. the co-cultivation time is 5d, remaining condition is equal
With the embodiment of the present invention 1.
Comparative example 4: lactobacillus inoculum amount is 12%, remaining condition is the same as the embodiment of the present invention 2；
Comparative example 5: lactobacillus inoculum amount is 15%, remaining condition is the same as the embodiment of the present invention 2；
Comparative example 6: lactobacillus inoculum amount is 20%, remaining condition is the same as the embodiment of the present invention 2.
Table 1
Note: control group 1 is not add lactic acid bacteria co-cultivation using only Pleurotus tuber-regium mycelium liquid state fermentation；Control group 2 is only
It is fermented using lactic acid bacteria single bacterium.Polysaccharide yield is to measure polyoses content therein using phend-sulphuric acid method to ethanol pellet
Afterwards, by the ratio of the polyoses content and fermentation liquid total volume.
The obtained exocellular polysaccharide low yield of the method for Comparison study example.As shown in Table 1, in Pleurotus tuber-regium mycelium and lactic acid
When bacterium co-cultures number of days greater than 4d, gained exocellular polysaccharide yield is relatively low compared with control group 1；In lactic acid bacteria access Pleurotus tuber-regium fermentation liquid
When inoculum concentration (volume ratio) is more than 10%, exocellular polysaccharide yield and the equal degradation of biomass.
In addition, at 28 DEG C we determine exocellular polysaccharide obtained by co-cultivation when reaching saturation state in 100g water institute it is molten
The quality of the polysaccharide of solution, i.e. solubility.Compared with Pleurotus tuber-regium mycelium ferments merely exocellular polysaccharide obtained by (control group), the present invention
Gained exocellular polysaccharide solubility is increased to 10.33g/L from 0.52g/L, and preliminary analysis may be reduced with wherein mannose content to be had
It closes.
The monosaccharide composition of gained Pleurotus tuber-regium exocellular polysaccharide of the invention, is shown in Table 2.
Table 2
Although the present invention has been described by way of example and in terms of the preferred embodiments, it is not intended to limit the invention, any to be familiar with this skill
The people of art can do various change and modification, therefore protection model of the invention without departing from the spirit and scope of the present invention
Enclosing subject to the definition of the claims.
Claims (10)
1. a kind of method of high yield Pleurotus tuber-regium exocellular polysaccharide, which is characterized in that micro- using lactic acid bacteria and two kinds of Pleurotus tuber-regium mycelium
Biology building co-culture system.
2. the method according to claim 1, wherein the following steps are included:
(1) Pleurotus tuber-regium mycelium is added to preculture in seed culture fluid；Lactic acid bacteria is added in fluid nutrient medium and is carried out
Preculture；
(2) culture solution obtained after Pleurotus tuber-regium mycelium preculture is linked into liquid state fermentation 7d in fermentation medium；
(3) when the fermentation of step (2) is carried out to 3-6d, the culture solution obtained after lactic acid bacteria preculture is inoculated into Pleurotus tuber-regium
In mycelium fermentation broth, co-cultured；
(4) it is centrifuged, filters after fermentation, gained filtrate is fermentation liquid, and ethyl alcohol is added into fermentation liquid, is stirred, after standing
Precipitating, dissolution precipitating are collected, freeze-drying obtains Pleurotus tuber-regium exocellular polysaccharide.
3. according to the method described in claim 2, it is characterized in that, the mycelial preculture of Pleurotus tuber-regium described in step (1)
It is that Pleurotus tuber-regium mycelia is added in seed culture fluid, the preculture 2-4d under the conditions of 160-200rpm, 26-32 DEG C.
4. according to the method described in claim 2, it is characterized in that, the preculture of lactic acid bacteria described in step (1) is will be newborn
Sour bacterium is added in MRS culture medium, the preculture 18-36h under the conditions of 100-120rpm, 30-40 DEG C.
5. according to the method in claim 2 or 3, which is characterized in that liquid state fermentation described in step (2) refers to Pleurotus tuber-regium
The inoculum concentration of Mycelium culture liquid is 5-10%, accesses fermentation medium, ferment 7d at 150-200rpm, 25-32 DEG C.
6. according to the method described in claim 2, it is characterized in that, in step (1) the mycelial seed culture fluid of Pleurotus tuber-regium and
Fermentative medium formula in step (2) are as follows: glucose 20-40g/L, yeast extract 3-5g/L, potassium dihydrogen phosphate (KH2PO4)0.5-
1.5g/L, epsom salt (MgSO4·7H2O)0.4-1.0g/L。
7. the method according to claim 2 or 6, which is characterized in that co-cultivation described in step (3), which refers to, is fermenting extremely
When 3-6d, by lactobacillus inoculum into Pleurotus tuber-regium mycelium fermentation broth, inoculum concentration 2.5-10%, in 150-200rpm, 26-
1-4d is co-cultured under the conditions of 35 DEG C.
8. according to the method described in claim 2, referring to that 3000-5000g is centrifuged 5- it is characterized in that, being centrifuged in step (4)
10min。
9. the method according to claim 2 or 8, which is characterized in that obtain the method for exocellular polysaccharide described in step (4)
Are as follows: 95% ethyl alcohol of 3-5 times of volume of fermentation liquid is added into centrifugation, filtered ferment filtrate, stirring stands 5-8h, will sink
Starch is freeze-dried after being dissolved with water.
10. method described in claim 1-9 is in the application of bioengineering, food, medicine or field of health care products.
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