CN102465160A - Method for breeding high-yield extracellular polysaccharide bacterial strain by ganoderma lucidum protoplast mutagenesis - Google Patents

Abstract

The invention relates to a preparation method of a mutagenic ganoderma polysaccharide, specifically, relates to a method for breeding a high-yield extracellular polysaccharide bacterial strain by ganoderma lucidum protoplast mutagenesis, and belongs to the technical field of the hereditism. The method utilizes ganoderma lucidum as a strain and comprises the following steps of carrying out activation by a potato dextrose agar medium, carrying out shake-flask culture in starch liquid, carrying out ganoderma lucidum mycelium hydrolysis by lywallzyme, helicase and cellulose, and carrying out shaking at a constant temperature to obtain a ganoderma lucidum protoplast. Through passage, the mutagenic high-yield extracellular polysaccharide bacterial strain has extracellular polysaccharide content which is 6 to 8 times higher than extracellular polysaccharide content of the original bacterial strain. The high-yield extracellular polysaccharide bacterial strain has stable hereditary, is an engineering strain and can be utilized for ganoderma lucidum polysaccharide industrial production.

Description

A kind of preparation method of mutagenesis red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains

Technical field

The present invention relates to a kind of preparation method of ganoderma polysaccharide, specifically relate to a kind of preparation method of mutagenesis red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains, belong to the genetic technique field.

Background technology

Present cultivation, development technique field edible mushrooms; Edible mushrooms various in style; Quality is different, and the red ganoderma polysaccharide is the effective constituent that tool exploitation is worth in the red ganoderma, has the human body of raising hypoxia-bearing capability, removes radical, enhance immunity power, pharmacologically active widely such as antitumor.But the red ganoderma polysaccharide yield is generally not high and be difficult to suitability for industrialized production.And the protoplastis induced-mutation technique is a kind of effective Microbial Breeding novel method.Li Gang waited and adopted ultraviolet mutagenesis glossy ganoderma protoplastis calendar year 2001; Selected the bacterial strain of two plant heights product intracellular polyse; Its polysaccharide content has improved 10.15% than the pilot plant test polysaccharide content that original strain has improved 39.29% and 26.92%, 3 ton of scale respectively than original strain.The physics ultraviolet mutagenesis is adopted in protoplastis mutagenesis at present basically, and it is lower to obtain mutagenic strain active substance output, and the mutagenesis effect is relatively poor.

Summary of the invention

The objective of the invention is for overcoming the weak point of above-mentioned prior art; A kind of preparation method of mutagenesis red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains is provided; It is bacterial classification with the red ganoderma, behind potato dextrose agar activation, starch liquid shake-flask culture, utilizes lywallzyme, Snailase and cellulase hydrolysis red ganoderma mycelium; Vibration under constant temperature, preparation red ganoderma protoplastis.After lithium chloride mutagenesis, exocellular polysaccharide content has improved 8～10 times than original strain.Mutagenic strain is through after going down to posterity, and exocellular polysaccharide content has improved 6～8 times than former bacterial strain, and inherited character is more stable, and this mutagenic strain can be used for the suitability for industrialized production ganoderan.

The present invention realizes with following technical scheme: a kind of preparation method who becomes red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains is characterized in that: it is bacterial classification with the red ganoderma, behind potato dextrose agar activation, starch liquid shake-flask culture; Utilize prozyme liquid hydrolysis Ganoderma mycelium, at 28～32 ℃ of 1.5～4h that vibrate down, preparation protoplastis; The lithium chloride that adopts 0.02%～0.3% concentration is as mutagenic compound; Join in the regeneration culture medium, mutagenesis glossy ganoderma protoplastis is through regenerating and culturing, shake bottle submerged fermentation and obtain an exocellular polysaccharide fermented liquid; Extract polysaccharide with alcohol deposition method, the phenolsulfuric acid method is measured polysaccharide content.

(1) bacterial classification inoculation activation 1～2 time on the potato dextrose agar substratum is got the bacterium piece and is inoculated in and shakes in bottle Zulkovsky starch liquid nutrient medium, and 27～29 ℃ were carried out shake-flask culture 4～5 days;

(2) get the mycelia of liquid culture, filter and collect mycelium pellet,, wash 2～3 times with N.F,USP MANNITOL again with aseptic water washing 2～3 times; Change in the mercaptoethanol, constant temperature shaking table vibration 30～60min filters and collects mycelium pellet; With N.F,USP MANNITOL flushing 2～3 times, spinning is got and is precipitated as mycelium, gets mycelium and adds prozyme liquid with 1: 10～20 ratio; Constant temperature is vibration enzymolysis 2～4h down, filters rear filtrate in the centrifugal 3～5min of 500～1000r/min, gets supernatant in 3000～4000r/min; Centrifugal 5～10min, deposition is protoplastis, dilutes 10 times to protoplastis with the N.F,USP MANNITOL flushing and with N.F,USP MANNITOL;

(3) will regenerate colony inoculation on potato dextrose agar; Cultivate under the constant temperature and won for mutagenic strain in 6～7 days, the potato dextrose agar that its inoculation is new, constant temperature are cultivated down and were got s-generation mutagenic strain in 6～7 days; And inoculate new potato dextrose agar; Cultivate under the constant temperature third generation mutagenic strain, the inoculation s-generation, third generation bacterium piece are in shaking bottle Zulkovsky starch liquid nutrient medium, the constant temperature shaking table is cultivated;

(4) get centrifuging and taking supernatant behind the filtering fermentation liquor, 80～90 ℃ of water-baths are condensed into 0.2～0.25 times of original volume, add 4～5 times of volume 95% ethanol, and 4 ℃ leave standstill, and liquid concentrator is centrifugal, with absolute ethanol washing deposition twice, dry to constant weight, crude extracellular polysaccharide.

Advantage of the present invention is: in nutrient solution, produce exocellular polysaccharide after adopting red ganoderma mutagenic strain that this technology obtains through submerged fermentation, after extracting, separating, can be used as pharmaceutical prod and foodstuff additive, be widely used in health care industry and foodstuffs industry.

Embodiment

Below in conjunction with embodiment the present invention is done further explain:

Embodiment 1,

The red ganoderma bacterial strain is 28 ℃ of following heat insulating culture activated spawn 7 days, and the bacterium piece of getting after the activation is inoculated in the starch culture medium of triangular flask, on shaking table, cultivates 5 days under 28 ℃ of temperature; Filter and collect mycelium, after the N.F,USP MANNITOL flushing, change in 0.3% mercaptoethanol; Vibration 40min; Filter and collect mycelium, use the N.F,USP MANNITOL washing and filtering, the centrifugal 8min of 1200r/min.Get the 0.5g mycelium and add prozyme liquid with 1: 15 ratio, at 28 ℃ of vibration enzymolysis mycelium 3h down, filtrating is with the centrifugal 3min of 1000r/min, 2 times.Supernatant adopts the centrifugal 10min of 3000r/min to make protoplastis.The lithium chloride of employing 0.02% adds in the regeneration culture medium, gets protoplastis 1ml and is coated on the regeneration culture medium, constant temperature culture 7 days.The bacterium piece of getting after the activation is inoculated in the triangular flask Zulkovsky starch liquid nutrient medium, places 28 ℃ of shaking tables to cultivate 5 days.Fermented liquid is centrifugal after filtering, gets supernatant and in 80 ℃ of water-baths, is concentrated into 0.2 times of original volume, adds 4 times of volume 95% ethanol; 4 ℃ leave standstill 12h, and the centrifugal 5min of 4500r/min precipitates 2 times with absolute ethanol washing then; Dry to constant weight for 70 ℃, get crude extracellular polysaccharide.Adopt the phenol sulfuric acid process to measure polysaccharide content.Mutagenic strain exocellular polysaccharide content improves 9 times than original strain, and polysaccharide content still can improve 7 times after going down to posterity.

Embodiment 2,

At 27 ℃ of following activation red ganoderma bacterial strains, the bacterium piece after the inoculation activation shakes in the bottle in 250ml, and 27 ℃ of shaking tables were cultivated 5 days.Filter and collect mycelium,, change in 0.2% mercaptoethanol with sterilized water, N.F,USP MANNITOL flushing, shaking table vibration 30min, 28 ℃ of temperature are filtered and are collected mycelium, the N.F,USP MANNITOL washing and filtering, mycelium is with the centrifugal 10min separation of mycelial of 1500r/min.Get the 1g mycelium and add prozyme liquid (lywallzyme+Snailase+cellulase) with 1: 20 ratio, at 30 ℃ of vibration enzymolysis 4h down, filtrating is with the centrifugal 5min of 600r/min, 2 times.Supernatant gets protoplastis with the centrifugal 5min of 4000r/min.The lithium chloride of 0.2% concentration is added in the regeneration culture medium, get protoplastis 1.5ml and be coated on the regeneration culture medium, cultivated 6 days for 27 ℃.Bacterium piece after the activation is inoculated in 250ml shakes in bottle Zulkovsky starch liquid nutrient medium, put 27 ℃ of shaking tables and cultivated 5 days.Fermented liquid filters with sterile gauze, and filtrating is got supernatant at the centrifugal 10min of 4000r/min, and 90 ℃ of water-baths are condensed into 0.25 times of original volume, add 5 times of volume 95% ethanol, and 4 ℃ are spent the night.Centrifugal back is precipitated 2 times with absolute ethanol washing, weighs after 60 ℃ of vacuum-dryings, gets crude extracellular polysaccharide, measures mutagenic fungi exocellular polysaccharide content and improves 10 times than original strain.

Claims (2)

1. the preparation method of a mutagenesis red ganoderma protoplastis breeding high-yield extracellular polysaccharide strains, it is characterized in that: it is bacterial classification with the red ganoderma, behind potato dextrose agar activation, starch liquid shake-flask culture; Utilize prozyme liquid hydrolysis Ganoderma mycelium; At 28～32 ℃ of 1.5～4h that vibrate down, the preparation protoplastis, the lithium chloride that adopts 0.02%～0.3% concentration is as mutagenic compound; Join in the regeneration culture medium; Mutagenesis glossy ganoderma protoplastis through regenerating and culturing, shake bottle submerged fermentation and obtain an exocellular polysaccharide fermented liquid, extracts polysaccharide with alcohol deposition method.

2. method for preparing the described mutagenesis red ganoderma of claim 1 protoplastis breeding high-yield extracellular polysaccharide strains is characterized in that: carry out as follows:

(1) bacterial classification inoculation activation 1～2 time on the potato dextrose agar substratum is got the bacterium piece and is inoculated in and shakes in bottle Zulkovsky starch liquid nutrient medium, and 27～29 ℃ were carried out shake-flask culture 4～5 days;

(2) get the mycelia of liquid culture, filter and collect mycelium pellet,, wash 2～3 times with N.F,USP MANNITOL again with aseptic water washing 2～3 times; Change in the mercaptoethanol, constant temperature shaking table vibration 30～60min filters and collects mycelium pellet; With N.F,USP MANNITOL flushing 2～3 times, spinning is got and is precipitated as mycelium, gets mycelium and adds prozyme liquid with 1: 10～20 ratio; Constant temperature is vibration enzymolysis 2～4h down, filters rear filtrate in the centrifugal 3～5min of 500～1000r/min, gets supernatant in 3000～4000r/min; Centrifugal 5～10min, deposition is protoplastis, dilutes 10 times to protoplastis with the N.F,USP MANNITOL flushing and with N.F,USP MANNITOL;

(3) will regenerate colony inoculation on potato dextrose agar; Cultivate under the constant temperature and won for mutagenic strain in 6～7 days, the potato dextrose agar that its inoculation is new, constant temperature are cultivated down and were got s-generation mutagenic strain in 6～7 days; And inoculate new potato dextrose agar; Cultivate under the constant temperature third generation mutagenic strain, the inoculation s-generation, third generation bacterium piece are in shaking bottle Zulkovsky starch liquid nutrient medium, the constant temperature shaking table is cultivated;

(4) get centrifuging and taking supernatant behind the filtering fermentation liquor, 80～90 ℃ of water-baths are condensed into 0.2～0.25 times of original volume, add 4～5 times of volume 95% ethanol, and 4 ℃ leave standstill, and liquid concentrator is centrifugal, with absolute ethanol washing deposition twice, dry to constant weight, crude extracellular polysaccharide.