CN103255085A - Preparation method of concentrated caproic acid bacterial liquid - Google Patents
Preparation method of concentrated caproic acid bacterial liquid Download PDFInfo
- Publication number
- CN103255085A CN103255085A CN2013101456271A CN201310145627A CN103255085A CN 103255085 A CN103255085 A CN 103255085A CN 2013101456271 A CN2013101456271 A CN 2013101456271A CN 201310145627 A CN201310145627 A CN 201310145627A CN 103255085 A CN103255085 A CN 103255085A
- Authority
- CN
- China
- Prior art keywords
- caproic acid
- preparation
- acid bacteria
- liquid
- adds
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention relates to a preparation method of concentrated caproic acid bacterial liquid, which comprises the following steps of: (1) inoculating caproic acid bacteria into a strain medium of the caproic acid bacteria, and performing activation culture to obtain seed liquid; (2) inoculating the seed liquid into a fermentation medium with introduced CO2, and performing fermentation culture to obtain fermentation liquid; and (3) filtering and concentrating the fermentation liquid through a 0.1-micron microporous membrane, and removing the filtrate to obtain the concentrated caproic acid bacterial liquid. The concentrated caproic acid bacterial liquid prepared by the method provided by the invention can be used for culturing pit mud, producing high-activity dehydrated pit mud functional bacteria and maintaining the pit; and moreover, since the bacterial concentration is 5-6 times higher than that in the fermentation liquid, the concentrated caproic acid bacterial liquid is convenient to transport as goods, and the transportation cost is reduced.
Description
Technical field
The present invention relates to a kind of preparation method of concentrated caproic acid bacteria solution, particularly a kind of method of utilizing microorganism culturing, separating concentration technique production caproic acid bacteria viable bacteria body concentrated solution belongs to fermentation engineering and microbial technology field.
Background technology
Caproic acid bacteria is a kind of clostridium, and that the earliest caproic acid fungas is studied is American Bei Qiangpu (Bechamp).Bark people such as (BarKer) in 1936 has found product caproic acid phenomenon again when separation and purification Ovshinsky methagen from black mud, and is separated to the clostridium that produces caproic acid, i.e. the carat Wa Shi shuttle shape sporeformer of name afterwards.Caproic acid bacteria is one of key factor that influences the aromatic Chinese spirit local flavor, the caproic acid bacteria fermentation produces caproic acid, caproic acid is ethyl hexanoate by other microbial transformations again, and ethyl hexanoate is the main aroma composition of aromatic Chinese spirit, is playing an important role aspect the quality of aroma daqu liquor.
Therefore, strengthening concentration and the metabolic activity thereof of caproic acid bacteria in the mud of cellar for storing things, improve it and produce the caproic acid amount, is the important channel of improving the aromatic Chinese spirit quality.
Prior art is cultivated in the enrichment culture of caproic acid bacteria and separation and purification, generally uses traditional Pasteur's substratum.The main component of Pasteur's substratum:
Ethanol, sodium acetate, ammonium sulfate, yeast extract powder, dipotassium hydrogen phosphate, calcium carbonate, sal epsom, calcium sulfate, ferrous sulfate, vitamin H, para-amino benzoic acid, distilled water etc.Caproic acid bacteria can utilize composition growth and breeding and synthesizing hexanoic acids such as ethanol in the substratum, sodium acetate, yeast extract powder.Vitamins such as VitB1, vitamin H is the growth important factor of caproic acid bacteria, and Nucleotide and amino acid play an important role in caproic acid bacteria growth and biosynthesizing caproic acid.
Chinese patent literature CN102260640A(application number 201110194432.7) cultural method that a kind of caproic acid bacteria and liquid are stored the mud mixed bacteria liquid is disclosed, this method at first adopts caproic acid bacteria substratum and liquid cellar for storing things mud substratum that caproic acid bacteria and liquid cellar for storing things mud bacterial classification are cultivated respectively, store mud bacterial classification inoculation Ka Shi jar, stainless steel seeding tank and stainless steel fermentor tank with 1% caproic acid bacterial classification, 9% liquid successively afterwards, thereby make caproic acid bacteria and liquid cellar for storing things mud mixed bacteria liquid.
Chinese patent literature CN102260639A(application number 201110194430.8) a kind of cellar for storing things mud special life fragrant function yeast liquid cultural method and substratum thereof are disclosed, each component and weight percent thereof that this substratum comprises are: lose poor immersion liquid: 3～5%, quality pit mud: 8～10%, the bottom unstrained spirits immersion liquid that is pickled with grains or in wine: 10%, K2HPO4:0.2～0.5%, NaAC:0.1～0.3%, MgSO4:0.005～0.01%, 95% alcohol: 2～4%, yellow water: 5～10%.
But these compositions mainly are the chemical industry series products, and the cost of producing caproic acid bacteria is higher, and the caproic acid bacteria poor growth, thereby have limited the output of liquor.
Summary of the invention
The present invention is directed to the deficiencies in the prior art, a kind of preparation method of concentrated caproic acid bacteria solution is provided, utilize this method to produce the caproic acid bacteria concentrated solution and can enhance productivity, improve advantages such as the concentration of somatic cells in the product, fermented liquid recycling.
The term explanation:
Ventilation: the vvm of unit refers to the volume ratio of the amount of gas volume that per minute feeds and fermented liquid.
Cell concn: refer in the fermented liquid or the caproic acid bacteria concentrated solution in the cell content of caproic acid bacteria.
Technical scheme of the present invention is as follows:
A kind of preparation method of concentrated caproic acid bacteria solution comprises the steps:
(1) caproic acid bacteria (Clostridium kluyveri) is inoculated in the caproic acid bacteria bacterium culture medium, 33～35 ℃ of activation culture 5～7 days, makes seed liquor;
(2) by volume the ratio of per-cent 5%～10% seed liquor that step (1) is made is inoculated in and feeds CO
2Fermention medium in, cultivated 6～8 days at 33～35 ℃ condition bottom fermentations, making cell concn is 1.0～1.5 * 10
8CFU/mL, caproic acid concentration are the fermented liquid of 5.5～9.4g/L;
(3) fermented liquid that step (2) is made concentrates through 0.1 μ m micro-pore-film filtration, except filtrate, makes concentrated caproic acid bacteria solution;
Fermention medium component in the described step (2) is as follows:
Sodium acetate 5～8g/L, zinc chloride 0.01～0.02g/L, potassium primary phosphate 0.2～0.4g/L, wheat bran ethanol fermentation stoste 200～280mL/L, Zein powder hydrolyzed solution 20～60mL/L, water is settled to 1L, pH6.5～7.0;
Above-mentioned wheat bran ethanol fermentation stoste prepares as follows:
The water that adds 3.5～4 times 85～90 ℃ in the Testa Tritici, Testa Tritici is with dry weight basis, add α-Dian Fenmei, after once being incubated enzymolysis, be cooled to 62～63 ℃, add saccharifying enzyme and Pullulanase, behind the secondary insulation enzymolysis, make enzymolysis solution, in enzymolysis solution, add aspartic protease, be cooled to 28～30 ℃, add high temperature resistant active dry yeast, heat-preservation fermentation, the waste molasses that adds gross weight 16～18% again continues heat-preservation fermentation, removes the gred through solid-liquid separation, behind the high-temperature sterilization, make wheat bran ethanol fermentation stoste;
Wheat bran is cheap and contain abundant VITAMIN, mineral substance growth has promoter action to caproic acid bacteria, thereby can save yeast powder or add the cost of trace element; And the wheat bran ethanol fermentation stoste behind yeast fermentation contains the ethanol of concentration 11.3～12.5ml/L, is conducive to the preservation of ethanol fermentation stoste; Simultaneously, the yeast fermentation process has produced vitamin substances, and these materials are high-quality nutritive ingredients of caproic acid bacteria growth.
Above-mentioned Zein powder hydrolyzed solution prepares as follows:
The water that adds 6～7 times 45～55 ℃ in the Zein powder, Zein powder is with dry weight basis, stir adding sulfuric acid, make the mass concentration of sulfuric acid reach 0.15～0.20%, be warming up to 105～110 ℃, insulation hydrolysis 3～4 hours, be cooled to 45～55 ℃, transfer pH6.0～6.5, remove the gred through solid-liquid separation, behind the high-temperature sterilization, make the Zein powder hydrolyzed solution.
Zein powder is the tankage of corn processed, and cheapness, and contain protein more than 60% becomes amino acid or little peptide can directly be utilized by caproic acid bacteria through the dilute sulphuric acid hydrolysis, thereby accelerates the caproic acid bacteria speed of growth and cell viability.
Preferred according to the present invention, in the described wheat bran ethanol fermentation stoste preparation process, the addition of α-Dian Fenmei is that every gram Testa Tritici adds 8～12U(unit of activity) α-Dian Fenmei, once being incubated enzymolysis time is 35～45min;
The addition of described saccharifying enzyme is that every gram Testa Tritici adds 115～125U(unit of activity) saccharifying enzyme, the addition of Pullulanase is that every gram Testa Tritici adds 4～6U(unit of activity) Pullulanase, secondary insulation enzymolysis time is 85～95min;
The addition of described aspartic protease is that every gram Testa Tritici adds 8～12U(unit of activity) aspartic protease;
The addition of described high temperature resistant active dry yeast is to add the high temperature resistant active dry yeast of 0.1～0.12g in every liter of enzymolysis solution; The heat-preservation fermentation time is 22～26h, and adding the follow-up continuation of insurance temperature of molasses fermentation time is 22～26h.
Above-mentioned enzyme and yeast are common commercially available prod, and those skilled in the art can select according to the function needs.
Preferred according to the present invention, described solid-liquid separation slagging-off all adopts Plate Filtration to carry out the solid-liquid separation slagging-off.
Preferred according to the present invention, described high-temperature sterilization is all at 103～108 ℃ of sterilization 30min.
Preferred according to the present invention, in the described Zein powder hydrolyzed solution preparation process, the pH regulator agent is ammoniacal liquor.
Preferred according to the present invention, every liter of component of described step (1) caproic acid bacteria bacterium culture medium is as follows:
Ethanol 20mL, sodium acetate 6g, ammonium sulfate 400～500mg, yeast extract powder 4～5g, adding distil water be to 1000mL, pH6.5～7.0.
Preferred according to the present invention, feed CO in the described step (2)
2Fermention medium be to make to fermention medium ventilation 10～15min with the ventilation of 0.1～0.2vvm.
Preferred according to the present invention, the feed pressure of 0.1 μ m micro-pore-film filtration is 0.2Mpa in the described step (3).This step can be with cell concentration from 1.0～1.5 * 10
8CFU/mL is concentrated to 5.0～7.5 * 10
8CFU/mL.
Preferred according to the present invention, the caproic acid bacteria in the described step (1) (Clostridium kluyveri) is available from Chinese industrial microbial strains preservation administrative center (CICC), and preserving number is 8023.
Preferred according to the present invention, also comprise the clear liquid after the distillation of filtrate in the step (3) through making behind distillation removal caproic acid, the butyric acid, mix the back with wheat bran ethanol fermentation stoste, Zein powder hydrolyzed solution, potassium primary phosphate and water and reuse as fermention medium;
Further preferred, above-mentioned fermention medium component is as follows, is mass percent:
Clear liquid 50～70% after the distillation, wheat bran ethanol fermentation stoste 18～22%, Zein powder hydrolyzed solution 2～4%, potassium primary phosphate 0.01%, the remainder water replenishes.
With from fermented liquid, the separating of compositions such as caproic acid, butyric acid, remove these compositions to the inhibition of caproic acid bacteria by distillation, be conducive to the fermented liquid reuse.
Beneficial effect
1, the concentrated caproic acid bacteria solution that makes of the present invention, can be used for cellar for storing things mud cultivation, produce highly active dehydration pit mud functional bacteria, maintenance Jiao Chi, and because cell concentration is high 5～6 times than cell concentration in the fermented liquid, be convenient to as the transportation of goods, reduce transportation cost.
2, the present invention is by wheat bran ethanol fermentation stoste that the wheat bran enzymic hydrolysis is made by yeast fermentation and the Zein powder hydrolyzed solution after the Zein powder hydrolysis main fermentation raw material as caproic acid bacteria, unnecessary employing straight alcohol, yeast extract powder, other nutritive salt etc., not only culture effect is better than existing synthetic medium, and has reduced production cost significantly;
3, adopt the caproic acid bacteria solution of membrane concentration technology production high density, than centrifugation, flocculation sediment traditional technology, save energy consumption, reduce the opportunities for contamination of caproic acid bacteria solution; And because caproic acid bacteria is anaerobion, membrane separation technique has effectively been isolated thalline and air touch opportunity;
4, can recycle through the membrane concentration filtrate filtered, thus blowdown, conservation reduced.
But 5, the present invention's biological fermentation synthesizing hexanoic acid and being applied in the foodstuff additive in Caproic Acid Bacteria Culture, it is safer to produce caproic acid than chemical synthesis.
Embodiment
Below in conjunction with embodiment technical scheme of the present invention is further elaborated, but these embodiment must not be used for explaining limiting the scope of the invention.
Raw material explanation `
Wheat bran is opened summer Jin Zhuan flour mill available from Jinan City's Changqing District; Main nutrient composition is as follows after measured: protein 16.2wt%, starch 32.0wt%, moisture 13wt%, ash content 6.3wt%, fatty 3.88wt%, vitamin H 210 μ g/kg.
Zein powder is available from Shandong Xiwang Group Co., Ltd., and main nutrient composition is as follows after measured:
Protein 61.2wt%, total reducing sugar 20.0wt%, moisture 11.4wt%, ash content 4.6wt%, other 2.8wt%.
α-Dian Fenmei is available from the grand mcroorganism Engineering Co., Ltd in Shandong; 90 ℃ of the suitableeest enzyme temperature alive, enzyme activity 20000U/mL
Saccharifying enzyme (originating from rhizopus) is available from the grand mcroorganism Engineering Co., Ltd in Shandong; 58～60 ℃ of the suitableeest enzyme temperature alive, the work of every gram enzyme is 100,000 U.
Pullulanase is available from the grand mcroorganism Engineering Co., Ltd in Shandong; 58～60 ℃ of the suitableeest enzyme temperature alive, the work of every gram enzyme is 20,000 U.
Aspartic protease is available from the grand mcroorganism Engineering Co., Ltd in Shandong; 45 ℃ of the suitableeest enzyme temperature alive, the work of every gram enzyme is 50,000 U.
High temperature resistant active dry yeast is available from Angel Yeast Co.,Ltd;
Molasses after testing, contain the total reducing sugar of 65wt% available from Shandong Xiwang Group Co., Ltd., and main component is glucose.
Embodiment 1
A kind of preparation method of concentrated caproic acid bacteria solution comprises the steps:
(1) caproic acid bacteria (Clostridium kluyveri) is inoculated in the caproic acid bacteria bacterium culture medium, 35 ℃ of activation culture 5 days, makes seed liquor;
(2) by volume the ratio of per-cent 5% seed liquor that step (1) is made is inoculated in and feeds CO
2Fermention medium in, CO
2Ventilation is 0.1vvm, and aeration time is 15min, cultivates 6 days at 35 ℃ condition bottom fermentations, and making cell concn is 1.5 * 10
8CFU/mL, caproic acid concentration are the fermented liquid of 5.8g/L;
(3) fermented liquid that step (2) is made concentrates through 0.1 μ m micro-pore-film filtration, feed pressure 0.2Mpa, input speed 3m
3/ h except filtrate, makes concentrated caproic acid bacteria solution; After testing, cell concn is 5.0 * 10
8CFU/mL.
Every liter of component of described step (1) caproic acid bacteria bacterium culture medium is as follows:
Ethanol 20mL, sodium acetate 6g, ammonium sulfate 500mg, yeast extract powder 4g, adding distil water be to 1000mL, pH6.5; Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Fermention medium component in the described step (2) is as follows:
Sodium acetate 5g/L, zinc chloride 0.01g/L, potassium primary phosphate 0.4g/L, wheat bran ethanol fermentation stoste 200mL/L, Zein powder hydrolyzed solution 20mL/L, water is settled to 1L, pH6.8.Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Above-mentioned wheat bran ethanol fermentation stoste prepares as follows:
With 1 ton in wheat bran (with dry weight basis), press the weight ratio of 1:4 and 90 ℃ water 4t mixing and stirring; Add α-Dian Fenmei, add α-Dian Fenmei 1.0 * 10
7U unit of enzyme (being equivalent to 100g), insulation 40min is cooled to 62～63 ℃, adds saccharifying enzyme 1.2 * 10
8U unit of enzyme (being equivalent to 1.2kg) adds Pullulanase 4.0 * 10
6The U unit of enzyme, insulation 90min adds aspartic protease 1.0 * 10
7The U unit of enzyme; Be cooled to 28～30 ℃ then, add high temperature resistant active dry yeast 500g(in advance with 3L, 40 ℃ of warm water activation 20min), heat-preservation fermentation 24h adds molasses 0.8t, continues 32～33 ℃ of fermentations of insulation 24 hours, and the fermented wine precision reaches: 11.3mL/L.Plate Filtration stays clear liquid, and 103～108 ℃ of sealing sterilizations make wheat bran ethanol fermentation stoste;
Above-mentioned Zein powder hydrolyzed solution prepares as follows:
Get Zein powder 100kg, add 55 ℃ hot water 600L, in the pyroreaction still, stir; Adding dilute sulphuric acid to concentration is 0.15wt%, is heated to 105 ℃, and insulation hydrolysis 4 hours is cooled to 54～55 ℃, transfers pH6.0～6.5 with weak ammonia, and Plate Filtration stays clear liquid, makes the Zein powder hydrolyzed solution.
The filtrate that step (3) makes is contained 9mL/L ethanol after testing, and the 5.8g/L caproic acid through the indirect steam distillation, can obtain the mixed fraction of ethanol, caproic acid and other volatile matter, the clear liquid after obtaining simultaneously distilling.After testing, the main effective constituent (wt%) of mixed fraction is as follows: ethanol 12.1%, caproic acid 5.2%, butyric acid 1.3%, acetic acid 0.3%.
Embodiment 2
A kind of preparation method of concentrated caproic acid bacteria solution comprises the steps:
(1) caproic acid bacteria (Clostridium kluyveri) is inoculated in the caproic acid bacteria bacterium culture medium, 35 ℃ of activation culture 5 days, makes seed liquor;
(2) by volume the ratio of per-cent 10% seed liquor that step (1) is made is inoculated in and feeds CO
2Fermention medium in, CO
2Ventilation is 0.1vvm, and aeration time is 10min, cultivates 7 days at 33 ℃ condition bottom fermentations, and making cell concn is 1.6 * 10
8CFU/mL, caproic acid concentration are the fermented liquid of 6.7g/L;
(3) fermented liquid that step (2) is made concentrates through 0.1 μ m micro-pore-film filtration, feed pressure 0.25Mpa, input speed 3m
3/ h except filtrate, makes concentrated caproic acid bacteria solution; After testing, cell concn is 6.6 * 10
8CFU/mL.
Every liter of component of described step (1) caproic acid bacteria bacterium culture medium is as follows:
Ethanol 20mL, sodium acetate 6g, ammonium sulfate 500mg, yeast extract powder 4g, adding distil water be to 1000mL, pH6.5; Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Fermention medium component in the described step (2) is as follows, all is weight percentage:
Clear liquid 70% after the distillation that embodiment 1 makes, wheat bran ethanol fermentation stoste 18%, Zein powder hydrolyzed solution 4%, potassium primary phosphate 0.01%, excess water, pH6.8.Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Above-mentioned wheat bran ethanol fermentation stoste prepares as follows:
With 1 ton in wheat bran (with dry weight basis), press the weight ratio of 1:3.5 and 3.5 tons of mixing and stirring of water of 90 ℃; Add α-Dian Fenmei, add α-Dian Fenmei 1.0 * 10
7U unit of enzyme (being equivalent to 100g), insulation 40min is cooled to 62～63 ℃, adds saccharifying enzyme 1.2 * 10
8U unit of enzyme (being equivalent to 1.2kg) adds Pullulanase 4.0 * 10
6The U unit of enzyme, insulation 90min adds aspartic protease 1.0 * 10
7The U unit of enzyme; Be cooled to 28～30 ℃ then, add high temperature resistant active dry yeast 600g(in advance with 3L40 ℃ of warm water activation 20min), heat-preservation fermentation 24h adds molasses 0.85t, continues 32～33 ℃ of fermentations of insulation 24 hours, and the fermented wine precision reaches: 12.5mL/L.Plate Filtration stays clear liquid, and 103～108 ℃ of sealing sterilizations make wheat bran ethanol fermentation stoste;
Above-mentioned Zein powder hydrolyzed solution prepares as follows:
Get Zein powder 100kg, add 45 ℃ hot water 600L, in the pyroreaction still, stir; Adding dilute sulphuric acid to concentration is 0.20wt%, is heated to 105 ℃, and insulation hydrolysis 3 hours is cooled to 45～46 ℃, transfers pH6.0～6.5 with weak ammonia, and Plate Filtration stays clear liquid, makes the Zein powder hydrolyzed solution.
The filtrate that step (3) makes is contained 11mL/L ethanol after testing, and the 6.7g/L caproic acid through the indirect steam distillation, can obtain the mixed fraction of ethanol, caproic acid and other volatile matter.After testing, the main effective constituent (wt%) of mixed fraction is as follows: ethanol 12.4%, caproic acid 5.5%, butyric acid 1.34%, acetic acid 0.33%.
Embodiment 3
A kind of preparation method of concentrated caproic acid bacteria solution comprises the steps:
(1) caproic acid bacteria (Clostridium kluyveri) is inoculated in the caproic acid bacteria bacterium culture medium, 35 ℃ of activation culture 5 days, makes seed liquor;
(2) by volume the ratio of per-cent 8% seed liquor that step (1) is made is inoculated in and feeds CO
2Fermention medium in, CO
2Ventilation is 0.2vvm, and aeration time is 10min, cultivates 8 days at 34 ℃ condition bottom fermentations, and making cell concn is 1.7 * 10
8CFU/mL, caproic acid concentration are the fermented liquid of 8.7g/L;
(3) fermented liquid that step (2) is made concentrates through 0.1 μ m micro-pore-film filtration, feed pressure 0.25Mpa, input speed 3m
3/ h except filtrate, makes concentrated caproic acid bacteria solution; After testing, cell concn is 7.0 * 10
8CFU/mL.
Every liter of component of described step (1) caproic acid bacteria bacterium culture medium is as follows:
Ethanol 20mL, sodium acetate 6g, ammonium sulfate 400mg, yeast extract powder 5g, adding distil water be to 1000mL, pH6.5; Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Fermention medium component in the described step (2) is as follows, all is weight percentage:
Clear liquid 50% after the distillation that embodiment 1 makes, wheat bran ethanol fermentation stoste 22%, Zein powder hydrolyzed solution 3%, potassium primary phosphate 0.01%, excess water, pH6.8.Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Above-mentioned wheat bran ethanol fermentation stoste prepares as follows:
With 1 ton in wheat bran (with dry weight basis), press the weight ratio of 1:3.7 and 90 ℃ water 3.7t mixing and stirring; Add α-Dian Fenmei, add α-Dian Fenmei 1.0 * 10
7U unit of enzyme (being equivalent to 100g), insulation 40min is cooled to 62～63 ℃, adds saccharifying enzyme 1.2 * 10
8U unit of enzyme (being equivalent to 1.2kg) adds Pullulanase 4.0 * 10
6The U unit of enzyme, insulation 90min adds aspartic protease 1.0 * 10
7The U unit of enzyme; Be cooled to 28～30 ℃ then, add high temperature resistant active dry yeast 600g(in advance with 3L, 40 ℃ of warm water activation 20min), heat-preservation fermentation 24h adds molasses 0.9t, continues 32～33 ℃ of fermentations of insulation 24 hours, and the fermented wine precision reaches: 12.3mL/L.Plate Filtration stays clear liquid, and 103～108 ℃ of sealing sterilizations make wheat bran ethanol fermentation stoste;
Above-mentioned Zein powder hydrolyzed solution prepares as follows:
Get Zein powder 100kg, add 55 ℃ hot water 700L, in the pyroreaction still, stir; Adding dilute sulphuric acid to concentration is 0.17wt%, is heated to 105 ℃, and insulation hydrolysis 3.5 hours is cooled to 50 ℃, transfers pH6.0～6.5 with weak ammonia, and Plate Filtration stays clear liquid, makes the Zein powder hydrolyzed solution.
The filtrate that step (3) makes is contained 10mL/L ethanol after testing, and the 7.9g/L caproic acid through the indirect steam distillation, can obtain the mixed fraction of ethanol, caproic acid and other volatile matter.After testing, the main effective constituent (wt%) of mixed fraction is as follows: ethanol 11.1%, caproic acid 4.3%, butyric acid 1.30%, acetic acid 0.41%.
Embodiment 4
A kind of preparation method of concentrated caproic acid bacteria solution comprises the steps:
(1) caproic acid bacteria (Clostridium kluyveri) is inoculated in the caproic acid bacteria bacterium culture medium, 35 ℃ of activation culture 5 days, makes seed liquor;
(2) by volume the ratio of per-cent 8% seed liquor that step (1) is made is inoculated in and feeds CO
2Fermention medium in, CO
2Ventilation is 0.2vvm, and aeration time is 10min, cultivates 8 days at 34 ℃ condition bottom fermentations, and making cell concn is 1.75 * 10
8CFU/mL, caproic acid concentration are the fermented liquid of 9.2g/L;
(3) fermented liquid that step (2) is made concentrates through 0.1 μ m micro-pore-film filtration, feed pressure 0.25Mpa, input speed 3m
3/ h except filtrate, makes concentrated caproic acid bacteria solution; After testing, cell concn is 7.5 * 10
8CFU/mL.
Every liter of component of described step (1) caproic acid bacteria bacterium culture medium is as follows:
Ethanol 20mL, sodium acetate 6g, ammonium sulfate 400mg, yeast extract powder 5g, adding distil water be to 1000mL, pH6.5; Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Fermention medium component in the described step (2) is as follows, all is weight percentage:
Clear liquid 55% after the distillation that embodiment 1 makes, wheat bran ethanol fermentation stoste 18%, Zein powder hydrolyzed solution 2.5%, potassium primary phosphate 0.01%, excess water, pH6.5.Above-mentioned substratum is heated to 105 ℃ of sealing sterilization 40min and can uses.
Above-mentioned wheat bran ethanol fermentation stoste prepares as follows:
With 1 ton in wheat bran (with dry weight basis), press the weight ratio of 1:3.6 and 90 ℃ water 3.6t mixing and stirring; Add α-Dian Fenmei, add α-Dian Fenmei 1.0 * 10
7U unit of enzyme (being equivalent to 100g), insulation 40min is cooled to 62～63 ℃, adds saccharifying enzyme 1.2 * 10
8U unit of enzyme (being equivalent to 1.2kg) adds Pullulanase 4.0 * 10
6The U unit of enzyme, insulation 90min adds aspartic protease 1.0 * 10
7The U unit of enzyme; Be cooled to 28～30 ℃ then, add high temperature resistant active dry yeast 550g(in advance with 3L40 ℃ of warm water activation 20min), heat-preservation fermentation 24h adds molasses 0.9t, continues 32～33 ℃ of fermentations of insulation 24 hours, and the fermented wine precision reaches: 12.5mL/L.Plate Filtration stays clear liquid, and 103～108 ℃ of sealing sterilizations make wheat bran ethanol fermentation stoste;
Above-mentioned Zein powder hydrolyzed solution prepares as follows:
Get Zein powder 100kg, add 55 ℃ hot water 650L, in the pyroreaction still, stir; Adding dilute sulphuric acid to concentration is 0.16wt%, is heated to 107 ℃, and insulation hydrolysis 3.5 hours is cooled to 53 ℃, transfers pH6.0～6.5 with weak ammonia, and Plate Filtration stays clear liquid, makes the Zein powder hydrolyzed solution.
The filtrate that step (3) makes is contained 10mL/L ethanol after testing, and the 9.2g/L caproic acid through the indirect steam distillation, can obtain the mixed fraction of ethanol, caproic acid and other volatile matter.After testing, the main effective constituent (wt%) of mixed fraction is as follows: ethanol 10.2%, caproic acid 6.3%, butyric acid 1.60%, acetic acid 0.48%.
Comparative Examples
According to Chinese patent literature CN102260640A(application number 201110194432.7) in the method for embodiment 1 record make caproic acid bacteria bacterium liquid, after testing, cell concn is 1.2 * 10
8CFU/mL, caproic acid concentration is 3.5g/L.
The test example
Get the concentrated caproic acid bacteria solution that embodiment 4 makes, after testing, concentrate the caproic acid bacteria main component: caproic acid bacteria sum (in anaerobic spore-bearing bacilli) is 7.5 * 10
8CFU//mL, total solid concentration is: 55.0g/L; Reducing sugar (with glucose meter) is 2.8g/L; Total amino acid is 1.1g/L; Caproic acid content is: 9.2g/L; Intestinal bacteria do not detect.
To concentrate caproic acid bacteria solution and preserve 7d in normal temperature (20 ℃) 25L food grade plastic bucket, after testing, caproic acid bacteria viable bacteria body is: 7.1 * 10
8CFU//mL; Continue to preserve 30d under the same conditions, caproic acid bacteria viable bacteria body is: 6.5 * 10
8CFU//mL.
Comparative analysis
The present invention is by concentrating fermented liquid feed pressure 0.25Mpa, input speed 3m through 0.1 μ m micro-pore-film filtration
3/ h, membrane filtration concentrates, and makes concentrated caproic acid bacteria solution, and cell concn is 5.0～7.5 * 10
8Between the CFU/mL, can find by contrast that the concentrated caproic acid bacteria solution that the present invention makes is higher than cell concentration in the bacterium liquid that Comparative Examples obtains, also be higher than the cell concentration of at present existing similar bacterium liquid.The concentrated caproic acid bacteria solution that utilizes the present invention to obtain simultaneously contains the concentration of caproic acid and concentration, the kind of other nutritive ingredients and all is higher than present prior art.These indexs are conducive to transportation, storage and the result of use of caproic acid bacteria solution.
Claims (10)
1. the preparation method of a concentrated caproic acid bacteria solution is characterized in that, comprises the steps:
(1) caproic acid bacteria (Clostridium kluyveri) is inoculated in the caproic acid bacteria bacterium culture medium, 33～35 ℃ of activation culture 5～7 days, makes seed liquor;
(2) by volume the ratio of per-cent 5%～10% seed liquor that step (1) is made is inoculated in and feeds CO
2Fermention medium in, cultivated 6～8 days at 33～35 ℃ condition bottom fermentations, making cell concn is 1.0～1.5 * 10
8CFU/mL, caproic acid concentration are the fermented liquid of 5.5～9.4g/L;
(3) fermented liquid that step (2) is made concentrates through 0.1 μ m micro-pore-film filtration, except filtrate, makes concentrated caproic acid bacteria solution;
Fermention medium component in the described step (2) is as follows:
Sodium acetate 5～8g/L, zinc chloride 0.01～0.02g/L, potassium primary phosphate 0.2～0.4g/L, wheat bran ethanol fermentation stoste 200～280mL/L, Zein powder hydrolyzed solution 20～60mL/L, water is settled to 1L, pH6.5～7.0;
Above-mentioned wheat bran ethanol fermentation stoste prepares as follows:
The water that adds 3.5～4 times 85～90 ℃ in the Testa Tritici, Testa Tritici is with dry weight basis, add α-Dian Fenmei, after once being incubated enzymolysis, be cooled to 62～63 ℃, add saccharifying enzyme and Pullulanase, behind the secondary insulation enzymolysis, make enzymolysis solution, in enzymolysis solution, add aspartic protease, be cooled to 28～30 ℃, add high temperature resistant active dry yeast, heat-preservation fermentation, the waste molasses that adds gross weight 16～18% again continues heat-preservation fermentation, removes the gred through solid-liquid separation, behind the high-temperature sterilization, make wheat bran ethanol fermentation stoste;
Above-mentioned Zein powder hydrolyzed solution prepares as follows:
The water that adds 6～7 times 45～55 ℃ in the Zein powder, Zein powder is with dry weight basis, stir adding sulfuric acid, make the mass concentration of sulfuric acid reach 0.15～0.20%, be warming up to 105～110 ℃, insulation hydrolysis 3～4 hours, be cooled to 45～55 ℃, transfer pH6.0～6.5, remove the gred through solid-liquid separation, behind the high-temperature sterilization, make the Zein powder hydrolyzed solution.
2. preparation method as claimed in claim 1 is characterized in that, in the described wheat bran ethanol fermentation stoste preparation process, the addition of α-Dian Fenmei is that every gram Testa Tritici adds 8～12U α-Dian Fenmei, and once being incubated enzymolysis time is 35～45min;
The addition of described saccharifying enzyme is that every gram Testa Tritici adds 115～125U saccharifying enzyme, and the addition of Pullulanase is that every gram Testa Tritici adds 4～6U Pullulanase, and secondary insulation enzymolysis time is 85～95min;
The addition of described aspartic protease is that every gram Testa Tritici adds 8～12U aspartic protease;
The addition of described high temperature resistant active dry yeast is to add the high temperature resistant active dry yeast of 0.1～0.12g in every liter of enzymolysis solution; The heat-preservation fermentation time is 22～26h, and continuing the heat-preservation fermentation time is 22～26h.
3. preparation method as claimed in claim 1 is characterized in that, described solid-liquid separation slagging-off all adopts Plate Filtration to carry out the solid-liquid separation slagging-off.
4. preparation method as claimed in claim 1 is characterized in that, described high-temperature sterilization is all at 103～108 ℃ of sterilization 30min.
5. preparation method as claimed in claim 1 is characterized in that, in the described Zein powder hydrolyzed solution preparation process, the pH regulator agent is ammoniacal liquor.
6. preparation method as claimed in claim 1 is characterized in that, every liter of component of described step (1) caproic acid bacteria bacterium culture medium is as follows:
Ethanol 20mL, sodium acetate 6g, ammonium sulfate 400～500mg, yeast extract powder 4～5g, adding distil water be to 1000mL, pH6.5～7.0.
7. preparation method as claimed in claim 1 is characterized in that, feeds CO in the described step (2)
2Fermention medium be to make to fermention medium ventilation 10～15min with the ventilation of 0.1～0.2vvm.
8. preparation method as claimed in claim 1 is characterized in that, the feed pressure of 0.1 μ m micro-pore-film filtration is 0.2Mpa in the described step (3).
9. preparation method as claimed in claim 1 is characterized in that, the caproic acid bacteria in the described step (1) (Clostridium kluyveri) is available from Chinese industrial microbial strains preservation administrative center (CICC), and preserving number is 8023.
10. preparation method as claimed in claim 1, it is characterized in that, also comprise the clear liquid after the distillation of filtrate in the step (3) through making behind distillation removal caproic acid, the butyric acid, mix the back with wheat bran ethanol fermentation stoste, Zein powder hydrolyzed solution, potassium primary phosphate and water and reuse as fermention medium.
Further preferred, above-mentioned fermention medium component is as follows, is mass percent:
Clear liquid 50～70% after the distillation, wheat bran ethanol fermentation stoste 18～22%, Zein powder hydrolyzed solution 2～4%, potassium primary phosphate 0.01%, the remainder water replenishes.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310145627.1A CN103255085B (en) | 2013-04-24 | 2013-04-24 | Preparation method of concentrated caproic acid bacterial liquid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310145627.1A CN103255085B (en) | 2013-04-24 | 2013-04-24 | Preparation method of concentrated caproic acid bacterial liquid |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN103255085A true CN103255085A (en) | 2013-08-21 |
| CN103255085B CN103255085B (en) | 2014-07-30 |
Family
ID=48959304
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310145627.1A Active CN103255085B (en) | 2013-04-24 | 2013-04-24 | Preparation method of concentrated caproic acid bacterial liquid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103255085B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567614A (en) * | 2016-03-10 | 2016-05-11 | 安徽金种子酒业股份有限公司 | Fermented pit mud-maintained fungicide and preparation method thereof |
| CN105567613A (en) * | 2016-03-10 | 2016-05-11 | 安徽大学 | Method for preparing manmade pit mud |
| CN105670971A (en) * | 2016-03-10 | 2016-06-15 | 安徽大学 | Clostridium kluyveri JZZ and application thereof |
| CN111394397A (en) * | 2020-03-17 | 2020-07-10 | 北京科技大学 | Method for producing caproic acid by fermenting kitchen waste |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004029193A1 (en) * | 2002-09-26 | 2004-04-08 | Novozymes North America, Inc. | Fermentation methods and compositions |
| CN102178036A (en) * | 2011-04-22 | 2011-09-14 | 河南宏翔生物科技有限公司 | Method for producing high-methionine and high-lysine feed by using composite bacteria fermented corn protein powder |
| CN102260638A (en) * | 2011-06-24 | 2011-11-30 | 泸州品创科技有限公司 | Pit mud functional bacteria and preparation method thereof |
-
2013
- 2013-04-24 CN CN201310145627.1A patent/CN103255085B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004029193A1 (en) * | 2002-09-26 | 2004-04-08 | Novozymes North America, Inc. | Fermentation methods and compositions |
| CN102178036A (en) * | 2011-04-22 | 2011-09-14 | 河南宏翔生物科技有限公司 | Method for producing high-methionine and high-lysine feed by using composite bacteria fermented corn protein powder |
| CN102260638A (en) * | 2011-06-24 | 2011-11-30 | 泸州品创科技有限公司 | Pit mud functional bacteria and preparation method thereof |
Non-Patent Citations (1)
| Title |
|---|
| 戴群 等: "己酸菌发酵条件研究", 《南京师范大学学报（工程技术版）》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105567614A (en) * | 2016-03-10 | 2016-05-11 | 安徽金种子酒业股份有限公司 | Fermented pit mud-maintained fungicide and preparation method thereof |
| CN105567613A (en) * | 2016-03-10 | 2016-05-11 | 安徽大学 | Method for preparing manmade pit mud |
| CN105670971A (en) * | 2016-03-10 | 2016-06-15 | 安徽大学 | Clostridium kluyveri JZZ and application thereof |
| CN111394397A (en) * | 2020-03-17 | 2020-07-10 | 北京科技大学 | Method for producing caproic acid by fermenting kitchen waste |
Also Published As
| Publication number | Publication date |
|---|---|
| CN103255085B (en) | 2014-07-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Singh Dhillon et al. | Recent advances in citric acid bio-production and recovery | |
| Anastassiadis et al. | Citric acid production patent review | |
| CN101457190B (en) | Method for preparing spirit flavoring wine or spirit flavour liquid mainly with fragrance | |
| CN101445776B (en) | Method of preparing liquor seasoning wine or liquor seasoning liquid mainly to taste | |
| US20100196994A1 (en) | Fungi cultivation on alcohol fermentation stillage for useful products and energy savings | |
| Djukić-Vuković et al. | Effect of different fermentation parameters on L-lactic acid production from liquid distillery stillage | |
| CN112725114B (en) | Method for increasing ethyl caproate content of Luzhou-flavor liquor | |
| CN101698817B (en) | Method for producing white wine by distilled grain continuous solid state fermentation | |
| AU2006202609A1 (en) | Use of corn with low gelatinization temperature for production of fermentation based products | |
| Liu et al. | Research on microbial lipid production from potato starch wastewater as culture medium by Lipomyces starkeyi | |
| CN103173340A (en) | Method for producing organic acid seasoning liquid for wine through utilizing white wine brewage byproduct | |
| CN103255085B (en) | Preparation method of concentrated caproic acid bacterial liquid | |
| CN106554931A (en) | One plant of Bai Shi carboxylics bacterium and its application | |
| Bogale | Microbial protein production from agro-industrial wastes as food and feed | |
| CN101617828B (en) | Method for preparing astaxanthin food | |
| CN111424058B (en) | Method for preparing erythritol by adopting continuous fermentation mode | |
| CN114958631B (en) | Method for producing single cell protein by using heavy phase lactic acid | |
| CN110004202A (en) | A kind of method of microbial co culture catalysis carbohydrate synthesizing hexanoic acid | |
| CN102492634B (en) | High-temperature resistant yeast and application thereof | |
| CN104561141A (en) | Acetic fermentation nutrient and use method thereof | |
| CN106834188B (en) | Low-cost culture medium composition for propagation of bean pulp fermentation strains in cold regions | |
| CN112538504A (en) | Method for producing 2-phenethyl alcohol by mixed fermentation | |
| Zeng et al. | Development of new strategies for the production of high-purity fructooligosaccharides using β-fructofuranosidase and a novel isolated Wickerhamomyces anomalus | |
| CN104711208A (en) | Lactic acid bacterium with high starch decomposition ability | |
| CN117210368B (en) | Slender lysine bacillus and application thereof in preparing seaweed liquid fertilizer by kelp degradation |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |