CN107022509A - A kind of separation method of lactic bacteria composition and its application - Google Patents
A kind of separation method of lactic bacteria composition and its application Download PDFInfo
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- CN107022509A CN107022509A CN201710347980.6A CN201710347980A CN107022509A CN 107022509 A CN107022509 A CN 107022509A CN 201710347980 A CN201710347980 A CN 201710347980A CN 107022509 A CN107022509 A CN 107022509A
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- lactic
- lactic acid
- acid bacteria
- aspergillus flavus
- bacteria
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- 241000894006 Bacteria Species 0.000 title claims abstract description 173
- 239000000203 mixture Substances 0.000 title claims abstract description 42
- 238000000926 separation method Methods 0.000 title claims abstract description 31
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 claims abstract description 134
- 239000004310 lactic acid Substances 0.000 claims abstract description 67
- 235000014655 lactic acid Nutrition 0.000 claims abstract description 67
- 241000228197 Aspergillus flavus Species 0.000 claims abstract description 58
- 229920001817 Agar Polymers 0.000 claims abstract description 37
- 239000008272 agar Substances 0.000 claims abstract description 37
- 241000193830 Bacillus <bacterium> Species 0.000 claims abstract description 26
- 235000014897 Streptococcus lactis Nutrition 0.000 claims abstract description 23
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
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- 244000061456 Solanum tuberosum Species 0.000 claims description 26
- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 26
- 239000001888 Peptone Substances 0.000 claims description 25
- 108010080698 Peptones Proteins 0.000 claims description 25
- 235000019319 peptone Nutrition 0.000 claims description 25
- 239000008103 glucose Substances 0.000 claims description 24
- 241000228212 Aspergillus Species 0.000 claims description 23
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- 238000012360 testing method Methods 0.000 claims description 13
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- NPFOYSMITVOQOS-UHFFFAOYSA-K iron(III) citrate Chemical compound [Fe+3].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NPFOYSMITVOQOS-UHFFFAOYSA-K 0.000 claims description 4
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- 235000007079 manganese sulphate Nutrition 0.000 claims description 4
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 4
- 235000009754 Vitis X bourquina Nutrition 0.000 claims description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 claims description 2
- 240000006365 Vitis vinifera Species 0.000 claims description 2
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- CMVQZRLQEOAYSW-UHFFFAOYSA-N 1,2-dichloro-3-nitrobenzene Chemical compound [O-][N+](=O)C1=CC=CC(Cl)=C1Cl CMVQZRLQEOAYSW-UHFFFAOYSA-N 0.000 claims 1
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- UMEAURNTRYCPNR-UHFFFAOYSA-N azane;iron(2+) Chemical compound N.[Fe+2] UMEAURNTRYCPNR-UHFFFAOYSA-N 0.000 description 1
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- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
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- 230000004048 modification Effects 0.000 description 1
- KGPQKNJSZNXOPV-UHFFFAOYSA-N moniliformin Chemical compound OC1=CC(=O)C1=O KGPQKNJSZNXOPV-UHFFFAOYSA-N 0.000 description 1
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- 235000016709 nutrition Nutrition 0.000 description 1
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- MBMQEIFVQACCCH-QBODLPLBSA-N zearalenone Chemical class O=C1O[C@@H](C)CCCC(=O)CCC\C=C\C2=CC(O)=CC(O)=C21 MBMQEIFVQACCCH-QBODLPLBSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
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- Virology (AREA)
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Abstract
The invention discloses a kind of separation method of lactic bacteria composition and its application, belong to microbial technology field, the lactic acid bacteria separates acquisition in the fermentation leaven in Xinjiang, and the lactic acid bacteria includes series bacillus, VREF and Lactococcus lactis；The present invention carries out the separation of lactic acid bacteria using laboratory culture-dependent method to the fermentation leaven in Xinjiang, and is filtered out using double-layer agar technique with the lactic acid bacteria for suppressing Aspergillus flavus.Series bacillus, VREF and the Lactococcus lactis screened in the present invention has obvious inhibitory action to Aspergillus flavus, and excellent strain is provided to improve ensilage quality.Therefore, the separation method of the lactic bacteria composition in the present invention and this kind of lactic bacteria composition are adapted to promote in microorganism field in the application of antibacterial aspect.
Description
Technical field
The present invention relates to separation method and its application of microbial technology field, more particularly to a kind of lactic bacteria composition.
Background technology
Fungi is the extremely strong putrefactive microorganisms of a class vitality, wherein the feed caused by saccharomycete and mould contamination
The existing a large amount of reports of loss.Have determined at present and have more than when 200 kinds of moulds grow in certain food, animal feed and can produce
The material being harmful to humans and animals, mainly there is aflatoxin class, moniliformin, conspicuous aspertoxin class, clavacin, list
Hold the mould alkenes of spore and the major class of zearalenone 6.It is also toxic to nerve, liver kidney because these toxin have carcinogenic teratogenesis
Effect, therefore these rot fungis, while huge economic losses are caused, also the health to the public constitutes great threat.Such as
Occur more than 10 ten thousand poults in " the turkey X disease " in 1960s Britain's chicken house, some months to die off, as a result 1963
Year confirms that this disease is that the aflatoxin B1 produced by Aspergillus flavus causes by Spensley.
Both at home and abroad numerous studies have been carried out to suppressing aflatoxin.But all there is certain disadvantage in these conventional methods
End, such as physical method can cause the loss of nutritional ingredient, and chemical preservative can then be brought to the health and environment of humans and animals
Harm.Therefore, it is badly in need of a kind of lactic bacteria composition for suppressing Aspergillus flavus at present.
The content of the invention
The technical problem to be solved in the present invention is to provide a kind of lactic acid bacteria combination that can suppress Aspergillus flavus and this kind of breast
The separation method of sour bacterium.The present invention is screened by using double-layer agar technique with the lactic acid bacteria for suppressing Aspergillus flavus activity, from
And provide excellent strain to improve ensilage quality.
In order to solve the above-mentioned technical problem, the invention provides a kind of separation method of lactic bacteria composition, the lactic acid
Bacterium separates acquisition in the fermentation leaven in Xinjiang, and the lactic acid bacteria includes series bacillus, VREF and Lactococcus lactis.
Further, the separation method of the lactic bacteria composition specifically includes following steps：
(1) 10g fermentation leavens are often weighed to be added in the container for filling 90mL sterile peptone water, and container sealing is put
In shaking 2h on shaking table with 120r/min, precipitation one and supernatant one are produced, 1mL supernatants one is drawn and is added to another fill
In the container of 9mL sterile peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacterium solution；
(2) take the bacterium solution prepared in 100 μ L steps (1) to be coated on lactic acid bacteria plating medium surface, and lactic acid bacteria is put down
Plate culture medium cultivates 48~72h under conditions of being placed in 30 DEG C, produces single bacterium colony；
(3) picking single bacterium colony is rule on lactic acid bacteria solid medium, and lactic acid bacteria solid medium is placed in into 30 DEG C
Under the conditions of cultivate 48~72h, repeat line be separately cultured 3 times, produce single strain, by single strain access lactic acid bacteria fluid nutrient medium
In, and lactic acid bacteria fluid nutrient medium is placed under conditions of 30 DEG C cultivates 24h, lactobacillus suspension is produced, by prepared lactic acid bacteria
After liquid and the mixing of isometric glycerine and encapsulate be placed in -70 DEG C under conditions of preserve, it is standby；
(4) the mouldy peanuts of 10g are weighed to be put into the container for filling 90mL sterile distilled waters, and container sealing is placed in shaking table
On 2h is shaken with 120r/min, produce precipitation two and supernatant two, draw 1mL supernatants two and be added to that another to fill 9mL sterile
In the container of peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacteria suspension；
(5) bacteria suspension prepared in 100 μ L steps (4) is taken to be coated on Aspergillus Solid agar culture surface, and by song
Mold agar solid medium is cultivated 4~7 days under conditions of being placed in 28 DEG C, produces aspergillus flavus bacterium colony, picking aspergillus flavus bacterium colony institute
The spore of production is rule separation on aspergillin Solid agar culture, produces aspergillus flavus strain；
(6) aspergillus flavus strain is seeded in potato glucose inclined-plane culture primary surface, and by potato glucose inclined-plane
Culture medium is cultivated 3~5 days under conditions of being placed in 28 DEG C, when aspergillus flavus strain produces a large amount of spores, adds 5mL sterile physiological salt
Water is placed in whirlpool concussion 5min on eddy mixer in the test tube equipped with potato glucose slant medium, and by test tube,
Aspergillus flavus suspension is produced, vegetative mycelium is filtered to remove with sterile gauze, and the concentration of aspergillus flavus suspension is adjusted to 105
Individual/mL, produces aspergillus spore liquid, standby；
(7) lactic acid bacteria solid medium is poured into culture dish as lower floor's flat board, after after its solidification, picked with oese
Standby lactobacillus suspension draws 2 2~3cm parallel lines on lower floor's flat board in step (3), and culture dish is placed in 37 DEG C of bar
24~36h is cultivated under part to pour into culture dish to growing lawn, then by aspergillus spore liquid standby in step (6), is covered in
48~72h is cultivated on lower floor's culture medium containing lactic acid bacteria lawn, and under conditions of culture dish is placed in into 28 DEG C, suppression is produced yellow
The lactic acid bacteria of Aspergillus.
Further, the lactic acid bacteria solid medium be by 10g peptone, 10g beef extract, 5g dusty yeast,
20g lemon acid diamine, 0.05g magnesium sulfate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 1g Tween 80,0.05g sulphur
The agar of sour manganese, 1g calcium carbonate and 15g is dissolved in after 1L deionized waters and the 15min that sterilized under conditions of 121 DEG C is made.
Further, the potato glucose slant medium is by the glucose and 8g of 200g potato, 20g
Agar be dissolved in after 1L deionized waters and under conditions of 121 DEG C sterilize 20min be made.
Further, the Aspergillus Solid agar culture be by 20g dusty yeast, 10g peptone, 0.5g lemon
Lemon acid iron ammonium, 1mL 0.2% Dichloran, 0.1g chloramphenicol and 15g agar are dissolved in after 1L deionized waters simultaneously
The 15min that sterilized under conditions of 121 DEG C is made.
Present invention also offers the lactic bacteria composition obtained by above-mentioned separation method in terms of Aspergillus flavus is suppressed
Using the lactic bacteria composition includes series bacillus, VREF and Lactococcus lactis.
Preferably, the lactic bacteria composition is any two kinds in series bacillus, VREF and Lactococcus lactis
Bacterium solution is with 1% inoculum concentration 1:1 combination of two is formed.
Preferably, the lactic bacteria composition by the bacterium solution of three kinds of series bacillus, VREF and Lactococcus lactis with
1% inoculum concentration 1:1 rhinoschisis is mixed.
Compared with prior art, the beneficial effect that the present invention is reached：
(1) series bacillus, VREF and the Lactococcus lactis screened in the present invention has suppression to Aspergillus flavus
Make and use, fungistatic effect is significantly improved after mixing two-by-two, wherein, the fungistatic effect mixed two-by-two preferably series bacillus liquid
With the combination of VREF liquid, antibacterial area's diameter can reach 5.3~5.4cm；By series bacillus liquid, VREF liquid and lactic acid breast
Fungistatic effect is optimal after coccus liquid three mixing, and antibacterial area's diameter reaches 6.2~6.4cm；The fungistatic effect of VREF liquid shows
The fungistatic effect higher than other single bacterium solutions is write, antibacterial area's diameter of VREF liquid reaches 4.5~5.0cm.
(2) series bacillus, VREF and the Lactococcus lactis in the present invention can give birth under the conditions of 40 DEG C and 45 DEG C
It is long, illustrate that the series bacillus in the present invention, VREF and Lactococcus lactis can grow under the high temperature conditions；In the present invention
Series bacillus, VREF and Lactococcus lactis can be grown in the case where NaCl concentration reaches 3% or 6.5%, explanation
The bacterial strain that the present invention is screened can salty area survival；The bacterial strain screened simultaneously can also be under conditions of acidity
Existence.
(3) it is separately cultured 3 times when preparing lactobacillus suspension, it is necessary to repeat line in the present invention；Repeat line culture be for
Determine without miscellaneous bacteria；After lactobacillus suspension and isometric glycerine are mixed in the present invention and encapsulate be placed in -70 DEG C under conditions of protect
Deposit；Glycerine is added into lactobacillus suspension, can make the freezing point of lactobacillus suspension reduces, and under the conditions of slow freezing, can make intracellular water
Part appears cell before freezing, so as to reduce the formation of ice crystal.
Brief description of the drawings
The present invention is described in further detail below in conjunction with the drawings and the specific embodiments.
Fig. 1 is the schematic diagram of aspergillus flavus bacterium colony in the present invention；
Fig. 2 is inhibition figure of the lactobacillus suspension to Aspergillus flavus in the present invention.
Embodiment
Following examples are merely to illustrate the present invention, but are not limited to the scope of the present invention.
The source of 1 material
1.1 sample source：In July, 2016 from Aksu, Xinjiang random acquisition fermentation leaven, mouldy peanut, mouldy jade
Rice and rotten ensiling sample.Sample is encapsulated in hermetic bag, it is labelled, indicate sampling time and place after transport laboratory back,
Under the conditions of 4 DEG C, saved backup by livestock technology key lab of Tarim University Livestock nutrient research room.
1.2 strain source：Separation obtains lactic acid bacteria from fermentation leaven sample, from mouldy peanut, mouldy corn and rotten
Separation obtains Aspergillus flavus in ensilage, is protected by livestock technology key lab of Tarim University Livestock nutrient research room
Deposit standby.
Embodiment 1
A kind of separation method of lactic bacteria composition, the lactic bacteria composition is separated in the fermentation leaven in Xinjiang and obtained
, the lactic bacteria composition includes series bacillus, VREF and Lactococcus lactis.
Specifically, the separation method step of above-mentioned lactic acid bacteria combination is as follows：
(1) 10g fermentation leavens are weighed to be added in the container for filling 90mL sterile peptone water, and container sealing is placed in
2h is shaken with 120r/min on shaking table, precipitation one and supernatant one is produced, 1mL supernatants one is drawn and is added to and another fill 9mL
In the container of sterile peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacterium solution；
(2) take the bacterium solution prepared in 100 μ L steps (1) to be coated on lactic acid bacteria plating medium surface, and lactic acid bacteria is put down
Plate culture medium cultivates 48h under conditions of being placed in 30 DEG C, produces single bacterium colony；
(3) picking single bacterium colony is rule on lactic acid bacteria solid medium, and lactic acid bacteria solid medium is placed in into 30 DEG C
Under the conditions of cultivate 48h, repeat line and be separately cultured 3 times, produce single strain, single strain is accessed in lactic acid bacteria fluid nutrient medium,
And lactic acid bacteria fluid nutrient medium be placed under conditions of 30 DEG C cultivate 24h, produce lactobacillus suspension, by prepared lactobacillus suspension and
After isometric glycerine mixing and encapsulate be placed in -70 DEG C under conditions of preserve, it is standby；
(4) the mouldy peanuts of 10g are weighed to be put into the container for filling 90mL sterile distilled waters, and container sealing is placed in shaking table
On 2h is shaken with 120r/min, produce precipitation two and supernatant two, draw 1mL supernatants two and be added to that another to fill 9mL sterile
In the container of peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacteria suspension；
(5) bacteria suspension prepared in 100 μ L steps (4) is taken to be coated on Aspergillus Solid agar culture surface, and by song
Mold agar solid medium is cultivated 4 days under conditions of being placed in 28 DEG C, produces aspergillus flavus bacterium colony, picking aspergillus flavus bacterium colony is produced
Spore is rule separation on aspergillin Solid agar culture, produces aspergillus flavus strain；
(6) aspergillus flavus strain is seeded in potato glucose inclined-plane culture primary surface, and by potato glucose inclined-plane
Culture medium is cultivated 3 days under conditions of being placed in 28 DEG C, when aspergillus flavus strain produces a large amount of spores, adds 5mL sterile salines
Whirlpool concussion 5min on eddy mixer is placed in the test tube equipped with potato glucose slant medium, and by test tube, i.e.,
Aspergillus flavus suspension is obtained, vegetative mycelium is filtered to remove with sterile gauze, and the concentration of aspergillus flavus suspension is adjusted to 105
Individual/mL, produces aspergillus spore liquid, standby；
(7) lactic acid bacteria solid medium is poured into culture dish as lower floor's flat board, after after its solidification, picked with oese
Standby lactobacillus suspension draws 2 2cm parallel lines on lower floor's flat board in step (3), and culture dish is placed in 37 DEG C of condition
Lower culture 24h is poured into culture dish to growing lawn, then by aspergillus spore liquid standby in step (6), is covered in containing breast
On lower floor's culture medium of sour bacterium lawn, and culture dish is placed under conditions of 28 DEG C cultivates 48h, produce the breast for suppressing Aspergillus flavus
Sour bacteria composition.
In the present embodiment, the lactic acid bacteria solid medium be by 10g peptone, 10g beef extract, 5g yeast
Powder, 20g lemon acid diamine, 0.05g magnesium sulfate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 1g Tween 80,0.05g
The agar of manganese sulfate, 1g calcium carbonate and 15g is dissolved in after 1L deionized waters and the 15min that sterilized under conditions of 121 DEG C is made.
In the present embodiment, the potato glucose slant medium be by 200g potato, 20g glucose with
And 8g agar is dissolved in after 1L deionized waters and the 20min that sterilized under conditions of 121 DEG C is made.
In the present embodiment, the Aspergillus Solid agar culture is by 20g dusty yeast, 10g peptone, 0.5g
Ferric citrate, 1mL 0.2% Dichloran, 0.1g chloramphenicol and 15g agar be dissolved in 1L deionized waters
The 15min that sterilizes afterwards and under conditions of 121 DEG C is made.
Embodiment 2
A kind of separation method of lactic bacteria composition, the lactic bacteria composition is separated in the fermentation leaven in Xinjiang and obtained
, the lactic bacteria composition includes series bacillus, VREF and Lactococcus lactis.
Specifically, the separation method step of above-mentioned lactic bacteria composition is as follows：
(1) 10g fermentation leavens are weighed to be added in the container for filling 90mL sterile peptone water, and container sealing is placed in
2h is shaken with 120r/min on shaking table, precipitation one and supernatant one is produced, 1mL supernatants one is drawn and is added to and another fill 9mL
In the container of sterile peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacterium solution；
(2) take the bacterium solution prepared in 100 μ L steps (1) to be coated on lactic acid bacteria plating medium surface, and lactic acid bacteria is put down
Plate culture medium cultivates 60h under conditions of being placed in 30 DEG C, produces single bacterium colony；
(3) picking single bacterium colony is rule on lactic acid bacteria solid medium, and lactic acid bacteria solid medium is placed in into 30 DEG C
Under the conditions of cultivate 60h, repeat line and be separately cultured 3 times, produce single strain, single strain is accessed in lactic acid bacteria fluid nutrient medium,
And lactic acid bacteria fluid nutrient medium be placed under conditions of 30 DEG C cultivate 24h, produce lactobacillus suspension, by prepared lactobacillus suspension and
After isometric glycerine mixing and encapsulate be placed in -70 DEG C under conditions of preserve, it is standby；
(4) the mouldy peanuts of 10g are weighed to be put into the container for filling 90mL sterile distilled waters, and container sealing is placed in shaking table
On 2h is shaken with 120r/min, produce precipitation two and supernatant two, draw 1mL supernatants two and be added to that another to fill 9mL sterile
In the container of peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacteria suspension；
(5) bacteria suspension prepared in 100 μ L steps (4) is taken to be coated on Aspergillus Solid agar culture surface, and by song
Mold agar solid medium is cultivated 6 days under conditions of being placed in 28 DEG C, produces aspergillus flavus bacterium colony, picking aspergillus flavus bacterium colony is produced
Spore is rule separation on aspergillin Solid agar culture, produces aspergillus flavus strain；
(6) aspergillus flavus strain is seeded in potato glucose inclined-plane culture primary surface, and by potato glucose inclined-plane
Culture medium is cultivated 4 days under conditions of being placed in 28 DEG C, when aspergillus flavus strain produces a large amount of spores, adds 5mL sterile salines
Whirlpool concussion 5min on eddy mixer is placed in the test tube equipped with potato glucose slant medium, and by test tube, i.e.,
Aspergillus flavus suspension is obtained, vegetative mycelium is filtered to remove with sterile gauze, and the concentration of aspergillus flavus suspension is adjusted to 105
Individual/mL, produces aspergillus spore liquid, standby；
(7) lactic acid bacteria solid medium is poured into culture dish as lower floor's flat board, after after its solidification, picked with oese
Standby lactobacillus suspension draws 2 2.5cm parallel lines on lower floor's flat board in step (3), and culture dish is placed in 37 DEG C of bar
30h is cultivated under part to pour into culture dish to growing lawn, then by aspergillus spore liquid standby in step (6), be covered in containing
On lower floor's culture medium of lactic acid bacteria lawn, and culture dish is placed under conditions of 28 DEG C cultivates 60h, produce and suppress Aspergillus flavus
Lactic bacteria composition.
In the present embodiment, the lactic acid bacteria solid medium be by 10g peptone, 10g beef extract, 5g yeast
Powder, 20g lemon acid diamine, 0.05g magnesium sulfate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 1g Tween 80,0.05g
The agar of manganese sulfate, 1g calcium carbonate and 15g is dissolved in after 1L deionized waters and the 15min that sterilized under conditions of 121 DEG C is made.
In the present embodiment, the potato glucose slant medium be by 200g potato, 20g glucose with
And 8g agar is dissolved in after 1L deionized waters and the 20min that sterilized under conditions of 121 DEG C is made.
In the present embodiment, the Aspergillus Solid agar culture is by 20g dusty yeast, 10g peptone, 0.5g
Ferric citrate, 1mL 0.2% Dichloran, 0.1g chloramphenicol and 15g agar be dissolved in 1L deionized waters
The 15min that sterilizes afterwards and under conditions of 121 DEG C is made.
Embodiment 3
A kind of separation method of lactic bacteria composition, the lactic bacteria composition is separated in the fermentation leaven in Xinjiang and obtained
, the lactic bacteria composition includes series bacillus, VREF and Lactococcus lactis.
Specifically, the separation method step of above-mentioned lactic bacteria composition is as follows：
(1) 10g fermentation leavens are weighed to be added in the container for filling 90mL sterile peptone water, and container sealing is placed in
2h is shaken with 120r/min on shaking table, precipitation one and supernatant one is produced, 1mL supernatants one is drawn and is added to and another fill 9mL
In the container of sterile peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacterium solution；
(2) take the bacterium solution prepared in 100 μ L steps (1) to be coated on lactic acid bacteria plating medium surface, and lactic acid bacteria is put down
Plate culture medium cultivates 72h under conditions of being placed in 30 DEG C, produces single bacterium colony；
(3) picking single bacterium colony is rule on lactic acid bacteria solid medium, and lactic acid bacteria solid medium is placed in into 30 DEG C
Under the conditions of cultivate 72h, repeat line and be separately cultured 3 times, produce single strain, single strain is accessed in lactic acid bacteria fluid nutrient medium,
And lactic acid bacteria fluid nutrient medium be placed under conditions of 30 DEG C cultivate 24h, produce lactobacillus suspension, by prepared lactobacillus suspension and
After isometric glycerine mixing and encapsulate be placed in -70 DEG C under conditions of preserve, it is standby；
(4) the mouldy peanuts of 10g are weighed to be put into the container for filling 90mL sterile distilled waters, and container sealing is placed in shaking table
On 2h is shaken with 120r/min, produce precipitation two and supernatant two, draw 1mL supernatants two and be added to that another to fill 9mL sterile
In the container of peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacteria suspension；
(5) bacteria suspension prepared in 100 μ L steps (4) is taken to be coated on Aspergillus Solid agar culture surface, and by song
Mold agar solid medium is cultivated 7 days under conditions of being placed in 28 DEG C, produces aspergillus flavus bacterium colony, picking aspergillus flavus bacterium colony is produced
Spore is rule separation on aspergillin Solid agar culture, produces aspergillus flavus strain；
(6) aspergillus flavus strain is seeded in potato glucose inclined-plane culture primary surface, and by potato glucose inclined-plane
Culture medium is cultivated 5 days under conditions of being placed in 28 DEG C, when aspergillus flavus strain produces a large amount of spores, adds 5mL sterile salines
Whirlpool concussion 5min on eddy mixer is placed in the test tube equipped with potato glucose slant medium, and by test tube, i.e.,
Aspergillus flavus suspension is obtained, vegetative mycelium is filtered to remove with sterile gauze, and the concentration of aspergillus flavus suspension is adjusted to 105
Individual/mL, produces aspergillus spore liquid, standby；
(7) lactic acid bacteria solid medium is poured into culture dish as lower floor's flat board, after after its solidification, picked with oese
Standby lactobacillus suspension draws 2 3cm parallel lines on lower floor's flat board in step (3), and culture dish is placed in 37 DEG C of condition
Lower culture 36h is poured into culture dish to growing lawn, then by aspergillus spore liquid standby in step (6), is covered in containing breast
On lower floor's culture medium of sour bacterium lawn, and culture dish is placed under conditions of 28 DEG C cultivates 72h, produce the breast for suppressing Aspergillus flavus
Sour bacteria composition.
In the present embodiment, the lactic acid bacteria solid medium be by 10g peptone, 10g beef extract, 5g yeast
Powder, 20g lemon acid diamine, 0.05g magnesium sulfate, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 1g Tween 80,0.05g
The agar of manganese sulfate, 1g calcium carbonate and 15g is dissolved in after 1L deionized waters and the 15min that sterilized under conditions of 121 DEG C is made.
In the present embodiment, the potato glucose slant medium be by 200g potato, 20g glucose with
And 8g agar is dissolved in after 1L deionized waters and the 20min that sterilized under conditions of 121 DEG C is made.
In the present embodiment, the Aspergillus Solid agar culture is by 20g dusty yeast, 10g peptone, 0.5g
Ferric citrate, 1mL 0.2% Dichloran, 0.1g chloramphenicol and 15g agar be dissolved in 1L deionized waters
The 15min that sterilizes afterwards and under conditions of 121 DEG C is made.
Experiment and result
The lactic bacteria composition and aspergillus flavus bacterium colony screened with embodiment 2, does experimental summary as follows：
1st, the aspergillus flavus bacterium colony screened from mouldy peanut is radial, and spore is in faint yellow；In potato grape
Grown on sugared slant medium, the culture medium back side is in bright orange, as shown in Figure 1.
2nd, it will find there are 3 strains of lactic acid bacteria to have suppression to Aspergillus flavus after the lactic bacteria composition the isolated screening of embodiment 2
Make and use, as shown in Fig. 2 wherein this 3 strains of lactic acid bacteria is respectively series bacillus, VREF and Lactococcus lactis, specific suppression
Bacterium effect is as shown in the table.
Inhibition table of the lactobacillus suspension of table 1 to Aspergillus flavus
Note：With marked after column data different lowercase letter indication differences significantly (P<0.05).
As shown in Table 1, series bacillus, VREF and the Lactococcus lactis that the present invention is screened are equal to Aspergillus flavus
There is inhibitory action, fungistatic effect is significantly improved after mixing two-by-two, wherein, the fungistatic effect mixed two-by-two preferably class gemma bar
Bacterium solution and the combination of VREF liquid, antibacterial area's diameter can reach 5.3~5.4cm；By series bacillus liquid, VREF liquid and breast
Fungistatic effect is optimal after yogurt coccus liquid three mixing, and antibacterial area's diameter reaches 6.2~6.4cm；The antibacterial effect of VREF liquid
Fruit is significantly higher than the fungistatic effect of other single bacterium solutions, and antibacterial area's diameter of VREF liquid reaches 4.5~5.0cm.
3rd, aerogenesis is carried out respectively to the series bacillus liquid with bacteriostatic activity, VREF liquid and Lactococcus lactis bacterium solution real
Test, temperature growth experiment and acid-fast alkali-proof are tested, as a result as shown in table 2 and table 3.
The lactobacillus suspension aerogenesis of table 2 is tested and temperature growth tests measurement result table
Note："+" represents growth, and "-" represents not grow, and " W " represents weak growth, similarly hereinafter.
As can be seen from Table 2,3 strains of lactic acid bacteria liquid glucose fermentations are unable to aerogenesis in aerogenesis experiment, and explanation is same
Type fermentative lactobacillus；In temperature growth experiment, 3 strains of lactic acid bacteria liquid can be grown under the conditions of 5 DEG C, and dung is removed under the conditions of 10 DEG C
Enterococcus liquid well-grown, remaining 2 plants of bacterium solution growth are weaker, and under the conditions of 40 DEG C and 45 DEG C, 3 strains of lactic acid bacteria liquid grow good
It is good.
The acidproof Salt tolerance measurement result of the lactic acid bacteria of table 3
As can be seen from Table 3, in acidproof Salt tolerance, when NaCl is 3% and 6.5%, 3 plants of bacterium can well grow；
Under conditions of pH=3,3 strains of lactic acid bacteria liquid can not grow；Under conditions of pH=4,3 plants of bacteria growings are weaker；It is 5 in pH value
When~7,3 plants of bacteria growings are in order.
4th, carbon source through fermentation reality is carried out to the series bacillus liquid with bacteriostatic activity, VREF liquid and Lactococcus lactis bacterium solution
Test, as a result as shown in table 4 and table 5.
The lactic acid bacteria carbon source through fermentation of table 4 is tested
The lactic acid bacteria carbon source through fermentation of table 5 is tested
From table 4 and the strains of lactic acid bacteria liquid of table 5,3 using D-Glucose, D-Maltose, D-MANNOSE, fiber two
Sugar, PEARLITOL 25C, D-Fructose, sucrose and D-glucitol；The unfermentable lactose of series bacillus liquid, D- galactolipins, L- sorboses,
D- melezitoses, xylitol, D- raffinoses, L- rhamnoses, D- xyloses and D- melibioses；The unfermentable L- sorbs of VREF liquid
Sugar, D- turanoses, xylitol, D- raffinoses and L- rhamnoses；Lactococcus lactis bacterium solution can not utilize L- sorboses, xylitol, D-
Raffinose, L- rhamnoses and D- xyloses.
In summary, series bacillus liquid, VREF liquid and the Lactococcus lactis bacterium solution screened in the present invention is to Huang
Aspergillus has obvious fungistatic effect, also, fungistatic effect after 3 plants of bacterium solutions mixing is optimal.
Those of ordinary skill in the art it should be appreciated that the embodiment of the above be intended merely to explanation the present invention,
And be not used as limitation of the invention, as long as in the spirit of the present invention, the change to embodiment described above
Change, modification will all fall in scope of the presently claimed invention.
Claims (8)
1. a kind of separation method of lactic bacteria composition, it is characterised in that the lactic bacteria composition by Xinjiang fermentation leaven
Middle separation is obtained, and the lactic bacteria composition that the separation is obtained includes series bacillus, VREF and Lactococcus lactis.
2. the separation method of a kind of lactic bacteria composition according to claim 1, it is characterised in that comprise the following steps that：
(1) often weigh 10g Xinjiang fermentation leaven to be added in the container for filling 90mL sterile peptone water, and container sealing is put
In shaking 2h on shaking table with 120r/min, precipitation one and supernatant one are produced, 1mL supernatants one is drawn and is added to another fill
In the container of 9mL sterile peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacterium solution；
(2) take the bacterium solution prepared in 100 μ L steps (1) to be coated on lactic acid bacteria plating medium surface, and lactic acid bacteria flat board is trained
Foster base cultivates 48~72h under conditions of being placed in 30 DEG C, produces single bacterium colony；
(3) picking single bacterium colony is rule on lactic acid bacteria solid medium, and lactic acid bacteria solid medium is placed in 30 DEG C of condition
48~72h of lower culture, repeats line and is separately cultured 3 times, produce single strain, single strain is accessed in lactic acid bacteria fluid nutrient medium,
And lactic acid bacteria fluid nutrient medium be placed under conditions of 30 DEG C cultivate 24h, produce lactobacillus suspension, by prepared lactobacillus suspension and
After isometric glycerine mixing and encapsulate be placed in -70 DEG C under conditions of preserve, it is standby；
(4) the mouldy peanuts of 10g are weighed to be put into the container for filling 90mL sterile distilled waters, and by container sealing be placed on shaking table with
120r/min shakes 2h, produces precipitation two and supernatant two, draws 1mL supernatants two and is added to another 9mL that fills without mycoprotein
In the container of peptone water, and container is placed in whirlpool concussion on eddy mixer, produces bacteria suspension；
(5) bacteria suspension prepared in 100 μ L steps (4) is taken to be coated on Aspergillus Solid agar culture surface, and by Aspergillus
Solid agar culture is cultivated 4~7 days under conditions of being placed in 28 DEG C, produces aspergillus flavus bacterium colony, picking aspergillus flavus bacterium colony is produced
Spore is rule separation on aspergillin Solid agar culture, produces aspergillus flavus strain；
(6) aspergillus flavus strain is seeded in potato glucose inclined-plane culture primary surface, and by potato glucose inclined-plane culture
Base be placed in 28 DEG C under conditions of cultivate 3~5 days, whne aspergillus flavus strain produce a large amount of spores when, add 5mL sterile salines in
In test tube equipped with potato glucose slant medium, and test tube is placed in whirlpool concussion 5min on eddy mixer, produced
Aspergillus flavus suspension, vegetative mycelium is filtered to remove with sterile gauze, and the concentration of aspergillus flavus suspension is adjusted into 105Individual/
ML, produces aspergillus spore liquid, standby；
(7) lactic acid bacteria solid medium is poured into culture dish as lower floor's flat board, after after its solidification, step is picked with oese
(3) standby lactobacillus suspension draws 2 2~3cm parallel lines on lower floor's flat board in, and culture dish is placed under conditions of 37 DEG C
24~36h is to growing lawn for culture, then aspergillus spore liquid standby in step (6) is poured into culture dish, be covered in containing
48~72h is cultivated on lower floor's culture medium of lactic acid bacteria lawn, and under conditions of culture dish is placed in into 28 DEG C, suppression aspergillus flavus is produced
The lactic acid bacteria of bacterium.
3. a kind of separation method of lactic bacteria composition according to claim 2, it is characterised in that the lactic acid bacteria solid
Culture medium is by 10g peptone, 10g beef extract, 5g dusty yeast, 20g lemon acid diamine, 0.05g magnesium sulfate, 2g
Dipotassium hydrogen phosphate, 5g sodium acetate, 1g Tween 80,0.05g manganese sulfate, 1g calcium carbonate and 15g agar is dissolved in
The 15min that sterilized after 1L deionized waters and under conditions of 121 DEG C is made.
4. a kind of separation method of lactic bacteria composition according to claim 2, it is characterised in that the potato grape
Sugared slant medium is that the agar of 200g potato, 20g glucose and 8g is dissolved in after 1L deionized waters and at 121 DEG C
Under conditions of sterilizing 20min be made.
5. a kind of separation method of lactic bacteria composition according to claim 2, it is characterised in that the aspergillus bacterio-agar
Solid medium be by 20g dusty yeast, 10g peptone, 0.5g ferric citrate, 1mL 0.2% dichloronitrobenzene
The agar of amine, 0.1g chloramphenicol and 15g is dissolved in after 1L deionized waters and the 15min that sterilized under conditions of 121 DEG C is made.
6. the lactic bacteria composition obtained by separation method as described in claim any one of 1-5 is in terms of Aspergillus flavus is suppressed
Application, it is characterised in that the lactic bacteria composition include series bacillus, VREF and Lactococcus lactis.
7. application of the lactic bacteria composition according to claim 6 in terms of Aspergillus flavus is suppressed, it is characterised in that described
Lactic bacteria composition in series bacillus, VREF and Lactococcus lactis any two kinds of bacterium solution with 1% inoculum concentration 1:1
Combination of two is formed.
8. application of the lactic bacteria composition according to claim 6 in terms of Aspergillus flavus is suppressed, it is characterised in that described
Lactic bacteria composition is by the bacterium solution of three kinds of series bacillus, VREF and Lactococcus lactis with 1% inoculum concentration 1:1 rhinoschisis is mixed
Conjunction is formed.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710347980.6A CN107022509B (en) | 2017-05-17 | 2017-05-17 | Separation method and application of lactobacillus composition |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710347980.6A CN107022509B (en) | 2017-05-17 | 2017-05-17 | Separation method and application of lactobacillus composition |
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| CN107022509B CN107022509B (en) | 2021-04-02 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885875A (en) * | 2019-11-14 | 2020-03-17 | 华东师范大学 | Method for high-throughput screening of mould-inhibiting lactic acid bacteria |
| CN111187729A (en) * | 2019-11-14 | 2020-05-22 | 华东师范大学 | Screening method of propionibacterium and lactobacillus combined bacteria with synergistic mildew inhibition activity |
| CN113265352A (en) * | 2021-05-11 | 2021-08-17 | 塔里木大学 | Preparation method and application of enterococcus faecium powder |
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| 唐雨蕊等: "乳酸菌对黄曲霉生长和产毒的抑制影响", 《中国畜牧医学会动物微生态学分会第四届第九次学术研讨会论文集（上册）》 * |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110885875A (en) * | 2019-11-14 | 2020-03-17 | 华东师范大学 | Method for high-throughput screening of mould-inhibiting lactic acid bacteria |
| CN111187729A (en) * | 2019-11-14 | 2020-05-22 | 华东师范大学 | Screening method of propionibacterium and lactobacillus combined bacteria with synergistic mildew inhibition activity |
| CN113265352A (en) * | 2021-05-11 | 2021-08-17 | 塔里木大学 | Preparation method and application of enterococcus faecium powder |
| CN113265352B (en) * | 2021-05-11 | 2023-08-25 | 塔里木大学 | Preparation method and application of enterococcus faecium powder |
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