CN109207396A - A kind of application of method and the strain that screening strain using squid viscera - Google Patents
A kind of application of method and the strain that screening strain using squid viscera Download PDFInfo
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- CN109207396A CN109207396A CN201811062276.7A CN201811062276A CN109207396A CN 109207396 A CN109207396 A CN 109207396A CN 201811062276 A CN201811062276 A CN 201811062276A CN 109207396 A CN109207396 A CN 109207396A
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Abstract
The present invention relates to field of biotechnology, disclose a kind of method using squid viscera screening strain and the application of the strain.The present invention is using squid viscera as sole substrate, by unknown bacterium solution together with squid viscera fermented and cultured, it is cultivated being applied on aseptic culture medium after the dilution of squid viscera fermentation liquid, the form of bacterium colony after observation culture, choose the bacterium colony of three kinds of most forms of colony counts, the three kinds of bacterium colonies that will be singled out are crossed in aseptic culture medium respectively to be isolated and purified, and three purified kind bacterial strain is just the dominant strain of degradation squid viscera.The logical mixed bacteria liquid to unknown constituent of the present invention carries out the screening of specified strain, separates the dominant bacteria that can efficiently utilize squid leftover bits and pieces waste.Squid viscera can be recycled by means of the present invention, extract nutriment therein, avoid wasting, while also avoiding environmental pollution.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of methods and the strain using squid viscera screening strain
Application.
Background technique
Marine product internal organ refer to the waste material generated during marine products processing and some leftover pieces, but contained nutrition
Ingredient is extremely abundant.Squid is also referred to as squid, sea mollusk, and the waste of about 20-25% is generated in process,
Its Hitting Viscera is in the great majority, followed by skin, eyes, cartilage and pure white.These by-products rich in taurine, protein, chondroitin,
The nutriments such as collagen, proteoglycan, chitin.Collagen is commonly applied in cosmetics and healthcare products, by glue
Former albumen is extracted from collagen solution can assist being used for pharmaceuticals industry.It is right but since current processing factory's technology is limited
The utility value of these by-products is also indefinite, and the mode of direct emergency burial is just taken to handle, or carries out some simple
Roughing, be made into fish-meal after having dried, do so many nutriments for not only wasting squid, also polluted ring
Border, it is more serious to cause economy and health problem.
The patent of Chinese patent application Publication No. CN104938604B discloses a kind of comprehensive benefit of aquatic products processing leftover bits and pieces
Use method.This method is to pass through cleaning, low temperature drying, freezing powder using the crustaceans such as fish, shrimp, crab processing fent as raw material
Broken, complex enzyme degradation, organic acid extraction, centrifuge separation, reverse osmosis dehydration, molecule embedding, high-pressure homogeneous, vacuum and low temperature freezing type
The full set technique such as drying, granulation, tabletting, automatic bottle packing production line, outer packing, will be all high attached in aquatic products processing leftover bits and pieces
Value added bioactive ingredients --- astaxanthin, protein, organic calcium and chitin and its derivative chitosan etc. extract together
Out.But this method is complicated for operation, and water consumption is big, waste water resource.Meanwhile at the unpowered ecological sewage in underground in the invention
Reason system is buried in ground end depths, and difficulty of construction is big.
Summary of the invention
In order to solve the above-mentioned technical problems, the present invention provides a kind of method using squid viscera screening strain and the bacterium
The application of kind.The present invention reprocesses squid viscera waste, acts in conjunction with microbial fermentation, recycles to it, extract it
Nutriment.The present invention carries out specified strain using squid viscera as sole substrate, to the mixed bacteria liquid of unknown constituent
Screening separates the dominant bacteria that can efficiently utilize leftover bits and pieces waste.Squid viscera waste can be recycled, be extracted
Nutriment therein avoids wasting, while also avoiding environmental pollution.
The specific technical proposal of the invention is: a kind of method using squid viscera screening strain, comprising the following steps:
(1) under gnotobasis, homogenate is semisolid after squid viscera is sterilized；
(2) under gnotobasis, by semisolid squid viscera, sterile water and mixed bacteria liquid together fermented and cultured, squid viscera is obtained
Fermentation liquid；
(3) under aseptic condition, squid viscera fermentation liquid is subjected to gradient dilution with sterile water, the squid viscera after dilution is fermented
Liquid, which is coated in aseptic culture medium, to be cultivated to there is apparent bacterium colony to grow；
(4) under aseptic condition, the bacterium colony of three kinds of most different shapes of picking quantity is successively named as X1, X2, X3, every kind of shape
The single colonie of state is crossed respectively cultivates in different aseptic culture mediums to there is apparent bacterium colony to grow；
(5) under aseptic condition, the scribing line respectively that will be cultivated through step (4) has the single bacterium of X1, X2, X3 in the culture medium of X1, X2, X3
It falls to be crossed in aseptic culture medium again and cultivate；Scribing line at least 3 times is repeated, the strain finally obtained is the bacterium after isolating and purifying
Kind X1, X2, X3.
The present invention using squid viscera as sole substrate, by unknown bacterium solution together with squid viscera fermented and cultured, by squid
It is applied on aseptic culture medium and cultivates after the dilution of internal organ fermentation liquid, the form of the bacterium colony after observation culture chooses colony counts most
The bacterium colony of three kinds of more forms, the three kinds of bacterium colonies that will be singled out are crossed in aseptic culture medium respectively to be isolated and purified, purifying
Three kinds of obtained bacterial strains are just the dominant strain of degradation squid viscera.The logical mixed bacteria liquid to unknown constituent of the present invention carries out
The screening of specified strain separates the dominant bacteria that can efficiently utilize leftover bits and pieces waste.By means of the present invention can
Squid viscera waste is recycled, nutriment therein is extracted, avoids wasting, while also avoiding environmental pollution.
Preferably, in step (2), the squid viscera, sterile water material-water ratio be 1:1~9, the addition of mixed bacteria liquid
Amount is the 3~25% of squid viscera suspension volume.
Preferably, the additive amount of the mixed bacteria liquid is the 3~8% of squid viscera suspension volume.Inventor is through excessive
The experiment of amount finds that, when the additive amount of mixed bacteria liquid is the 3~8% of squid viscera suspension volume, the effect of fermented and cultured is most
It is good.
Due to squid viscera by homogenate at semi-solid, mixed bacteria liquid is easy to be uniformly mixed with squid viscera, fermented and cultured
Shi Tianjia less mixed bacteria liquid can reach good degradation effect.When the amount of mixed bacteria liquid is very few, fermenting speed is slow, drop
Solution is incomplete.When the amount of mixed bacteria liquid is excessive, it will lead to bacterium solution and reach the stage of stable development in a short time, and then enter decline quickly
Phase causes colony counts to reduce, and is also unfavorable for the progress of fermentation.Inventor is by a large amount of experiment discovery, when mixed bacteria liquid
When additive amount is the 3~8% of crab leftover bits and pieces suspension volume, the effect of fermented and cultured is best.
Preferably, the flora in the mixed bacteria liquid includes photosynthetic bacteria group, yeast flora, Gram positive actinomycetes group
With the Filamentous flora of fermentation system, the mass percentage concentration of mixed bacteria liquid is 0.4~1%.
Mixed bacteria liquid is the silk for being of business comprising photosynthetic bacteria group, yeast flora, Gram positive actinomycetes group and fermentation
The bacterium solution of shape flora certainly exists the dominant colony of certain several fermentation squid viscera, by it when fermenting together with squid viscera
Isolate and purify the dominant colony that energy efficient degradation squid viscera can be obtained.When the concentration of mixed bacteria liquid is 0.4~1wt%, mix
The bacterium colony for the different shape in squid viscera fermentation liquid that combined bacteria liquid ferments together with squid viscera is easy to distinguish, and is easy to pick out
The bacterium colony of advantage.When the excessive concentration of mixed bacteria liquid, the bacterium colony of different shape is not easy to distinguish in squid viscera fermentation liquid, excellent
Gesture bacterial strain isolates and purifies difficulty.When the concentration of mixed bacteria liquid is too low, fermentation time is too long.
Preferably, the temperature of the fermented and cultured is 28~30 DEG C in step (2), shaking speed is 100~120r/
Min, incubation time are 1~2d.Under condition of culture of the invention, fermented and cultured effect is best.
Preferably, the squid viscera fermentation liquid after the dilution is three gradients, and respectively gradient is dilute in step (3)
The 10 of concentration before releasing-4、10-5、10-6Squid viscera fermentation liquid again, the condition of the culture and step (2) middle condition cultivated
It is identical.
Preferably, step (3), (4), in (5), the aseptic culture medium is to be purchased from Qingdao sea to win biotechnology limited
The nutrient agar of company sterilizes what 30min was obtained through 121 DEG C.
Preferably, identifying the strain X 1 after being isolated and purified described in step (5), X2, X3, identification method
Are as follows: strain X 1, X2, X3 after isolating and purifying are crossed to respectively in nutrient broth solid medium tablets, and nutrient broth is consolidated
The bacterium colony to grow on body culture medium flat plate observes colonial morphology after carrying out Gram's staining under the microscope；Meanwhile through meticulous
The extraction of bacterium genome, special primer expand the 16SrDNA that 16SrDNA sequence, purified pcr product, DNA sequencing obtain X1, X2, X3
Sequence, the 16SrDNA sequence of obtained X1, X2, X3 is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 institute
Show.
The 16SrDNA nucleotide sequence of strain X 1 are as follows: CCCTCTAGATGCATGCTCGAGGGAGAGTTTGATCAGGCTC
AGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAGAGCTTGCTCTTATGAAGTTAGCG
GCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGAT
AACATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTATGGATGGACCCGCGTCGCATTAGC
TAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGAGTGATCGGCCACACTGGGACTGA
GACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGGCGGAGCAACGCCG
CGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAGTGCTAGTTGAATAAGCTGGCACC
TTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATC
CGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAAGCCCACGGCTCAACCGTGGAGGG
TCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGAAAAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAGAGATAT
GGAGGAACACCAGTGGCGAAGACGACTTTCTGGTCTGTAACTGACACTGAGGCGCGAAAGCATGGGGAGCAAACAGG
ATTAGATACCCTGG
The 16SrDNA nucleotide sequence of strain X 2 are as follows: GGAGAGTTTGATCTGGCTCAGATTGAACGCTGGCGGCAGGCCTA
ACACATGCAAGTCGAGCGGATGAAAGGAGCTTGCTCCTGGATTCAGCGGCGGACGGGTGAGTAATGCCTAGGAATCT
GCCTGGTAGTGGGGGACAACGTTTCGAAAGGAACGCTAATACCGCATACGTCCTACGGGAGAAAGCAGGGGACCTTC
GGGCCTTGCGCTATCAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCGT
AACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACGGTTCAGACTCCTACGGGAGGCAGCAGTGGGGAA
TATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAA
GTTGGGAGGAAGGGTTGTAGATTAATACTCTGCAATTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGC
CAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTA
AGTTGGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTATGGTAGAGGGTGGT
GGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGAACACCAGTGGCGAAGGCGACCACCTGGACTGATA
CTGACACTGAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
The 16SrDNA nucleotide sequence of strain X 3 are as follows:
GGAGAGTTTGATCAGGCTCAGGATGAACGCTGGCGGCGTGCCTAATACATGCAAGTCGAGCGAATGGATTAAG
AGCTTGCTCTTATGAAGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCCATAAGACTGGGATAACTCCG
GGAAACCGGGGCTAATACCGGATAATATTTTGAACCGCATGGTTCGAAATTGAAAGGCGGCTTCGGCTGTCACTTAT
GGATGGACCCGCGTCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGG
GTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGAC
GAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGCTTTCGGGTCGTAAAACTCTGTTGTTAGGGAAGAACAAG
TGCTAGTTGAATAAGCTGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTA
ATACGTAGGTGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCGCGCGCAGGTGGTTTCTTAAGTCTGATGTGAAA
GCCCACGGCTCAACCGTGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAAAGTGGAATTCCATGTGT
AGCGGTGAAATGCGTAGAGATATGGAGGAACACCAGTGGCGAAGGCGACTTTCTGGTCTGTAACTGACACTGAGGCG
CGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGG
It can be concluded that strain X 1 belongs to bacillus cereus category (Bacillus according to strain X 1, the 16SrDNA sequence of X2, X3
Cereus), the sequence homology of strain X 1 and Bacillus cereus reach 99%；Strain X 2 belongs to pseudomonas
The sequence homology of (Acidovorax sp), strain X 2 and Acidovorax temperans strain Cl-08 reach
99%；Strain X 3 belongs to bacillus (Bacillus sp), strain X 3 and Bacillus sp.enrichment culture
The sequence homology of clone Jlu BC reaches 100%.
A kind of strain that sieves of method using squid viscera screening strain degradation squid viscera application, including it is following
Step:
(a) actication of culture: respectively by single strain X1, the bacterium solution of X2, X3 are inoculated into different sterile vegetative broth agar culture mediums
In, at 28~30 DEG C, 23~25h of isothermal vibration culture under the conditions of 100~120r/min obtains the activation bacterium solution of single strain；
(b) widened strain X 1, X2 will be activated, X3 carries out fermenting experiment, squid viscera and activation bacterium solution to squid viscera respectively
Solid-liquid ratio be 1:1~9, fermentation pH be 5~9, fermentation temperature be 20~40 DEG C, fermentation time be 1~5d, with Folin- phenol method
Soluble protein content in fermentation liquid is surveyed, albumen conversion ratio is finally calculated.
Preferably, in step (a), the single strain X1, bacterium solution and the sterile vegetative broth agar culture medium of X2, X3
Volume ratio is 1:50~100；The bacterium solution of single strain X1, X2, X3 are single strain X1, and X2, X3 are respectively in different sterile vegetative meat
In soup agar medium, at 28~30 DEG C, 4~18h of isothermal vibration culture is obtained under the conditions of 100~120r/min bacterium solution.
To strain X 1, X2, X3 has found inventor during being cultivated, and strain bacterium X1, X2, X3 grow slow in 0-2h
Slowly, OD value is begun to ramp up after 2h, it is meant that bacterial strain initially enters exponential phase of growth；Bacterial strain quantity is gradually after culture is to 18h
It is intended to steadily, bacterial strain is in the growth stage of stable development, and the nutrient in culture medium is persistently consumed at this time, and strain metabolic waste gradually increases
More, 20h or so bacterial strain quantity is begun to decline, into the decline phase.Therefore, the bacterium solution that should choose culture 4-18h or so is activated
Inoculation.
It is compared with the prior art, the beneficial effects of the present invention are: can be filtered out efficiently using method provided by the invention
Using the dominant bacteria of squid viscera waste, squid viscera of efficiently degrading is conducive to extract the nutrients in squid viscera
Matter avoids waste and environmental pollution.
Specific embodiment
The present invention will be further described with reference to the examples below.Related device, connection structure in the present invention
And method, if being device well known in the art, connection structure and method without refering in particular to.
Flora in mixed bacteria liquid includes the silk that photosynthetic bacteria group, yeast flora, Gram positive actinomycetes group and fermentation are
Shape flora.Aseptic culture medium is the nutrient agar for being purchased from Qingdao Hai Bo Bioisystech Co., Ltd, at 121 DEG C of sterilizings
Manage 30min.
Embodiment 1
A method of strain is screened using squid viscera, comprising the following steps:
(1) under gnotobasis, homogenate is semisolid after squid viscera is sterilized.
(2) under gnotobasis, by semisolid squid viscera, sterile water and mixed bacteria liquid together temperature be 30 DEG C, shake
Bed revolving speed is fermented and cultured 2d under conditions of 120r/min, obtains squid viscera fermentation liquid.Wherein, the material of squid viscera, sterile water
Water ratio is 1:7, and the additive amount of mixed bacteria liquid is the 5% of squid viscera suspension volume, and the mass percentage concentration of mixed bacteria liquid is
1%.
(3) under aseptic condition, squid viscera fermentation liquid is subjected to ten times of dilutions with sterile water, is successively diluted to 10-6Times, will
Extension rate is 10-4、10-5、10-6Squid viscera fermentation liquid be coated in aseptic culture medium, temperature be 30 DEG C, shaking table turn
Speed is cultivated under conditions of being 120r/min to there is apparent bacterium colony to grow.
(4) under aseptic condition, the bacterium colony of three kinds of most different shapes of picking quantity is successively named as X1, X2, X3, often
The single colonie of kind of form is crossed respectively cultivates in different aseptic culture mediums to there is apparent bacterium colony to grow；
(5) under aseptic condition, the scribing line respectively that will be cultivated through step (4) has the single bacterium of X1, X2, X3 in the culture medium of X1, X2, X3
It falls to be crossed in aseptic culture medium again and cultivate；Scribing line at least 3 times is repeated, the strain finally obtained is the bacterium after isolating and purifying
Kind X1, X2, X3.
Embodiment 2
A method of strain is screened using squid viscera, comprising the following steps:
(1) under gnotobasis, homogenate is semisolid after squid viscera is sterilized.
(2) under gnotobasis, by semisolid squid viscera, sterile water and mixed bacteria liquid together temperature be 29 DEG C, shake
Bed revolving speed is fermented and cultured 1d under conditions of 110r/min, obtains squid viscera fermentation liquid.Wherein, the material of squid viscera, sterile water
Water ratio is 1:9, and the additive amount of mixed bacteria liquid is the 8% of squid viscera suspension volume, and the mass percentage concentration of mixed bacteria liquid is
0.6%.
(3) under aseptic condition, squid viscera fermentation liquid is subjected to ten times of dilutions with sterile water, is successively diluted to 10-6Times, will
Extension rate is 10-4、10-5、10-6Squid viscera fermentation liquid be coated in aseptic culture medium, temperature be 29 DEG C, shaking table turn
Speed is cultivated under conditions of being 110r/min to there is apparent bacterium colony to grow.
(4) under aseptic condition, the bacterium colony of three kinds of most different shapes of picking quantity is successively named as X1, X2, X3, often
The single colonie of kind of form is crossed respectively cultivates in different aseptic culture mediums to there is apparent bacterium colony to grow；
(5) under aseptic condition, the scribing line respectively that will be cultivated through step (4) has the single bacterium of X1, X2, X3 in the culture medium of X1, X2, X3
It falls to be crossed in aseptic culture medium again and cultivate；Scribing line at least 3 times is repeated, the strain finally obtained is the bacterium after isolating and purifying
Kind X1, X2, X3.
Strain X 1, X2, X3 after isolating and purifying described in the step of to embodiment 1 (5) identify, identification method
Are as follows: strain X 1, X2, X3 after isolating and purifying are crossed to respectively in nutrient broth solid medium tablets, and nutrient broth is consolidated
The bacterium colony to grow on body culture medium flat plate observes colonial morphology after carrying out Gram's staining under the microscope；Meanwhile through meticulous
The extraction of bacterium genome, special primer expand the 16SrDNA that 16SrDNA sequence, purified pcr product, DNA sequencing obtain X1, X2, X3
Sequence, the 16SrDNA sequence of obtained X1, X2, X3 is respectively such as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 institute
Show.
It can be concluded that strain X 1 belongs to bacillus cereus category according to strain X 1, the 16SrDNA sequence of X2, X3
The sequence homology of (Bacillus cereus), strain X 1 and Bacillus cereus reach 99%；Strain X 2 belongs to false single
The sequence homology of born of the same parents Pseudomonas (Acidovorax sp), strain X 2 and Acidovorax temperans strain Cl-08 reaches
To 99%；Strain X 3 belongs to bacillus (Bacillus sp), strain X 3 and Bacillus sp.enrichment
The sequence homology of culture clone Jlu BC reaches 100%.
The strain filtered out is carried out to the evaluation of degradation squid viscera by following steps:
Embodiment 3
A kind of method for the strain degradation squid viscera that the method using squid viscera screening strain is sieved, comprising the following steps:
(a) actication of culture: the bacterium solution of single strain X1 is inoculated into different sterile vegetative broth agar culture mediums respectively, single bacterium
The bacterium solution of strain X1 and the volume ratio of sterile vegetative broth agar culture medium are 1:70, and at 30 DEG C, constant temperature shakes under the conditions of 120r/min
It swings culture for 24 hours, obtains the activation bacterium solution of single strain.Wherein the bacterium solution of single strain X1 is single strain X1 in different sterile vegetative meat soup
In agar medium, isothermal vibration culture 12h is obtained under the conditions of 30 DEG C, 120r/min bacterium solution.
(b) widened strain X 1 will be activated, fermenting experiment, squid viscera and activation bacterium solution is carried out to squid viscera respectively
Solid-liquid ratio is 1:5, and fermentation pH is 5, and fermentation temperature is 30 DEG C, fermentation time 1d, is surveyed with Folin- phenol method solvable in fermentation liquid
Property protein content, finally calculate albumen conversion ratio.
Embodiment 4
Embodiment 4 and embodiment 3 the difference is that: the bacterium solution of single strain X1 and sterile vegetative bouillon agar in step (a)
The volume ratio of culture medium is 1:50；In step (b), fermentation pH is 6, and fermentation temperature is 35 DEG C, fermentation time 2d.Other with
Embodiment 3 is identical.
Embodiment 5
Embodiment 5 and embodiment 3 the difference is that: in step (b), fermentation pH is 7, and fermentation temperature is 40 DEG C, when fermentation
Between be 3d.Other are same as Example 3.
Embodiment 6
Embodiment 6 and embodiment 3 the difference is that: in step (b), the solid-liquid ratio of squid viscera and activation bacterium solution is 1:7,
Fermentation temperature is 35 DEG C, fermentation time 3d.Other are same as Example 3.
Embodiment 7
Embodiment 7 and embodiment 3 the difference is that: in step (b), the solid-liquid ratio of squid viscera and activation bacterium solution is 1:7,
The pH that ferments is 6, and fermentation temperature is 40 DEG C.Other are same as Example 3.
Embodiment 8
Embodiment 8 and embodiment 3 the difference is that: in step (b), the solid-liquid ratio of squid viscera and activation bacterium solution is 1:7,
The pH that ferments is 7, fermentation time 2d.Other are same as Example 3.
Embodiment 9
Embodiment 9 and embodiment 3 the difference is that: in step (b), the solid-liquid ratio of squid viscera and activation bacterium solution is 1:9,
Fermentation temperature is 40 DEG C, fermentation time 2d.Other are same as Example 3.
Embodiment 10
Embodiment 10 and embodiment 3 the difference is that: in step (b), the solid-liquid ratio of squid viscera and activation bacterium solution is 1:
9, fermentation pH is 6, fermentation time 3d.Other are same as Example 3.
Embodiment 11
Embodiment 11 and embodiment 3 the difference is that: in step (b), the solid-liquid ratio of squid viscera and activation bacterium solution is 1:
9, fermentation pH is 7, and fermentation temperature is 35 DEG C.Other are same as Example 3.
Embodiment 12
Embodiment 12 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:3, and fermentation pH is 7.Other are same as Example 3.
Embodiment 13
Embodiment 13 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:3, and fermentation pH is 8, and fermentation temperature is 35 DEG C, fermentation time 2d.Other are same as Example 3.
Embodiment 14
Embodiment 14 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:3, and fermentation pH is 9, and fermentation temperature is 40 DEG C, fermentation time 3d.Other are same as Example 3.
Embodiment 15
Embodiment 15 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), fermentation pH is 7, fermentation
Temperature is 35 DEG C, fermentation time 3d.Other are same as Example 3.
Embodiment 16
Embodiment 16 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), fermentation pH is 8, fermentation
Temperature is 40 DEG C.Other are same as Example 3.
Embodiment 17
Embodiment 17 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), fermentation pH is 9, fermentation
Time is 2d.Other are same as Example 3.
Embodiment 18
Embodiment 18 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:7, and fermentation pH is 7, and fermentation temperature is 40 DEG C, fermentation time 2d.Other are same as Example 3.
Embodiment 19
Embodiment 19 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:7, and fermentation pH is 8, fermentation time 3d.Other are same as Example 3.
Embodiment 20
Embodiment 20 and embodiment 3 the difference is that: bacterial strain uses therefor is X2 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:7, and fermentation pH is 9, and fermentation temperature is 35 DEG C.Other are same as Example 3.
Embodiment 21
Embodiment 21 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), fermentation pH is 7.Other
It is same as Example 3.
Embodiment 22
Embodiment 22 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), fermentation pH is 8, fermentation
Temperature is 35 DEG C, fermentation time 2d.Other are same as Example 3.
Embodiment 23
Embodiment 23 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), fermentation pH is 9, fermentation
Temperature is 40 DEG C, fermentation time 3d.Other are same as Example 3.
Embodiment 24
Embodiment 24 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:7, and fermentation pH is 7, and fermentation temperature is 35 DEG C, fermentation time 3d.Other are same as Example 3.
Embodiment 25
Embodiment 25 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:7, and fermentation pH is 8, and fermentation temperature is 40 DEG C.Other are same as Example 3.
Embodiment 26
Embodiment 26 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:7, and fermentation pH is 9, fermentation time 2d.Other are same as Example 3.
Embodiment 27
Embodiment 27 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:9, and fermentation pH is 7, and fermentation temperature is 40 DEG C, fermentation time 2d.Other are same as Example 3.
Embodiment 28
Embodiment 28 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:9, and fermentation pH is 8, fermentation time 3d.Other are same as Example 3.
Embodiment 29
Embodiment 29 and embodiment 3 the difference is that: bacterial strain uses therefor is X3 bacterial strain；In step (b), squid viscera and activation
The solid-liquid ratio of bacterium solution is 1:9, and fermentation pH is 9, and fermentation temperature is 35 DEG C.Other are same as Example 3.
Evaluation result: strain X 1, X2, X3 fermentation squid viscera orthogonal experiments are shown in Table 1, table, 2 and in embodiment 3~29
Table 3.
Table 1
Table 2
Table 3
By the orthogonal experiments of table 1 it is found that the optimum condition of the fermentation squid viscera of strain X 1: solid-liquid ratio 1: 9, pH 5, temperature
Degree is 30 DEG C, fermentation time 3d.Analysis R value can obtain the influence size of four factors are as follows: solid-liquid ratio > pH > time > temperature.By
The optimum condition of orthogonal experiments squid viscera it is found that strain X 2 is fermented of table 2 are as follows: solid-liquid ratio 1: 7, pH 9, temperature are
40 DEG C, time 1d.Solid-liquid ratio and incubation time are the principal elements for influencing the fermentation of X2 bacterium, and the influence of pH and temperature is unobvious.
Analysis R value can obtain the influence size of four factors are as follows: solid-liquid ratio > time > pH > temperature.It can by the orthogonal experiments of table 3
Know, the optimum condition of X3 bacterium fermentation squid viscera are as follows: solid-liquid ratio 1: 7, pH 8, temperature are 35 DEG C, time 2d.Analyze R value
The influence size of four factors can be obtained are as follows: solid-liquid ratio > pH > time > temperature.
The present invention, without apparent regularity, cannot pass through limited trials in the optimal conditions of screening degradation squid viscera
It obtains, there is blindness when screening best degradation condition, therefore, the present invention is in the optimal conditions of screening degradation squid viscera
Needs make the creative labor.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention
Technical spirit any simple modification, change and equivalent structure transformation to the above embodiments, still fall within skill of the present invention
The protection scope of art scheme.
Sequence table
<110>Zhejiang Ocean university
<120>a kind of application for the method and the strain that strain is screened using squid viscera
<130> 2018
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 824
<212> DNA
<213>strain X 1 (Bacillus cereus)
<400> 1
ccctctagat gcatgctcga gggagagttt gatcaggctc aggatgaacg ctggcggcgt 60
gcctaataca tgcaagtcga gcgaatggat taagagcttg ctcttatgaa gttagcggcg 120
gacgggtgag taacacgtgg gtaacctgcc cataagactg ggataactcc gggaaaccgg 180
ggctaatacc ggataacatt ttgaaccgca tggttcgaaa ttgaaaggcg gcttcggctg 240
tcacttatgg atggacccgc gtcgcattag ctagttggtg aggtaacggc tcaccaaggc 300
aacgatgcgt agccgacctg agagagtgat cggccacact gggactgaga cacggcccag 360
actcctacgg gaggcagcag tagggaatct tccgcaatgg acgaaagtct ggcggagcaa 420
cgccgcgtga gtgatgaagg ctttcgggtc gtaaaactct gttgttaggg aagaacaagt 480
gctagttgaa taagctggca ccttgacggt acctaaccag aaagccacgg ctaactacgt 540
gccagcagcc gcggtaatac gtaggtggca agcgttatcc ggaattattg ggcgtaaagc 600
gcgcgcaggt ggtttcttaa gtctgatgtg aaagcccacg gctcaaccgt ggagggtcat 660
tggaaactgg gagacttgag tgcagaagag aaaagtggaa ttccatgtgt agcggtgaaa 720
tgcgtagaga tatggaggaa caccagtggc gaagacgact ttctggtctg taactgacac 780
tgaggcgcga aagcatgggg agcaaacagg attagatacc ctgg 824
<210> 2
<211> 788
<212> DNA
<213>strain X 2 (Acidovorax sp)
<400> 2
ggagagtttg atctggctca gattgaacgc tggcggcagg cctaacacat gcaagtcgag 60
cggatgaaag gagcttgctc ctggattcag cggcggacgg gtgagtaatg cctaggaatc 120
tgcctggtag tgggggacaa cgtttcgaaa ggaacgctaa taccgcatac gtcctacggg 180
agaaagcagg ggaccttcgg gccttgcgct atcagatgag cctaggtcgg attagctagt 240
tggtgaggta atggctcacc aaggcgacga tccgtaactg gtctgagagg atgatcagtc 300
acactggaac tgagacacgg ttcagactcc tacgggaggc agcagtgggg aatattggac 360
aatgggcgaa agcctgatcc agccatgccg cgtgtgtgaa gaaggtcttc ggattgtaaa 420
gcactttaag ttgggaggaa gggttgtaga ttaatactct gcaattttga cgttaccgac 480
agaataagca ccggctaact ctgtgccagc agccgcggta atacagaggg tgcaagcgtt 540
aatcggaatt actgggcgta aagcgcgcgt aggtggtttg ttaagttgga tgtgaaatcc 600
ccgggctcaa cctgggaact gcatccaaaa ctggcaagct agagtatggt agagggtggt 660
ggaatttcct gtgtagcggt gaaatgcgta gatataggaa ggaacaccag tggcgaaggc 720
gaccacctgg actgatactg acactgaggt gcgaaagcgt ggggagcaaa caggattaga 780
taccctgg 788
<210> 3
<211> 803
<212> DNA
<213>strain X 3 (Bacillus sp)
<400> 3
ggagagtttg atcaggctca ggatgaacgc tggcggcgtg cctaatacat gcaagtcgag 60
cgaatggatt aagagcttgc tcttatgaag ttagcggcgg acgggtgagt aacacgtggg 120
taacctgccc ataagactgg gataactccg ggaaaccggg gctaataccg gataatattt 180
tgaaccgcat ggttcgaaat tgaaaggcgg cttcggctgt cacttatgga tggacccgcg 240
tcgcattagc tagttggtga ggtaacggct caccaaggca acgatgcgta gccgacctga 300
gagggtgatc ggccacactg ggactgagac acggcccaga ctcctacggg aggcagcagt 360
agggaatctt ccgcaatgga cgaaagtctg acggagcaac gccgcgtgag tgatgaaggc 420
tttcgggtcg taaaactctg ttgttaggga agaacaagtg ctagttgaat aagctggcac 480
cttgacggta cctaaccaga aagccacggc taactacgtg ccagcagccg cggtaatacg 540
taggtggcaa gcgttatccg gaattattgg gcgtaaagcg cgcgcaggtg gtttcttaag 600
tctgatgtga aagcccacgg ctcaaccgtg gagggtcatt ggaaactggg agacttgagt 660
gcagaagagg aaagtggaat tccatgtgta gcggtgaaat gcgtagagat atggaggaac 720
accagtggcg aaggcgactt tctggtctgt aactgacact gaggcgcgaa agcgtgggga 780
gcaaacagga ttagataccc tgg 803
Claims (10)
1. a kind of method using squid viscera screening strain, it is characterised in that the following steps are included:
(1) under gnotobasis, homogenate is semisolid after squid viscera is sterilized；
(2) under gnotobasis, by semisolid squid viscera, sterile water and mixed bacteria liquid together fermented and cultured, squid viscera is obtained
Fermentation liquid；
(3) under aseptic condition, squid viscera fermentation liquid is subjected to gradient dilution with sterile water, the squid viscera after dilution is fermented
Liquid, which is coated in aseptic culture medium, to be cultivated to there is apparent bacterium colony to grow；
(4) under aseptic condition, the bacterium colony of three kinds of most different shapes of picking quantity is successively named as X1, X2, X3, every kind of shape
The single colonie of state is crossed respectively cultivates in different aseptic culture mediums to there is apparent bacterium colony to grow；
(5) under aseptic condition, the scribing line respectively that will be cultivated through step (4) has the single bacterium of X1, X2, X3 in the culture medium of X1, X2, X3
It falls to be crossed in aseptic culture medium again and cultivate；Scribing line at least 3 times is repeated, the strain finally obtained is the bacterium after isolating and purifying
Kind X1, X2, X3.
2. a kind of method using squid viscera screening strain as described in claim 1, it is characterised in that: in step (2), institute
State squid viscera, the material-water ratio of sterile water is 1:1 ~ 9, the additive amount of mixed bacteria liquid is the 3 ~ 25% of squid viscera suspension volume.
3. a kind of method using squid viscera screening strain as claimed in claim 2, it is characterised in that: the mixed bacteria liquid
Additive amount be squid viscera suspension volume 3 ~ 8%.
4. a kind of method using squid viscera screening strain as claimed in claim 3, it is characterised in that: the mixed bacteria liquid
In flora include photosynthetic bacteria group, yeast flora, Gram positive actinomycetes group and fermentation system Filamentous flora, mixed bacteria liquid
Mass percentage concentration is 0.4 ~ 1%.
5. a kind of method using squid viscera screening strain as described in claim 1, it is characterised in that: in step (2), institute
The temperature for stating fermented and cultured is 28 ~ 30 DEG C, and shaking speed is 100 ~ 120r/min, and incubation time is 1 ~ 2d.
6. a kind of method using squid viscera screening strain as described in claim 1, it is characterised in that: in step (3), institute
Squid viscera fermentation liquid after stating dilution is three gradients, the 10 of concentration respectively before gradient dilution-4、10-5、10-6Squid again
Internal organ fermentation liquid, the condition of the culture are identical as the condition cultivated in step (2).
7. a kind of method using squid viscera screening strain as described in claim 1, it is characterised in that: step (3), (4),
(5) in, the aseptic culture medium is to be purchased from the nutrient agar of Qingdao Hai Bo Bioisystech Co., Ltd to go out through 121 DEG C
Bacterium 30min is obtained.
8. a kind of method using squid viscera screening strain as described in claim 1, it is characterised in that: in step (5)
It is described isolate and purify after strain X 1, X2, X3 identified, identification method are as follows: the strain X 1 after isolating and purifying, X2, X3
It is crossed in nutrient broth solid medium tablets respectively, the bacterium colony to grow in nutrient broth solid medium tablets is carried out
Colonial morphology is observed after Gram's staining under the microscope；Meanwhile it being extracted by bacterial genomes, special primer amplification
16SrDNA sequence, purified pcr product, DNA sequencing obtain the 16SrDNA sequence of X1, X2, X3, obtained X1, X2, X3's
16SrDNA sequence is respectively as shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3.
9. a kind of application of strain sieved using method as described in claim 1 in degradation squid viscera, it is characterised in that
The following steps are included:
(a) actication of culture: respectively by single strain X1, the bacterium solution of X2, X3 are inoculated into different sterile vegetative broth agar culture mediums
In, at 28 ~ 30 DEG C, 23 ~ 25h of isothermal vibration culture under the conditions of 100 ~ 120r/min obtains the activation bacterium solution of single strain；
(b) widened strain X 1, X2 will be activated, X3 carries out fermenting experiment, squid viscera and activation bacterium solution to squid viscera respectively
Solid-liquid ratio be 1:1 ~ 9, fermentation pH be 5 ~ 9, fermentation temperature be 20 ~ 40 DEG C, fermentation time be 1 ~ 5d, with Folin- phenol method survey send out
Soluble protein content in zymotic fluid finally calculates albumen conversion ratio.
10. method as claimed in claim 9, it is characterised in that: in step (a), the single strain X1, the bacterium solution of X2, X3 with
The volume ratio of sterile vegetative broth agar culture medium is 1:50 ~ 100；The bacterium solution of single strain X1, X2, X3 are single strain X1, X2, X3
Respectively in different sterile vegetative broth agar culture mediums, at 28 ~ 30 DEG C, isothermal vibration culture under the conditions of 100 ~ 120r/min
The bacterium solution that 4 ~ 18h is obtained.
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| KR20180095402A (en) * | 2017-02-17 | 2018-08-27 | 한진열 | Quick frozen squid processing method on shipboard |
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| CN101974460A (en) * | 2010-09-28 | 2011-02-16 | 中国科学院南海海洋研究所 | Ocean source Bacillus barbaricus SCSIO 02429 and method for preparing squid small peptide by using same |
| CN106343257A (en) * | 2016-08-29 | 2017-01-25 | 梁小荣 | Feed capable of promoting molt of crayfish and preparation method thereof |
| KR20180095402A (en) * | 2017-02-17 | 2018-08-27 | 한진열 | Quick frozen squid processing method on shipboard |
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