CN102363754A - Clear liquid fermentation medium for clostridium butyricum and fermentation culture method thereof - Google Patents
Clear liquid fermentation medium for clostridium butyricum and fermentation culture method thereof Download PDFInfo
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Abstract
The invention relates to a clear liquid fermentation medium for clostridium butyricum. The clear liquid fermentation medium, the pH value of which is 7-8, is made from glucose, tryptone, a yeast extract powder, ammonium sulfate, sodium bicarbonate, a maize liquid powder and the like. The clear liquid fermentation medium for clostridium butyricum contains metal salts for promoting the growth of gemma, wherein the metal salts are manganese sulfate, magnesium sulfate and ferrous sulphate. The enrichment medium of the clear liquid fermentation medium for clostridium butyricum contains yeast extract, beef extract, tryptone, glucose, soluble starch, sodium chloride, sodium acetate trihydrate, cysteine hydrochloride, methylene blue at the concentration of 0.5% and distilled water; and the pH is adjusted to 7.1. A fermentation culture method of the clear liquid fermentation medium for clostridium butyricum comprises the following steps of: glycerin tube refrigerated strain activation, heating and optimization, Erlenmeyer flask first-order seed culture, liquid fermentation and spray drying. The production of clostridium butyricum has advantages of simple operation, high amount of zymocyte, high gemma yield, simple post-treatment, less impurities in a bacterial powder sample and high amount of effective live bacteria, and saves cost at large.
Description
Technical field
The present invention relates to a kind of liquor fermentation substratum and fermentation culture method thereof of clostridium butylicum, belong to the microbial fermentation technology field.
Background technology
Clostridium butylicum (
Clostridium butyricum), promptly clostridium butyricum has another name called Butylic acid bacteria.It is a kind in the shuttle shape gemma Pseudomonas, mainly is present in the enteron aisle and ight soil of cheese, natural sour milk, people and animal, in the physical environment such as some leaf, soil.Clostridium butylicum is a kind of straight or crooked Gram-positive anaerobism endogenous spore bacillus, and environmental change is had certain resistance, and these characteristics make clostridium butylicum have the potentiality as good probiotic bacterium.On the other hand, when clostridium butylicum passed through animal intestinal, ability stomach juice-resistant and bile salt had guaranteed the performance of its performance well.Being widely used in drug for controlling intestinal function thing, protective foods, fodder additives, microbial fertilizer etc. aspect clinical medicine and the livestock industry, be a kind of comparatively ideal probiotics with extensive exploitation prospect.
Unite the 52nd Expert C-on Food Additive that holds by Food and Argriculture OrganizationFAO (FAO) and The World Health Organization (WHO) in February, 1999 and point out emphatically the normal entero-bacte that clostridium butylicum is the people.After this, people pay close attention to and have studied morphological specificity, physilogical characteristics, biochemical characteristics and the commercial applications thereof of clostridium butyricum widely.1940, clostridium butylicum was realized commercially producing and has been applied to clinical in Japan.At first, it is used as medicines for relieving intestinal disorders, is widely used in alteration of intestinal flora, acute and chronic diarrhea, and irritable bowel syndrome, treatment of diseases such as microbiotic dependency enteritis have obtained significant curative effect.After this, it is widely-used that it successively is used as prescription drugs, nonprescription drugs, veterinary drug, fodder additives, foodstuff additive etc.In Korea S, clostridium butyricum also has been used to the fodder additives of ox, pig and poultry, and clostridium butylicum can be separately uses as fodder additives, also can with the composite uses of probiotic bacterium such as milk-acid bacteria, sporeformer, bifidus bacillus, can strengthen its effect.
Probiotic bacterium class probiotics has obtained generally acknowledging in the active effect that substitutes microbiotic, prevents and treats livestock and poultry and improve aspects such as environment, but the high quality probiotics product that at present real accords with production requires also seldom.Clostridium butylicum has the result of treatment of highly significant clinically, and because of it has the gemma form, higher utility is arranged owing to biological characteristics.
Following patent CN1144873C, CN1184308C, US5.292 523 (1994), US4.892 731 (1990), JP63-146825 (1986), JP61-6625 (1984), JP06-166825 (1994), JP05-227898 (1993) relate to the fermentation and the preparation method of clostridium butylicum; There are some shortcomings in these prior aries;: 1, with regard to training method, use complex medium in a large number, perhaps other contain a large amount of insoluble composition substratum; Cause the aftertreatment difficulty; With high costs, unit bacterium powder viable bacteria content is low, and impurity is more; 2, with regard to culture effect, fermented liquid bacterium number is less, and the gemma rate is lower.
Summary of the invention
The present invention is the shortcoming that overcomes prior art, and a kind of liquor fermentation substratum and fermentation culture method thereof of clostridium butylicum is provided, and what all add is soluble carbon nitrogenous source and metal-salt; No substratum deposition; Utilization ratio is high, and the aftertreatment of bacterium liquid is simple, and bacterium powder effective content is high; The convenient preservation significantly reduced processing cost on the whole.
The technical scheme of the liquor fermentation substratum of a kind of clostridium butylicum of the present invention; Comprise following component: glucose 0.8～2.5% (w/v); Tryptones 0.6～2.3% (w/v), yeast soak powder 1.1～2.8% (w/v), ammonium sulfate 0.05～0.2 % (w/v); Sodium hydrogencarbonate 0.05～0.25 % (w/v), and corn starch or cereal starch or soybean starch 0.2～0.45% (w/v); PH value is 7～8.
Further technical scheme is:
The technical scheme of the liquor fermentation substratum of described clostridium butylicum; Component is: glucose 0.8% (w/v); Tryptones 0.6% (w/v), yeast soak powder 1.1% (w/v), ammonium sulfate 0.05 % (w/v); Sodium hydrogencarbonate 0.05 % (w/v), and corn starch or cereal starch or soybean starch 0.2% (w/v); PH value 7.
The liquor fermentation substratum of described clostridium butylicum, component is: glucose 2.5% (w/v), Tryptones 2.3% (w/v); Yeast soaks powder 2.8% (w/v); Ammonium sulfate 0.2 % (w/v), sodium hydrogencarbonate 0.25 % (w/v), corn starch or cereal starch or soybean starch 0.45% (w/v); PH value 8.
The liquor fermentation substratum of described clostridium butylicum, component is: glucose 1.5% (w/v), Tryptones 1.3% (w/v), yeast soak powder 1.8% (w/v), ammonium sulfate 0.1 % (w/v), sodium hydrogencarbonate 0.12% (w/v), corn starch 0.33% (w/v); PH value 7.5.
The metal-salt that a kind of short gemma of liquor fermentation substratum that is used for clostridium butylicum of the present invention generates comprises following component: manganous sulfate 0.01～0.1% (w/v), sal epsom 0.01～0.1% (w/v), ferrous sulfate 0.01～0.08% (w/v).
Further technical scheme is:
The metal-salt that the short gemma of the liquor fermentation substratum of described clostridium butylicum generates, component is: manganous sulfate 0.05% (w/v), sal epsom 0.05% (w/v), ferrous sulfate 0.03% (w/v).
A kind of proliferated culture medium (RCM) that is used for the liquor fermentation substratum of clostridium butylicum of the present invention is formed and weight part is: yeast extract 2～4, beef extract 8～12, Tryptones 8～12, glucose 3～8, Zulkovsky starch 0.5～2, sodium-chlor 3～8, sodium acetate trihydrate 1～6, cysteine hydrochloride 0.1～1.5,0.5% methylene blue, 0.1～3mL, zero(ppm) water 800～1200mL; Regulate pH7.1 ± 0.1.
Further technical scheme is:
The proliferated culture medium (RCM) of the liquor fermentation substratum of described clostridium butylicum, prescription consists of: yeast extract 3g, beef extract 10 g, Tryptones 10 g, glucose 5 g, Zulkovsky starch 1 g, sodium-chlor 5 g, sodium acetate trihydrate 3 g, cysteine hydrochloride 0.5 g, 0.5% methylene blue 0.2mL, zero(ppm) water 1000mL; Regulate pH7.1 ± 0.1.
The fermentation culture method of the liquor fermentation substratum of a kind of clostridium butylicum of the present invention may further comprise the steps:
(1) glycerine pipe refrigeration actication of culture: get glycerine pipe refrigeration bacterial classification inoculation in the test tube that proliferated culture medium (RCM) is housed, on cover whiteruss, substratum is sterilized in advance, leaves standstill then to cultivate to form gemma;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in the water-bath handles;
(3) the triangular flask first order seed is cultivated: respectively above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out, cultivating logarithmic phase can be inoculated in the fermention medium as seed liquor again; Fermention medium with metal-salt of short gemma generation comprises glucose, Tryptones, and yeast soaks powder, ammonium sulfate, sodium hydrogencarbonate, corn starch or cereal starch or soybean starch, manganous sulfate, sal epsom, ferrous sulfate, and keep certain pH value;
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted fermentation cylinder for fermentation;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and the control spraying drying can make high-purity bacterium powder.
Further technical scheme is:
The fermentation culture method of the liquor fermentation substratum of described clostridium butylicum, in said step:
(1) glycerine pipe refrigeration actication of culture: glycerine pipe bacterial classification 180～230 μ L that get-25～-18 ℃ of refrigerations are inoculated in the test tube that 6～12mL proliferated culture medium (RCM) is housed; On cover 1～3cm whiteruss; Substratum is sterilized in advance, leaves standstill under 33～38 ℃ of conditions to cultivate 40～50h to form gemma; Said proliferated culture medium (RCM) comprises following composition weight part: yeast extract 2～4, beef extract 8～12, Tryptones 8～12, glucose 3～8, Zulkovsky starch 0.5～2, sodium-chlor 3～8, sodium acetate trihydrate 1～6, cysteine hydrochloride 0.1～1.5,0.5% methylene blue, 0.1～3mL, zero(ppm) water 800～1200mL; Regulate pH7.1 ± 0.1,100～120 ℃, 20 min sterilization is subsequent use;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in 70～90 ℃ of water-baths handles 9～12min;
(3) the triangular flask first order seed is cultivated: above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out with 1～3% inoculum size respectively, cultivates 16-20h and can be inoculated in the fermention medium as seed liquor to logarithmic phase;
Said fermention medium with metal-salt of short gemma generation; Prescription consists of: glucose 0.8～2.5% (w/v); Tryptones 0.6～2.3% (w/v), yeast soak powder 1.1～2.8% (w/v), ammonium sulfate 0.05～0.2 % (w/v); Sodium hydrogencarbonate 0.05～0.25 % (w/v), and corn starch or cereal starch or soybean starch 0.2～0.45% (w/v); PH value is 7～8; Manganous sulfate 0.01～0.1% (w/v), sal epsom 0.01～0.1% (w/v), ferrous sulfate 0.01～0.08% (w/v);
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted in the fermentor tank inoculum size 3%～5%, 33～40 ℃ of temperature controls; Mixing speed 45～60rpm; The PH value is reduced to below 6.5 in the fermenting process, and adding 18～25% yellow soda ash control pH value is 6.5, up to fermentation ends; And fermentation 24h adds carbon source with interior stream, and stream adds 0.55～1.5% (w/v) carbon source;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and EAT is 130～160 ℃ in the control spraying drying, and 50～75 ℃ of temperature outs can make high-purity bacterium powder.
The fermentation culture method of the liquor fermentation substratum of described clostridium butylicum, in said step:
(1) glycerine is guaranteed the Tibetan actication of culture: the glycerine pipe bacterial classification 200 μ L that get-20 ℃ of preservations are inoculated in the test tube that 9mL proliferated culture medium (RCM) is housed, on cover the 2cm whiteruss, substratum is sterilized in advance, leaves standstill under 37 ℃ of conditions to cultivate 48h to form gemma; Said RCM culture medium prescription is following: yeast extract 3 g, beef extract 10 g, Tryptones 10g, glucose 5g, Zulkovsky starch 1g, sodium-chlor 5g, sodium acetate trihydrate 3g, cysteine hydrochloride 0.5g, 0.5% methylene blue 0.2mL, zero(ppm) water 1000mL; Regulate pH7.1 ± 0.1,115 ℃, 20 min sterilization is subsequent use;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in 80 ℃ of water-baths handles 10min; Heating can promote gemma to sprout, and helps the thalli growth consistence and prevents living contaminants;
(3) the triangular flask first order seed is cultivated: above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out with 3% inoculum size respectively again; Cultivating 16-20h can be inoculated in the fermention medium as seed liquor to logarithmic phase: glucose 1.5% (w/v), and Tryptones 1.3% (w/v), yeast soak powder 1.8% (w/v); Ammonium sulfate 0.1 % (w/v); Sodium hydrogencarbonate 0.124% (w/v), and corn starch 0.33% (w/v), manganous sulfate 0.05% (w/v); Sal epsom 0.05% (w/v), ferrous sulfate 0.03% (w/v); PH value 7.5;
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted in the fermentor tank inoculum size 3%～5%, 37 ℃ of temperature controls; Mixing speed 50rpm; The PH value is reduced to below 6.5 in the fermenting process, and adding 20% yellow soda ash control pH value is 6.5, up to fermentation ends; And fermentation 24h adds carbon source with interior stream, and stream adds 1% (w/v) carbon source;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and EAT is 150 ℃ in the control spraying drying, and 70 ℃ of temperature outs can make high-purity bacterium powder.
Major advantage of the present invention is following:
(1) in the fermentation culture method of the liquor fermentation substratum of clostridium butylicum of the present invention, adds soluble carbon nitrogenous source and metal-salt, no substratum deposition; Utilization ratio is high, and the aftertreatment of bacterium liquid is simple, and bacterium powder effective content is high; The convenient preservation reduces cost on the whole greatly.Anaerobically fermenting is through covering paraffin on substratum, but the utilization of paraffin recirculation need not feed rare gas element, environmental protection and saving.(2) segmentation feeding culture mode further improves butyric bacteria content, and then improves the gemma rate of formation, adopts the present invention to produce clostridium butylicum, improves zymophyte number and gemma yield greatly, and fermented liquid bacterium number can reach 2.3 * 10
9Cfu/ml, the gemma yield is more than 95%.(3) heating preferred seed substratum mode makes thalli growth consistent.(4) use the spraying drying mode after the fermentation ends, can rapidly and efficiently obtain a large amount of high-content bacterium powder.
Embodiment
Embodiment 1:The liquor fermentation media components of clostridium butylicum of the present invention is: glucose 1.5% (w/v), and Tryptones 1.3% (w/v), yeast soak powder 1.8% (w/v), ammonium sulfate 0.1 % (w/v), sodium hydrogencarbonate 0.12% (w/v), corn starch 0.33% (w/v); PH value 7.5.
Embodiment 2:The liquor fermentation media components of clostridium butylicum of the present invention is: glucose 0.8% (w/v); Tryptones 0.6% (w/v), yeast soak powder 1.1% (w/v), ammonium sulfate 0.05 % (w/v); Sodium hydrogencarbonate 0.05 % (w/v), and corn starch or cereal starch or soybean starch 0.2% (w/v); PH value 7.
Embodiment 3:The liquor fermentation media components of clostridium butylicum of the present invention is: glucose 2.5% (w/v); Tryptones 2.3% (w/v), yeast soak powder 2.8% (w/v), ammonium sulfate 0.2 % (w/v); Sodium hydrogencarbonate 0.25 % (w/v), and corn starch or cereal starch or soybean starch 0.45% (w/v); PH value 8.
Embodiment 4:The metal-salt that a kind of short gemma of liquor fermentation substratum that is used for clostridium butylicum of the present invention generates comprises following component: manganous sulfate 0.01～0.1% (w/v), sal epsom 0.01～0.1% (w/v), ferrous sulfate 0.01～0.08% (w/v).The present embodiment component is: manganous sulfate 0.05% (w/v), sal epsom 0.05% (w/v), ferrous sulfate 0.03% (w/v).
Embodiment 5:A kind of proliferated culture medium (RCM) that is used for the liquor fermentation substratum of clostridium butylicum of the present invention is formed and weight part is: yeast extract 2～4, beef extract 8～12, Tryptones 8～12, glucose 3～8, Zulkovsky starch 0.5～2, sodium-chlor 3～8, sodium acetate trihydrate 1～6, cysteine hydrochloride 0.1～1.5,0.5% methylene blue, 0.1～3mL, zero(ppm) water 800～1200mL; Regulate pH7.1 ± 0.1.The present embodiment prescription consists of: yeast extract 3g, beef extract 10 g, Tryptones 10 g, glucose 5 g, Zulkovsky starch 1 g, sodium-chlor 5 g, sodium acetate trihydrate 3 g, cysteine hydrochloride 0.5 g, 0.5% methylene blue 0.2mL, zero(ppm) water 1000mL; Regulate pH7.1 ± 0.1.
Embodiment 6:The fermentation culture method of the liquor fermentation substratum of a kind of clostridium butylicum of the present invention has following steps:
(1) glycerine pipe refrigeration actication of culture: get glycerine pipe refrigeration bacterial classification inoculation in the test tube that proliferated culture medium (RCM) is housed, on cover whiteruss, substratum is sterilized in advance, leaves standstill then to cultivate to form gemma;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in the water-bath handles; Heating can promote gemma to sprout, and helps the thalli growth consistence and prevents living contaminants;
(3) the triangular flask first order seed is cultivated: respectively above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out, cultivates logarithmic phase and be inoculated in the fermention medium as seed liquor; Fermention medium with metal-salt of short gemma generation comprises glucose, Tryptones, and yeast soaks powder, ammonium sulfate, sodium hydrogencarbonate, corn starch or cereal starch or soybean starch, manganous sulfate, sal epsom, ferrous sulfate, and keep certain pH value;
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted fermentation cylinder for fermentation;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and the control spraying drying can make high-purity bacterium powder.
The fermentation culture method of the liquor fermentation substratum of clostridium butylicum of the present invention, in said step, parameter choosing value scope is:
(1) glycerine pipe refrigeration actication of culture: glycerine pipe bacterial classification 180～230 μ L that get-25～-18 ℃ of refrigerations are inoculated in the test tube that 6～12mL proliferated culture medium (RCM) is housed; On cover 1～3cm whiteruss; Substratum is sterilized in advance, leaves standstill under 33～38 ℃ of conditions to cultivate 40～50h to form gemma; It is following that said proliferated culture medium (RCM) is formed weight part: yeast extract 2～4, beef extract 8～12, Tryptones 8～12, glucose 3～8, Zulkovsky starch 0.5～2, sodium-chlor 3～8, sodium acetate trihydrate 1～6, cysteine hydrochloride 0.1～1.5,0.5% methylene blue, 0.1～3mL, zero(ppm) water 800～1200mL; Regulate pH7.1 ± 0.1,100～120 ℃, 20 min sterilization is subsequent use;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in 70～90 ℃ of water-baths handles 9～12min;
(3) the triangular flask first order seed is cultivated: above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out with 1～3% inoculum size respectively, cultivates 16-20h and can be inoculated in the fermention medium as seed liquor to logarithmic phase;
Said fermention medium with metal-salt of short gemma generation; Comprise following component: glucose 0.8～2.5% (w/v), Tryptones 0.6～2.3% (w/v), yeast soak powder 1.1～2.8% (w/v); Ammonium sulfate 0.05～0.2 % (w/v); Sodium hydrogencarbonate 0.05～0.25 % (w/v), (w/v), pH value is 7～8 for corn starch or cereal starch or soybean starch 0.2～0.45%; Manganous sulfate 0.01～0.1% (w/v), sal epsom 0.01～0.1% (w/v), ferrous sulfate 0.01～0.08% (w/v);
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted in the fermentor tank inoculum size 3%～5%, 33～40 ℃ of temperature controls; Mixing speed 45～60rpm; The PH value is reduced to below 6.5 in the fermenting process, and adding 18～25% yellow soda ash control pH value is 6.5, up to fermentation ends; And fermentation 24h adds carbon source with interior stream, and stream adds 0.55～1.5% (w/v) carbon source;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and EAT is 130～160 ℃ in the control spraying drying, and 50～75 ℃ of temperature outs can make high-purity bacterium powder.
Specific embodiments is: the fermentation culture method of the liquor fermentation substratum of described clostridium butylicum, in said step:
(1) glycerine is guaranteed the Tibetan actication of culture: the glycerine pipe bacterial classification 200 μ L that get-20 ℃ of preservations are inoculated in the test tube that 9mL proliferated culture medium (RCM) is housed; On cover 2cm whiteruss secluding air; Paraffin and substratum sterilize in advance (115 ℃ sterilization 20 minutes); Preparation four pipes leave standstill under 37 ℃ of conditions and cultivate 48h to form gemma; Said proliferated culture medium (RCM) prescription is formed as follows: yeast extract 3 g, beef extract 10 g, Tryptones 10g, glucose 5g, Zulkovsky starch 1g, sodium-chlor 5g, sodium acetate trihydrate 3g, cysteine hydrochloride 0.5g, 0.5% methylene blue 0.2mL, zero(ppm) water 1000mL; Regulate pH7.1 ± 0.1;
(2) heating is preferred: select the robust growth sample, with above-mentioned nutrient solution separately mixing be placed in 80 ℃ of water-baths and handle 10min; Heating can promote gemma to sprout, and helps the thalli growth consistence and prevents living contaminants;
(3) the triangular flask first order seed is cultivated: with 3% inoculum size (being 6ml) above-mentioned bacterium liquid is transferred to respectively and 200ml is housed had gone out in the 250ml triangular flask of substratum after the optimization of bacterium (on cover whiteruss), inoculate 3 bottles altogether, cultivate 16-20h for 37 ℃ and can be inoculated in the fermention medium as seed liquor to logarithmic phase: glucose 1.5% (w/v); Tryptones 1.3% (w/v); Yeast soaks powder 1.8% (w/v), ammonium sulfate 0.1 % (w/v), sodium hydrogencarbonate 0.124% (w/v); Corn starch 0.33% (w/v); Manganous sulfate 0.05% (w/v), sal epsom 0.05% (w/v), ferrous sulfate 0.03% (w/v); PH value 7.5;
(4) liquid fermenting: will cultivate 16h left and right sides triangular flask first order seed substratum and insert in the fermentor tank, the canned liquid coefficient 70% that ferments, on cover 2 ml left and right sides whiterusss, secluding air; Inoculum size 350 ml (3%～5%), 37 ℃ of temperature controls, mixing speed 50rpm, the PH value is reduced to below 6.5 in the fermenting process; Adding 20% yellow soda ash (massfraction) control pH value is 6.5, and up to fermentation ends, and fermentation 24h adds carbon source with interior stream; Stream adds 1% (w/v) carbon source, i.e. glucose 70g is after the 30h; Every at a distance from the dyeing of 2h microscopy, observe gemma and form more than 95%, put jar;
(5) spraying drying: fermented liquid is with the pre-treatment of clarifixator homogeneous, and EAT is 150 ℃ in the control spraying drying, and 70 ℃ of temperature outs can make high-purity bacterium powder, collects the bacterium powder and preserves.
Claim protection domain of the present invention is not limited only to the foregoing description.
Claims (11)
1. the liquor fermentation substratum of a clostridium butylicum; It is characterized in that, comprise following component: glucose 0.8～2.5% (w/v), Tryptones 0.6～2.3% (w/v); Yeast soaks powder 1.1～2.8% (w/v); Ammonium sulfate 0.05～0.2 % (w/v), sodium hydrogencarbonate 0.05～0.25 % (w/v), corn starch or cereal starch or soybean starch 0.2～0.45% (w/v); PH value is 7～8.
2. the liquor fermentation substratum of clostridium butylicum according to claim 1; It is characterized in that component is: glucose 0.8% (w/v), Tryptones 0.6% (w/v); Yeast soaks powder 1.1% (w/v); Ammonium sulfate 0.05 % (w/v), sodium hydrogencarbonate 0.05 % (w/v), corn starch or cereal starch or soybean starch 0.2% (w/v); PH value 7.
3. the liquor fermentation substratum of clostridium butylicum according to claim 1; It is characterized in that component is: glucose 2.5% (w/v), Tryptones 2.3% (w/v); Yeast soaks powder 2.8% (w/v); Ammonium sulfate 0.2 % (w/v), sodium hydrogencarbonate 0.25 % (w/v), corn starch or cereal starch or soybean starch 0.45% (w/v); PH value 8.
4. the liquor fermentation substratum of clostridium butylicum according to claim 1; It is characterized in that component is: glucose 1.5% (w/v), Tryptones 1.3% (w/v); Yeast soaks powder 1.8% (w/v); Ammonium sulfate 0.1 % (w/v), sodium hydrogencarbonate 0.12% (w/v), corn starch 0.33% (w/v); PH value 7.5.
5. a liquor fermentation substratum that is used for the described clostridium butylicum of claim 1 is urged the metal-salt that gemma generates; It is characterized in that; Comprise following component: manganous sulfate 0.01～0.1% (w/v), sal epsom 0.01～0.1% (w/v), ferrous sulfate 0.01～0.08% (w/v).
6. the metal-salt of the short gemma generation of the liquor fermentation substratum of clostridium butylicum according to claim 5 is characterized in that component is: manganous sulfate 0.05% (w/v), sal epsom 0.05% (w/v), ferrous sulfate 0.03% (w/v).
7. proliferated culture medium (RCM) that is used for the liquor fermentation substratum of the described clostridium butylicum of claim 1; It is characterized in that composition and weight part are: yeast extract 2～4, beef extract 8～12, Tryptones 8～12, glucose 3～8, Zulkovsky starch 0.5～2, sodium-chlor 3～8, sodium acetate trihydrate 1～6, cysteine hydrochloride 0.1～1.5,0.5% methylene blue, 0.1～3mL, zero(ppm) water 800～1200mL; Regulate pH7.1 ± 0.1.
8. the proliferated culture medium (RCM) of the liquor fermentation substratum of clostridium butylicum according to claim 7; It is characterized in that prescription consists of: yeast extract 3g, beef extract 10 g, Tryptones 10 g, glucose 5 g, Zulkovsky starch 1 g, sodium-chlor 5 g, sodium acetate trihydrate 3 g, cysteine hydrochloride 0.5 g, 0.5% methylene blue 0.2mL, zero(ppm) water 1000mL; Regulate pH7.1 ± 0.1.
9. the fermentation culture method of the liquor fermentation substratum of a clostridium butylicum is characterized in that, may further comprise the steps:
(1) glycerine pipe refrigeration actication of culture: get glycerine pipe refrigeration bacterial classification inoculation in the test tube that proliferated culture medium (RCM) is housed, on cover whiteruss, substratum is sterilized in advance, leaves standstill then to cultivate to form gemma;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in the water-bath handles;
(3) the triangular flask first order seed is cultivated: respectively above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out, cultivating logarithmic phase can be inoculated in the fermention medium as seed liquor again; Fermention medium with metal-salt of short gemma generation comprises glucose, Tryptones, and yeast soaks powder, ammonium sulfate, sodium hydrogencarbonate, corn starch or cereal starch or soybean starch, manganous sulfate, sal epsom, ferrous sulfate, and keep certain pH value;
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted fermentation cylinder for fermentation;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and the control spraying drying can make high-purity bacterium powder.
10. the fermentation culture method of the liquor fermentation substratum of clostridium butylicum according to claim 9 is characterized in that, in said step:
(1) glycerine pipe refrigeration actication of culture: glycerine pipe bacterial classification 180～230 μ L that get-25～-18 ℃ of refrigerations are inoculated in the test tube that 6～12mL proliferated culture medium (RCM) is housed; On cover 1～3cm whiteruss; Substratum is sterilized in advance, leaves standstill under 33～38 ℃ of conditions to cultivate 40～50h to form gemma; Said proliferated culture medium (RCM) is formed and weight part is: yeast extract 2～4, beef extract 8～12, Tryptones 8～12, glucose 3～8, Zulkovsky starch 0.5～2, sodium-chlor 3～8, sodium acetate trihydrate 1～6, cysteine hydrochloride 0.1～1.5,0.5% methylene blue, 0.1～3mL, zero(ppm) water 800～1200mL; Regulate pH7.1 ± 0.1,100～120 ℃, 20 min sterilization is subsequent use;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in 70～90 ℃ of water-baths handles 9～12min;
(3) the triangular flask first order seed is cultivated: above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out with 1～3% inoculum size respectively, cultivates 16-20h and can be inoculated in the fermention medium as seed liquor to logarithmic phase;
Said fermention medium with metal-salt of short gemma generation; Component is: glucose 0.8～2.5% (w/v); Tryptones 0.6～2.3% (w/v), yeast soak powder 1.1～2.8% (w/v), ammonium sulfate 0.05～0.2 % (w/v); Sodium hydrogencarbonate 0.05～0.25 % (w/v), and corn starch or cereal starch or soybean starch 0.2～0.45% (w/v); PH value is 7～8; Manganous sulfate 0.01～0.1% (w/v), sal epsom 0.01～0.1% (w/v), ferrous sulfate 0.01～0.08% (w/v);
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted in the fermentor tank inoculum size 3%～5%, 33～40 ℃ of temperature controls; Mixing speed 45～60rpm; The PH value is reduced to below 6.5 in the fermenting process, and adding 18～25% yellow soda ash control pH value is 6.5, up to fermentation ends; And fermentation 24h adds carbon source with interior stream, and stream adds 0.55～1.5% (w/v) carbon source;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and EAT is 130～160 ℃ in the control spraying drying, and 50～75 ℃ of temperature outs can make high-purity bacterium powder.
11. the fermentation culture method according to the liquor fermentation substratum of claim 9 or 10 described clostridium butylicums is characterized in that, in said step:
(1) glycerine is guaranteed the Tibetan actication of culture: the glycerine pipe bacterial classification 200 μ L that get-20 ℃ of preservations are inoculated in the test tube that 9mL proliferated culture medium (RCM) is housed, on cover the 2cm whiteruss, substratum is sterilized in advance, leaves standstill under 37 ℃ of conditions to cultivate 48h to form gemma; Said proliferated culture medium (RCM) prescription is formed as follows: yeast extract 3 g, beef extract 10 g, Tryptones 10g, glucose 5g, Zulkovsky starch 1g, sodium-chlor 5g, sodium acetate trihydrate 3g, cysteine hydrochloride 0.5g, 0.5% methylene blue 0.2mL, zero(ppm) water 1000mL; Regulate pH7.1 ± 0.1,115 ℃, 20 min sterilization is subsequent use;
(2) heating is preferred: above-mentioned nutrient solution mixing is placed in 80 ℃ of water-baths handles 10min; Heating can promote gemma to sprout, and helps the thalli growth consistence and prevents living contaminants;
(3) the triangular flask first order seed is cultivated: above-mentioned bacterium liquid is transferred in the triangular flask of substratum after the optimization of the bacterium of going out with 1～3% inoculum size respectively again; Cultivating 16-20h can be inoculated in the fermention medium as seed liquor to logarithmic phase: glucose 1.5% (w/v), and Tryptones 1.3% (w/v), yeast soak powder 1.8% (w/v); Ammonium sulfate 0.1 % (w/v); Sodium hydrogencarbonate 0.124% (w/v), and corn starch 0.33% (w/v), manganous sulfate 0.05% (w/v); Sal epsom 0.05% (w/v), ferrous sulfate 0.03% (w/v); PH value 7.5;
(4) liquid fermenting: cultured triangular flask first order seed substratum is inserted in the fermentor tank inoculum size 3%～5%, 37 ℃ of temperature controls; Mixing speed 50rpm; The PH value is reduced to below 6.5 in the fermenting process, and adding 20% yellow soda ash control pH value is 6.5, up to fermentation ends; And fermentation 24h adds carbon source with interior stream, and stream adds 1% (w/v) carbon source;
(5) spraying drying: fermented liquid is used the clarifixator homogeneous, and EAT is 150 ℃ in the control spraying drying, and 70 ℃ of temperature outs can make high-purity bacterium powder.
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