CN109234199A - A kind of caproic acid bacterium culture medium containing liquor fermentation process raw material and its application - Google Patents
A kind of caproic acid bacterium culture medium containing liquor fermentation process raw material and its application Download PDFInfo
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Abstract
The present invention provides a kind of caproic acid bacterium culture medium containing liquor fermentation process raw material, by yeast leaching liquor, sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor group at；The yeast leaching liquor the preparation method comprises the following steps: yeast is mixed with water, 1~3h of ultrasound, then vibrate 10~14h, filtering adjusts the pH value of liquid phase to 7.0 ± 0.2, and logical nitrogen boils deoxygenation, sterilizing；The yellow mud leaching liquor the preparation method comprises the following steps: the yellow mud for being used to make pit mud is mixed with water, 1~3h of ultrasound, then vibrate 10~14h, filtering, logical nitrogen boils deoxygenation, sterilizes.The present invention also provides application of the above-mentioned caproic acid bacterium culture medium in the caproic acid bacteria of screening high yield caproic acid.Caproic acid bacterium culture medium provided by the invention, closer to brewed spirit environment, is conducive to the production caproic acid ability for studying caproic acid bacteria, and improve adaptability of the caproic acid bacteria in pit mud in nutritional ingredient, moreover it is possible to promote pit mud aging or improve pit mud character.
Description
Technical field
The invention belongs to field of microbial culture technology, are related to a kind of caproic acid bacteria culture containing liquor fermentation process raw material
Base and its application.
Background technique
White wine is the distinctive Spirit of China.Unique production technology makes China white wine solely set one in world's Spirit
Flag.According to fragrant liquor and style difference, Luzhou-flavor, Maotai-flavor, delicate fragrance type, rice-fragrant type and other odor types can be classified as (such as
Medicine odor type, sesame-flavor, fermented soya beans type) etc. 12 major class.Luzhou-flavor liquo is with " mud cellar solid state fermentation, continuous poor ingredient, mixed steam are mixed
The technique of burning ", is allowed to form the master of " based on ethyl hexanoate, mixing a small amount of ethyl acetate, ethyl butyrate and ethyl lactate "
Body note gas, and these mixing fragrance components there is Luzhou-flavor liquo " cellar aroma flavoring is strong, soft sweet ice-cold, smell coordination, tail remainder
The style of length ".
Ethyl hexanoate is the main aromatic components of Luzhou-flavor liquo, and caproic acid is as its precursor substance, mainly by can generation
The microorganism for thanking to the one kind referred to as " caproic acid bacteria " for generating caproic acid generates, and caproic acid bacteria is mostly anaerobism or facultative anaerobic bacteria.In environment
The caproic acid bacteria known includes clostridium klebsi, clostridium scatologenes, Rhodospirillum rubrum, mucus Eubacterium and Emhorn megacoccus etc..It grinds at present
Study carefully and shows that clostridium klebsi is the main caproic acid bacteria for producing caproic acid in pit mud.Although the report about caproic acid bacteria is more, at present about
It is nearly all using liquid Pasteur culture medium or the aging pit mud culture base of solid-state in the research of caproic acid bacteria.Wu Yan is mediocre et al. to be used
Liquid Pasteur culture medium filters out Lu wine clostridium from Luzhou Old Cellar pit mud, and production caproic acid ability is 3080mg/L, old from five-Grain Liquor
It is 10000mg/L that the caproic acid bacteria w-1 isolated in cellar, which produces caproic acid ability,；Century-old pit pit mud of the Xue Zhengkai from Wan Bin Wine Company
In filter out one plant of caproic acid bacteria, production caproic acid ability is 5470mg/L；It is high it is white and pure et al. using liquid Pasteur culture medium from affluence
Brewery filters out the bacterial strain that a plant height produces caproic acid, and caproic acid yield is 5400mg/L.But both methods is carried out to caproic acid bacteria
All have the defects that during research and application certain.For example, such as that the caproic acid bacteria Jing Guo liquid Pasteur culture medium culture is straight
Connecing to be added in pit mud will lead to wherein caproic acid bacteria and can not adapt to pit mud environment very well；Though using solid medium Caproic Acid Bacteria Culture
It can be so enriched with caproic acid bacteria, but the growth and metabolism that are difficult in solid-state environment to caproic acid bacteria are studied.
Therefore, if the liquid caproic acid bacterium culture medium containing liquor fermentation process raw material can be developed, in nutritional ingredient
Closer to brewed spirit environment, this is not only advantageous to the production caproic acid ability of research caproic acid bacteria, caproic acid bacteria is studied on metaboilic level
Product acid activity, and can increase adaptability of the caproic acid bacteria in pit mud, so that it is more particularly for pit mud production, promote pit mud old
Ripe or improvement aged pit mud.
Summary of the invention
It is an object of the invention to overcome the deficiencies in the prior art, an object of the present invention is to provide containing liquor fermentation
The caproic acid bacterium culture medium of process raw material, so that caproic acid bacterium culture medium closer brewed spirit environment in nutritional ingredient, with advantageous
In the production caproic acid ability of research caproic acid bacteria, and adaptability of the caproic acid bacteria in pit mud is improved, the second object of the present invention is to mention
For application of the caproic acid bacterium culture medium in the caproic acid bacteria of screening high yield caproic acid, to promote pit mud aging or improve pit mud
Shape.
For realize foregoing invention purpose, what the present invention was realized using the technical solution that following technical measures are constituted.
Caproic acid bacterium culture medium provided by the invention containing liquor fermentation process raw material, by yeast leaching liquor, sodium acetate, nothing
Water-ethanol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor group at；The yeast leaching liquor the preparation method comprises the following steps: by yeast and water
According to 1:(5~8) mass ratio mixing, then 1~3h of ultrasound vibrates 10~14h, filters, adjust the pH value of liquid phase to 7.0 ±
0.2, logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C；The yellow mud leaching liquor the preparation method comprises the following steps: will be used to make cellar
The yellow mud of mud is with water according to 1:(2~4) mass ratio mix, then 1~3h of ultrasound vibrates 10~14h, filtering, logical nitrogen boils
Deoxygenation is boiled, then is sterilized at 115~121 DEG C.
In the technical solution of the above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material, based on yeast leaching liquor
Culture medium, sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, the additive amount of urea and yellow mud leaching liquor in basal medium according to
Practical application request is determined and adjusts, for example, mainly according to practical application when the type of caproic acid bacteria cultivated, caproic acid flora
Factors, the factors such as mixed bacterial including caproic acid bacteria or caproic acid flora such as composition be determined and adjust.Preferably, acetic acid
The additive amount of sodium, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor in basal medium be followed successively by 5~10g/L,
15~25mL/L, 15~25mL/L, 0.1~0.5g/L, 1~3g/L and 0.5~1.5mL/L.It is highly preferred that sodium acetate, nothing
The additive amount of water-ethanol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor in basal medium be followed successively by 8~10g/L, 15~
25mL/L, 15~25mL/L, 0.4~0.5g/L, 1~2.5g/L and 0.5~1.5mL/L.
In the technical solution of the above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material, the system of the yeast leaching liquor
Preparation Method is preferred are as follows: by yeast with water according to 1:(5~6) mass ratio mix, 1~3h of ultrasound, then vibrate 10~14h, mistake
Filter adjusts the pH value of liquid phase to 7.0 ± 0.2, and logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C.
In the technical solution of the above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material, the system of the yellow mud leaching liquor
Preparation Method is preferred are as follows: by the yellow mud for being used to make pit mud with water according to 1:(2~3) mass ratio mix, 1~3h of ultrasound, then
10~14h, filtering are vibrated, logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C.
The above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material the preparation method is as follows:
(1) by yeast with water according to 1:(5~8) mass ratio mix, 1~3h of ultrasound, then vibrate 10~14h, filtering,
The pH value of liquid phase is adjusted to 7.0 ± 0.2, logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C, obtains yeast leaching liquor；
By the yellow mud for being used to make pit mud with water according to 1:(2~4) mass ratio mix, then 1~3h of ultrasound vibrates 10
~14h, filtering, logical nitrogen boil deoxygenation, then in 115~121 DEG C of sterilizings to get yellow mud leaching liquor；
It (2) is basic culture medium with yeast leaching liquor, by sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud
Leaching liquor is added in basal medium, mixes to obtain the final product.
The formula of the above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material is to screen to obtain by the following method
:
(1) preparation of yeast leaching liquor and yellow mud leaching liquor
By yeast with water according to 1:(5~8) mass ratio mix, 1~3h of ultrasound, then vibrate 10~14h, filter, adjust
The pH value of liquid phase is saved to 7.0 ± 0.2, logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C, obtains yeast leaching liquor；
By the yellow mud for being used to make pit mud with water according to 1:(2~4) mass ratio mix, then 1~3h of ultrasound vibrates 10
~14h, filtering, logical nitrogen boil deoxygenation, then in 115~121 DEG C of sterilizings to get yellow mud leaching liquor；
(2) screening of carbon source
It is basic culture medium with yeast leaching liquor, different amounts of sodium acetate, ethyl alcohol and liquor tailing is added into basal medium
Multiple groups carbon source recipe determination group is formed, the additive amount of sodium acetate in 0~20g/L and forms at least 5 in each carbon source recipe determination group
A concentration gradient, dehydrated alcohol additive amount be 0~50mL/L and form at least five concentration gradient, the additive amount of liquor tailing be 0~
50mL/L simultaneously forms at least five concentration gradient, using natural ph or adjusts pH value to 7.0 ± 0.2, then to each carbon source formula
It is inoculated with the caproic acid flora of equivalent in screening group, is cultivated 12~15 days at 37 ± 0.5 DEG C, the condition of culture of each carbon source recipe determination group
Identical, in detection gained fermentation liquid caproic acid content, is formed with the corresponding carbon source of carbon source recipe determination group that caproic acid bacterial content is high
And concentration is as carbon source formula；
(3) screening of nitrogen source
Be basic culture medium with yeast leaching liquor, add different amounts of ammonium sulfate into basal medium and urea formed it is more
Group nitrogen source recipe determination group, in each nitrogen source recipe determination group, the additive amount of ammonium sulfate in 0~2g/L and forms at least five concentration
Gradient, urea additive amount be 0~5g/L and form at least five concentration gradient, using natural ph or adjust pH value to 7.0 ±
0.2, it is then inoculated with the caproic acid flora of equivalent into each nitrogen source recipe determination group, is cultivated 12~15 days at 37 ± 0.5 DEG C, each nitrogen source
The condition of culture of recipe determination group is identical, the caproic acid content in detection gained fermentation liquid, with the high nitrogen source formula of caproic acid bacterial content
The corresponding nitrogen source composition of screening group and concentration are as nitrogen source formula；
(4) growth factor is screened
It is basic culture medium with yeast leaching liquor, different amounts of yellow mud leaching liquor is added into basal medium and forms multiple groups
Growth factor screening group, in each growth factor screening group, the additive amount of yellow mud leaching liquor is 0~5mL/L and to form at least five dense
Gradient is spent, using natural ph or pH value is adjusted to 7.0 ± 0.2, oneself of equivalent is then inoculated with into each growth factor screening group
Sour flora is cultivated 12~15 days at 37 ± 0.5 DEG C, and the condition of culture of each growth factor recipe determination group is identical, and detection caproic acid produces
Amount, using the corresponding growth factor concentration of the highest growth factor recipe determination group of caproic acid bacteria yield as growth factor formula.
During screening the formula of the above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material, the caproic acid that uses
Bacterium can be single caproic acid bacteria strain, be also possible to the caproic acid bacteria flora of a variety of caproic acid bacteria strain compositions, for example, guarantor can be used
It is hidden in the caproic acid flora J of Sichuan University's light textile Yu Foodstuffs Academy food ecological engineering and biotechnology research room30, caproic acid flora
J30It is Sichuan University's light textile and Foodstuffs Academy is filtered out from pit mud containing there are many caproic acid flora of caproic acid bacteria bacterial strain, it can also be with
Using other caproic acid bacteria strains or caproic acid flora, such as clostridium klebsi etc..
The present invention also provides a kind of above-mentioned caproic acid bacterium culture medium containing liquor fermentation process raw material screening high yield oneself
Application in the caproic acid bacteria of acid.In practical application, the caproic acid bacteria of high yield caproic acid can be filtered out from pit mud, and screening is obtained
Caproic acid bacteria in pit mud, for promoting pit mud aging or improving pit mud character.
By taking aging pit mud as an example, illustrate to utilize the caproic acid bacterium culture medium provided by the invention containing liquor fermentation process raw material
Application in the caproic acid bacteria of screening high yield caproic acid, steps are as follows.
(1) prepared by spore solution
Aging pit mud is taken, deoxygenation sterile saline is added, is shaken up in 37 ± 0.5 DEG C of oscillations, is subsequently placed in 80 DEG C of water-baths
Middle holding a period of time to remove non-gemma viable bacteria therein, obtains gemma liquid.
(2) example enrichment culture
Take gemma liquid be added deoxygenation sterilizing caproic acid bacterium culture medium provided by the invention in, 37 ± 0.5 DEG C cultivate 12~
15 days, gained culture solution is subjected to secondary culture, enrichment several times, obtains pregnant solution.
(3) pregnant solution dilution spread
Gained pregnant solution is subjected to gradient dilution in deoxygenation sterile saline, plate is inverted in 37 by spread plate
It is cultivated in ± 0.5 DEG C of anaerobic box.Single colonie is grown to plate, random picking individual colonies simultaneously continue scribing line purifying culture, select list
Colony inoculation carries out liquid state fermentation into caproic acid bacterium culture medium provided by the invention, and fermentation liquid is taken to carry out caproic acid content detection, choosing
The fermentation liquid that caproic acid content is high is taken, continues to isolate and purify until screening pure bacterial strain.
Caproic acid bacterium culture medium provided by the invention is mainly utilized the raw material of liquor fermentation process, for example, yeast, for making
The liquor tailing that the yellow mud and liquor production process for making pit mud generate, passes through flooding for yeast and the yellow mud for being used to make pit mud
Yeast leaching liquor and yellow mud leaching liquor is made, then the carbon source and nitrogen source that cooperate concentration suitable, forms caproic acid of the present invention
Bacterium culture medium.For existing solid-state caproic acid bacterium culture medium, caproic acid bacterium culture medium provided by the invention is a kind of liquid
Culture medium, and be more advantageous to research caproic acid bacteria close to brewed spirit environment in nutritional ingredient and produce caproic acid ability, in metaboilic level
Upper research caproic acid bacteria produces caproic acid performance, for existing liquid Pasteur culture medium, since caproic acid bacteria provided by the invention is trained
The raw material containing liquor fermentation process in base is supported, closer to brewed spirit environment in nutritional ingredient, this can increase caproic acid bacteria
Adaptability in pit mud.Based on caproic acid bacterium culture medium provided by the invention, can be filtered out from pit mud high yield oneself
The caproic acid bacteria of acid, for promoting pit mud aging or improving pit mud character, so as to improve pit mud quality and brewing high-quality giving off a strong fragrance
Type white wine.
Compared with prior art, present invention produces technical effects beneficial below:
1. the present invention provides a kind of caproic acid bacterium culture mediums containing liquor fermentation process raw material, due to the caproic acid bacteria culture
Base is a kind of liquid culture medium, and the raw material of the liquor fermentations processes such as yeast, yellow mud for making pit mud is utilized, also added
The liquor tailing that liquor production process generates, based on yeast leaching liquor, using yellow mud leaching liquor as growth factor source, then with dense
The suitable carbon source of degree and nitrogen source combination form, thus the caproic acid bacterium culture medium that constructs of the present invention in nutritional ingredient closer to white wine
Environment is made, is on the one hand conducive to enhance adaptability of the caproic acid bacteria in pit mud, is on the other hand conducive to study caproic acid bacteria production
Caproic acid ability studies caproic acid bacteria on metaboilic level and produces caproic acid performance.
2. the formula and processing technology of the caproic acid bacterium culture medium provided by the invention containing liquor fermentation process raw material is simple,
The raw material of use is conventional raw material, is easy to obtain, low in cost, these features are all conducive to its popularization and application.
3. the present invention also provides a kind of caproic acid bacterium culture mediums containing liquor fermentation process raw material in screening high yield caproic acid
Caproic acid bacteria in application, can more effectively filter out the bacterial strain of high yield caproic acid using the caproic acid bacterium culture medium and be used for storing
In mud, promote pit mud aging or improve aged pit mud, so as to improve pit mud quality and brewing high-quality Luzhou-flavor liquo.
Specific embodiment
Caproic acid bacterium culture medium and its sieve to provided by the invention containing liquor fermentation process raw material by the following examples
Preparation method is selected to be described further with application.It is necessary to note that following embodiment is served only for, the invention will be further described,
It should not be understood as limiting the scope of the invention, one of ordinary skill in the art are the present invention according to foregoing invention content
Some nonessential modifications and adaptations are embodied out, still fall within the range of invention protection.
The caproic acid flora J being related in following embodiment and comparative example30It is preserved in Sichuan University's light textile and Foodstuffs Academy is eaten
Product ecological engineering and biotechnology research room, can provide, caproic acid flora J to the public30It is Sichuan University's light textile and Foodstuffs Academy
The caproic acid flora of caproic acid bacteria bacterial strain containing there are many filtered out from pit mud.
Liquor tailing in following embodiment derives from Sichuan Mianzhu Jiannanchur brewery limited company.
Embodiment 1
In the present embodiment, the formula of the caproic acid bacterium culture medium containing liquor fermentation process raw material is screened, using what is filtered out
Caproic acid bacterium culture medium Caproic Acid Bacteria Culture group J30, and caproic acid content in test broth.
(1) caproic acid bacteria culture medium prescription is screened, steps are as follows:
1. the preparation of yeast leaching liquor
Yeast is mixed with water according to the mass ratio of 1:5, then ultrasonic 3h vibrates 10h, filtering adjusts the pH value of liquid phase
To 7.0 ± 0.2, logical nitrogen boils deoxygenation, then 121 DEG C of sterilizing 20min to get.
2. the preparation of yellow mud leaching liquor
The yellow mud for being used to make pit mud is mixed with water according to the mass ratio of 1:2, then ultrasonic 2h vibrates 12h, filtering,
Logical nitrogen boils deoxygenation, then 121 DEG C of sterilizing 20min to get.
3. the screening of carbon source
1. the yeast leaching liquor prepared with step is basic culture medium, added into basal medium the sodium acetate that do not measure,
Dehydrated alcohol and liquor tailing form 10 groups of carbon source recipe determination groups, in each carbon source recipe determination group the additive amount of sodium acetate 0~
20g/L and form 10 concentration gradients, the additive amount of dehydrated alcohol is 0~50mL/L and forms 10 concentration gradients, liquor tailing
Additive amount is 0~50mL/L and forms 10 concentration gradients, adjusts pH value to 7.0 ± 0.2, connects into each carbon source recipe determination group
The caproic acid flora J of kind equivalent30, cultivated 15 days at 37 ± 0.5 DEG C, the condition of culture of each carbon source recipe determination group is identical, detects institute
Caproic acid content in fermentation liquid, the carbon source recipe determination group corresponding carbon source high using caproic acid bacterial content forms and concentration is as carbon
Source formula.
4. the screening of nitrogen source
It is basic culture medium with 1. yeast leaching liquor that step is prepared, different amounts of ammonium sulfate is added into basal medium
10 groups of nitrogen source recipe determination groups are formed with urea, in each nitrogen source recipe determination group, the additive amount of ammonium sulfate in 0~2g/L and is formed
10 concentration gradients, urea additive amount be 0~5g/L and form 10 concentration gradients, pH value is adjusted to 7.0 ± 0.2, to each
The caproic acid flora J of equivalent is inoculated in nitrogen source recipe determination group30, cultivated 15 days at 37 ± 0.5 DEG C, the training of each nitrogen source recipe determination group
The condition of supporting is identical, and the caproic acid content in detection gained fermentation liquid, the nitrogen source high using caproic acid bacterial content forms and concentration is as nitrogen source
Formula.The corresponding carbon source composition of carbon source recipe determination group and concentration are as carbon source formula.
5. the screening of growth factor
It is basic culture medium with 1. yeast leaching liquor that step is prepared, different amounts of step is added into basal medium 2.
The yellow mud leaching liquor of preparation forms 10 groups of growth factor screening groups, in each growth factor screening group, the additive amount of yellow mud leaching liquor
For 0~5mL/L and 10 concentration gradients are formed, pH value is adjusted to 7.0 ± 0.2, is inoculated with equivalent into each growth factor screening group
Caproic acid flora J30, cultivated 15 days at 37 ± 0.5 DEG C, the condition of culture of each growth factor recipe determination group is identical, detection gained
Caproic acid content in fermentation liquid, the yellow mud leaching liquor concentration high using caproic acid bacterial content is as growth factor formula.
According to step 3.~caproic acid content testing result 5., determine caproic acid bacteria culture medium prescription are as follows:
1. yeast leaching liquor, sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea that caproic acid bacterium culture medium is prepared by step
The yellow mud leaching liquor group 2. prepared with step is at sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor are in base
Additive amount in basal culture medium is followed successively by 8g/L, 15mL/L, 25mL/L, 0.5g/L, 2g/L, 0.5mL/L.
(2) caproic acid bacterium culture medium is prepared
According to the caproic acid bacteria culture medium prescription that step (1) determines, trained based on the yeast leaching liquor that step (1) is 1. prepared
Base is supported, the yellow mud that sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and step (1) are 3. prepared is added into basal medium
Leaching liquor simultaneously mixes to obtain the final product.
(3) caproic acid content in caproic acid bacteria culture and test broth
Using anaerobic operation, it is inoculated with the caproic acid flora J of 10% anaerobism preservation30In the caproic acid bacterium culture medium of step (2) preparation
In, the caproic acid bacterial content in 37 ± 0.5 DEG C of culture 15min, sample detection fermentation liquid, result 2902.2mg/L.
Above-mentioned steps (1) 3.~5. with step (3) in detection fermentation liquid in caproic acid content method are as follows:
The fermentation liquid obtained after culture is centrifuged 10min through 10000 × g, upper liquid is taken, adjusts the pH value of supernatant extremely
2.0, using caproic acid content in GC-FID measurement fermentation liquid, using 2- ethyl n-butyric acie as internal standard.By sample and internal standard 1:1 before sample introduction
It mixes, sampling volume is 0.5 μ L.Detector temperature is 260 DEG C, and injector temperature is 250 DEG C, split ratio 50:1.
Embodiment 2
In the present embodiment, the formula of the caproic acid bacterium culture medium containing liquor fermentation process raw material is screened, using what is filtered out
Caproic acid bacterium culture medium Caproic Acid Bacteria Culture group J30, and caproic acid content in test broth.
(1) caproic acid bacteria culture medium prescription is screened, steps are as follows:
1. the preparation of yeast leaching liquor
Yeast is mixed with water according to the mass ratio of 1:6, then ultrasonic 2h vibrates 12h, filtering adjusts the pH value of liquid phase
To 7.0 ± 0.2, logical nitrogen boils deoxygenation, then 121 DEG C of sterilizing 20min to get.
2. the preparation of yellow mud leaching liquor
The yellow mud for being used to make pit mud is mixed with water according to the mass ratio of 1:3, then ultrasonic 3h vibrates 10h, filtering,
Logical nitrogen boils deoxygenation, then 121 DEG C of sterilizing 20min to get.
3. the screening of carbon source
1. the yeast leaching liquor prepared with step is basic culture medium, added into basal medium the sodium acetate that do not measure,
Dehydrated alcohol and liquor tailing form 10 groups of carbon source recipe determination groups, in each carbon source recipe determination group the additive amount of sodium acetate 0~
20g/L and form 10 concentration gradients, the additive amount of dehydrated alcohol is 0~50mL/L and forms 10 concentration gradients, liquor tailing
Additive amount is 0~50mL/L and forms 10 concentration gradients, adjusts pH value to 7.0 ± 0.2, connects into each carbon source recipe determination group
The caproic acid flora J of kind equivalent30, cultivated 15 days at 37 ± 0.5 DEG C, the condition of culture of each carbon source recipe determination group is identical, detects institute
Caproic acid content in fermentation liquid, the carbon source recipe determination group corresponding carbon source high using caproic acid bacterial content forms and concentration is as carbon
Source formula.
4. the screening of nitrogen source
It is basic culture medium with 1. yeast leaching liquor that step is prepared, different amounts of ammonium sulfate is added into basal medium
10 groups of nitrogen source recipe determination groups are formed with urea, in each nitrogen source recipe determination group, the additive amount of ammonium sulfate in 0~2g/L and is formed
10 concentration gradients, urea additive amount be 0~5g/L and form 10 concentration gradients, pH value is adjusted to 7.0 ± 0.2, to each
The caproic acid flora J of equivalent is inoculated in nitrogen source recipe determination group30, cultivated 15 days at 37 ± 0.5 DEG C, the training of each nitrogen source recipe determination group
The condition of supporting is identical, and the caproic acid content in detection gained fermentation liquid, the nitrogen source high using caproic acid bacterial content forms and concentration is as nitrogen source
Formula.The corresponding carbon source composition of carbon source recipe determination group and concentration are as carbon source formula.
5. the screening of growth factor
It is basic culture medium with 1. yeast leaching liquor that step is prepared, different amounts of step is added into basal medium 2.
The yellow mud leaching liquor of preparation forms 10 groups of growth factor screening groups, in each growth factor screening group, the additive amount of yellow mud leaching liquor
For 0~5mL/L and 10 concentration gradients are formed, pH value is adjusted to 7.0 ± 0.2, is inoculated with equivalent into each growth factor screening group
Caproic acid flora J30, cultivated 15 days at 37 ± 0.5 DEG C, the condition of culture of each growth factor recipe determination group is identical, detection gained
Caproic acid content in fermentation liquid, the yellow mud leaching liquor concentration high using caproic acid bacterial content is as growth factor formula.
According to step 3.~caproic acid content testing result 5., determine caproic acid bacteria culture medium prescription are as follows:
1. yeast leaching liquor, sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea that caproic acid bacterium culture medium is prepared by step
The yellow mud leaching liquor group 2. prepared with step is at sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor are in base
Additive amount in basal culture medium is followed successively by 10g/L, 25mL/L, 15mL/L, 0.5g/L, 1g/L, 0.75mL/L.
(2) caproic acid bacterium culture medium is prepared
According to the caproic acid bacteria culture medium prescription that step (1) determines, trained based on the yeast leaching liquor that step (1) is 1. prepared
Base is supported, the yellow mud that sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and step (1) are 3. prepared is added into basal medium
Leaching liquor simultaneously mixes to obtain the final product.
(3) caproic acid content in caproic acid bacteria culture and test broth
Using anaerobic operation, it is inoculated with the caproic acid flora J of 10% anaerobism preservation30In the caproic acid bacterium culture medium of step (2) preparation
In, the caproic acid bacterial content in 37 ± 0.5 DEG C of culture 15min, sample detection fermentation liquid, result 3184.3mg/L.
Above-mentioned steps (1) 3.~5. with step (3) in detection fermentation liquid in caproic acid content method are as follows:
The fermentation liquid obtained after culture is centrifuged 10min through 10000 × g, upper liquid is taken, adjusts the pH value of supernatant extremely
2.0, using caproic acid content in GC-FID measurement fermentation liquid, using 2- ethyl n-butyric acie as internal standard.By sample and internal standard 1:1 before sample introduction
It mixes, sampling volume is 0.5 μ L.Detector temperature is 260 DEG C, and injector temperature is 250 DEG C, split ratio 50:1.
Embodiment 3
In the present embodiment, the formula of the caproic acid bacterium culture medium containing liquor fermentation process raw material is screened, using what is filtered out
Caproic acid bacterium culture medium Caproic Acid Bacteria Culture group J30, and caproic acid content in test broth.
(1) caproic acid bacteria culture medium prescription is screened, steps are as follows:
1. the preparation of yeast leaching liquor
Yeast is mixed with water according to the mass ratio of 1:8, then ultrasonic 1h vibrates 14h, filtering adjusts the pH value of liquid phase
To 7.0 ± 0.2, logical nitrogen boils deoxygenation, then 121 DEG C of sterilizing 20min to get.
2. the preparation of yellow mud leaching liquor
The yellow mud for being used to make pit mud is mixed with water according to the mass ratio of 1:4, then ultrasonic 1h vibrates 14h, filtering,
Logical nitrogen boils deoxygenation, then 121 DEG C of sterilizing 20min to get.
3. the screening of carbon source
1. the yeast leaching liquor prepared with step is basic culture medium, added into basal medium the sodium acetate that do not measure,
Dehydrated alcohol and liquor tailing form 10 groups of carbon source recipe determination groups, in each carbon source recipe determination group the additive amount of sodium acetate 0~
20g/L and form 10 concentration gradients, the additive amount of dehydrated alcohol is 0~50mL/L and forms 10 concentration gradients, liquor tailing
Additive amount is inoculated with equivalent into each carbon source recipe determination group using natural ph for 10 concentration gradients of 0~50mL/L and formation
Caproic acid flora J30, cultivated 12 days at 37 ± 0.5 DEG C, the condition of culture of each carbon source recipe determination group is identical, detection gained fermentation
Caproic acid content in liquid, the carbon source recipe determination group corresponding carbon source high using caproic acid bacterial content forms and concentration is matched as carbon source
Side.
4. the screening of nitrogen source
It is basic culture medium with 1. yeast leaching liquor that step is prepared, different amounts of ammonium sulfate is added into basal medium
10 groups of nitrogen source recipe determination groups are formed with urea, in each nitrogen source recipe determination group, the additive amount of ammonium sulfate in 0~2g/L and is formed
10 concentration gradients, urea additive amount be 0~5g/L and formed 10 concentration gradients matched using natural ph to each nitrogen source
The caproic acid flora J of equivalent is inoculated in square screening group30, cultivated 12 days at 37 ± 0.5 DEG C, the condition of culture of each nitrogen source recipe determination group
Identical, in detection gained fermentation liquid caproic acid content, the nitrogen source high using caproic acid bacterial content forms and concentration is as nitrogen source formula.Carbon
The corresponding carbon source composition of source recipe determination group and concentration are as carbon source formula.
5. the screening of growth factor
It is basic culture medium with 1. yeast leaching liquor that step is prepared, different amounts of step is added into basal medium 2.
The yellow mud leaching liquor of preparation forms 10 groups of growth factor screening groups, in each growth factor screening group, the additive amount of yellow mud leaching liquor
For 0~5mL/L and 10 concentration gradients are formed, using natural ph, the caproic acid of equivalent is inoculated with into each growth factor screening group
Flora J30, cultivated 12 days at 37 ± 0.5 DEG C, the condition of culture of each growth factor recipe determination group is identical, detection gained fermentation liquid
In caproic acid content, the yellow mud leaching liquor concentration high using caproic acid bacterial content is as growth factor formula.
According to step 3.~caproic acid content testing result 5., determine caproic acid bacteria culture medium prescription are as follows:
1. yeast leaching liquor, sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea that caproic acid bacterium culture medium is prepared by step
The yellow mud leaching liquor group 2. prepared with step is at sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor are in base
Additive amount in basal culture medium is followed successively by 5g/L, 15mL/L, 15mL/L, 0.1g/L, 3g/L, 1.5mL/L.
(2) caproic acid bacterium culture medium is prepared
According to the caproic acid bacteria culture medium prescription that step (1) determines, trained based on the yeast leaching liquor that step (1) is 1. prepared
Base is supported, the yellow mud that sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and step (1) are 3. prepared is added into basal medium
Leaching liquor simultaneously mixes to obtain the final product.
(3) caproic acid content in caproic acid bacteria culture and test broth
Using anaerobic operation, it is inoculated with the caproic acid flora J of 10% anaerobism preservation30In the caproic acid bacterium culture medium of step (2) preparation
In, the caproic acid bacterial content in 37 ± 0.5 DEG C of culture 15min, sample detection fermentation liquid, result 2656.6mg/L.
Above-mentioned steps (1) 3.~5. with step (3) in detection fermentation liquid in caproic acid content method are as follows:
The fermentation liquid obtained after culture is centrifuged 10min through 10000 × g, upper liquid is taken, adjusts the pH value of supernatant extremely
2.0, using caproic acid content in GC-FID measurement fermentation liquid, using 2- ethyl n-butyric acie as internal standard.By sample and internal standard 1:1 before sample introduction
It mixes, sampling volume is 0.5 μ L.Detector temperature is 260 DEG C, and injector temperature is 250 DEG C, split ratio 50:1.
Comparative example 1
In this comparative example, caproic acid bacteria culture is carried out using existing caproic acid bacterium culture medium-Pasteur's culture medium and tests fermentation
Caproic acid content in liquid, steps are as follows:
(1) preparation of Pasteur's culture medium
The formula and preparation method of Pasteur's culture medium: sodium acetate 5.0g/L, K2HPO40.4g/L, MgSO40.2g/L,
(NH4)2SO40.5g/L, yeast extract 2.0g/L, CaCO310.0g/L, dehydrated alcohol 20mL/L, by sodium acetate, K2HPO4,
MgSO4, (NH4)2SO4, yeast extract and CaCO3Relatively mixed according to aforementioned, adjustment pH value to 7.0, logical nitrogen boil after deoxygenation
121 DEG C of sterilizing 20min, add dehydrated alcohol before the use.
(2) caproic acid bacteria culture
Using anaerobic operation, it is inoculated with the caproic acid flora J of 10% anaerobism preservation30In Pasteur's culture medium of step (1) preparation,
Caproic acid bacterial content in 37 ± 0.5 DEG C of culture 15min, sample detection fermentation liquid, result 1676mg/L.
The method of caproic acid content in fermentation liquid are as follows: be centrifuged the fermentation liquid obtained after culture through 10000 × g
10min takes upper liquid, adjusts the pH value of supernatant to 2.0, measures caproic acid content in fermentation liquid using GC-FID, just with 2- ethyl
Butyric acid is internal standard.Sample and internal standard 1:1 are mixed before sample introduction, sampling volume is 0.5 μ L.Detector temperature is 260 DEG C, injection port
Temperature is 250 DEG C, split ratio 50:1.
Embodiment 4
In the present embodiment, the formula of the caproic acid bacterium culture medium containing liquor fermentation process raw material.
The caproic acid bacterium culture medium containing liquor fermentation process raw material provided in the present embodiment, by yeast leaching liquor, acetic acid
Sodium, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor group at, be basic culture medium with yeast leaching liquor, sodium acetate,
The additive amount of dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor in basal medium be followed successively by 9g/L, 22mL/L,
20mL/L、0.4g/L、2.5g/L、0.8mL/L。
The yeast leaching liquor the preparation method comprises the following steps: yeast is mixed with water according to the mass ratio of 1:5.5, ultrasonic 2h, so
After vibrate 12h, filter, adjust the pH value of liquid phase to 7.0 ± 0.2, logical nitrogen boils deoxygenation, then in 115 DEG C of sterilizing 30min；Institute
State yellow mud leaching liquor the preparation method comprises the following steps: the yellow mud for being used to make pit mud is mixed with water according to the mass ratio of 1:2.5, ultrasound
Then 2h vibrates 12h, filtering, logical nitrogen boils deoxygenation, then in 115 DEG C of sterilizing 30min.
Embodiment 5
In the present embodiment, illustrate screening using the caproic acid bacterium culture medium of the present invention containing liquor fermentation process raw material
Application in the caproic acid bacteria of high yield caproic acid is specifically screened from aging pit mud using the caproic acid bacterium culture medium that embodiment 2 provides
The caproic acid bacteria strain of high yield caproic acid.
(1) prepared by spore solution
The aging pit mud of 10g is taken, 10mL deoxygenation sterile saline is added and shakes up, is subsequently placed in 37 DEG C of oscillation 10min
It keeps 10min to remove non-gemma viable bacteria therein in 80 DEG C of water-baths, obtains gemma liquid.
(2) example enrichment culture
Gemma liquid obtained by 10mL step (1) is taken, 90mL deoxygenation sterilizing caproic acid bacterium culture medium is added, 37 DEG C are cultivated 15 days, will
Gained culture solution carries out secondary culture, and enrichment twice, obtains pregnant solution.
(3) pregnant solution dilution spread
Pregnant solution obtained by step (2) is subjected to gradient dilution, spread plate, by flat-plate inverted in deoxygenation sterile saline
It is placed in 37 DEG C of anaerobic box and cultivates.Single colonie is grown to plate, random picking individual colonies simultaneously continue scribing line purifying culture, select list
Colony inoculation carries out liquid state fermentation into caproic acid bacterium culture medium, and fermentation liquid is taken to carry out caproic acid content detection, and it is high to choose caproic acid content
Fermentation liquid, continue to isolate and purify until screen pure bacterial strain.
(4) bacterial strain is identified
Biotech firm is sent to carry out sequencing identification the bacterial strain that step (3) finally screens, the results showed that the present embodiment sieve
The caproic acid bacteria strain for selecting obtained high yield caproic acid is clostridium klebsi.
(5) fermenting property of the caproic acid bacteria strain filtered out measures
Using anaerobic operation, the caproic acid bacteria strain that the step of being inoculated with 10% anaerobism preservation (4) identifies is in caproic acid bacteria culture
In base, caproic acid bacterial content in 37 ± 0.5 DEG C of culture 15min, sample detection fermentation liquid, result 10951mg/L.
The method of caproic acid content in fermentation liquid are as follows: be centrifuged the fermentation liquid obtained after culture through 10000 × g
10min takes upper liquid, adjusts the pH value of supernatant to 2.0, measures caproic acid content in fermentation liquid using GC-FID, just with 2- ethyl
Butyric acid is internal standard.Sample and internal standard 1:1 are mixed before sample introduction, sampling volume is 0.5 μ L.Detector temperature is 260 DEG C, injection port
Temperature is 250 DEG C, split ratio 50:1.
Claims (5)
1. a kind of caproic acid bacterium culture medium containing liquor fermentation process raw material, which is characterized in that by yeast leaching liquor, sodium acetate,
Dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor group at；The yeast leaching liquor the preparation method comprises the following steps: by yeast with
Water is according to 1:(5~8) mass ratio mixing, then 1~3h of ultrasound vibrates 10~14h, filters, adjust the pH value of liquid phase to 7.0
± 0.2, logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C；The yellow mud leaching liquor the preparation method comprises the following steps: will be used to make
The yellow mud of pit mud is with water according to 1:(2~4) mass ratio mix, 1~3h of ultrasound, then vibrate 10~14h, filtering, lead to nitrogen
Deoxygenation is boiled, then is sterilized at 115~121 DEG C.
2. the caproic acid bacterium culture medium according to claim 1 containing liquor fermentation process raw material, which is characterized in that the caproic acid
Bacterium culture medium is basic culture medium, sodium acetate, dehydrated alcohol, liquor tailing, ammonium sulfate, urea and yellow mud leaching liquor with yeast leaching liquor
Additive amount in basal medium is followed successively by 5~10g/L, 15~25mL/L, 15~25mL/L, 0.1~0.5g/L, 1~3g/
L and 0.5~1.5mL/L.
3. the caproic acid bacterium culture medium according to claim 2 containing liquor fermentation process raw material, which is characterized in that described big
Bent leaching liquor the preparation method comprises the following steps: by yeast with water according to 1:(5~6) mass ratio mix, then 1~3h of ultrasound vibrates 10
~14h, filtering adjust the pH value of liquid phase to 7.0 ± 0.2, and logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C.
4. the caproic acid bacterium culture medium according to claim 2 containing liquor fermentation process raw material, which is characterized in that the Huang
Mud leaching liquor the preparation method comprises the following steps: by the yellow mud for being used to make pit mud with water according to 1:(2~3) mass ratio mix, ultrasound 1~
Then 3h vibrates 10~14h, filtering, logical nitrogen boils deoxygenation, then sterilizes at 115~121 DEG C.
5. the caproic acid bacterium culture medium containing liquor fermentation process raw material described in any one of Claims 1-4 claim
Application in the caproic acid bacteria of screening high yield caproic acid.
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| CN115161242A (en) * | 2022-07-26 | 2022-10-11 | 宜宾五粮液股份有限公司 | Method for directionally enriching and culturing clostridium microorganisms |
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