CN106754464B - One plant of bacillus cereus and its application - Google Patents

One plant of bacillus cereus and its application Download PDF

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CN106754464B
CN106754464B CN201611004340.7A CN201611004340A CN106754464B CN 106754464 B CN106754464 B CN 106754464B CN 201611004340 A CN201611004340 A CN 201611004340A CN 106754464 B CN106754464 B CN 106754464B
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bacillus cereus
milk
plant
application
cow
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CN106754464A (en
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张娟
陈坚
汤恒
堵国成
马浩
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Jiangnan University
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Jiangnan University
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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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C12R2001/085—Bacillus cereus
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
    • A23C9/00—Milk preparations; Milk powder or milk powder preparations
    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/1203—Addition of, or treatment with, enzymes or microorganisms other than lactobacteriaceae
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
    • C12N9/54—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea bacteria being Bacillus

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Abstract

The invention discloses one plant of bacillus cereus and its applications, belong to technical field of food biotechnology.Bacillus cereus CCTCC NO:M 2016530 of the present invention is that a large amount of soil samples are acquired from cattle farm, and the protease of the hydrolyzable cow's milk protein obtained by high flux screening secretes bacterial strain.The fermented supernatant fluid of the bacterial strain can effectively degrade αS1Casein, beta lactoglobulin, thus can be used for removing the anaphylactogen in cow's milk, prepare dairy produce, including formula milk, reduce the risk of baby, adult edible dairy produce allergy.

Description

One plant of bacillus cereus and its application
Technical field
The present invention relates to one plant of bacillus cereus and its applications, belong to technical field of food biotechnology.
Background technique
As industrialized Microbial cell factories, bacillus cereus is widely used to food, the fields such as fermentation, but At present in dairy products field using relatively fewer.Dairy products are the important components of infant food, but infant's cream system Existing main problem is protein allergies before items.The incidence of whole world children's Milk allergy is 0.3%-7.5%, lactoprotein Allergy is mainly the I type hypersensitivity mediated by specific IgE antibody, can cause respiratory tract, alimentary canal, skin or whole body mistake It is quick, it is mainly shown as the gastrointestinal discomforts such as vomiting, abdominal pain and diarrhea.Especially for baby, the casein that is mainly contained in cow's milk, α-lactalbumin, beta lactoglobulin etc. are used as foreign protei, it is possible to become anaphylactogen.Cow's milk protein is broadly divided into lactalbumin (whey) and chymotrypsin (curd) two parts, chymotrypsin account for 80%, α of lactoprotein total amountS1Casein is in chymotrypsin A most important anaphylactogen, while the main composition of lactalbumin is beta lactoglobulin.
Its antigenicity is effectively reduced while retaining lactoprotein nutritive value and flavor and allergenicity remains one A problem, and proteinase hydrolization method will not influence the nutritive value of product while being effectively reduced and eliminating anaphylactogen, compare It, not only can be with hydrolyzable moiety allergy in other several desensitization methods, such as heat treatment, glycosylation and fermented by lactic acid bacteria Source can also be produced with the polypeptide for adjusting other physiological functions such as immune function, anticoagulation, blood pressure lowering.
Summary of the invention
Technical problem to be solved by the invention is to provide one plant of bacillus cereus (Bacillus cereus) S1, China typical culture collection center was preserved on September 27th, 2016, deposit number is CCTCC NO:M 2016530, ground Location: China, Wuhan, Wuhan University.
The bacillus cereus CCTCC NO:M 2016530 is that a large amount of soil samples are acquired from cattle farm, passes through high throughput The protease for screening the hydrolyzable cow's milk protein obtained secretes bacterial strain.The fermented supernatant fluid of the bacterial strain can effectively degrade αS1Junket egg White, beta lactoglobulin, thus can be used for removing the anaphylactogen in cow's milk protein, prepare dairy produce, including formula milk, reduce baby Youngster, the adult risk for eating dairy produce allergy.
The statement of " cow's milk protein " is understood to refer to include at least a kind of whey or a kind of casein group The water-containing medium divided.As the unrestricted example of milk substrate, the milk referred to, milk concentrates, cream system food can be Product matrix, lactic substrate etc..
Biomaterial preservation
Bacillus cereus (Bacillus cereus) S1, was preserved in Chinese Typical Representative culture on September 27th, 2016 Collection, preservation address are Wuhan, China, and Wuhan University, deposit number is CCTCC NO:M 2016530.
Specific embodiment
Skimmed milk power culture medium: skimmed milk power 10g.L-1。
LB culture medium: tryptone 10.0g.L-1, yeast extract 5g.L-1, sodium chloride 10g.L-1。
CSN1S1 integrated enzyme reaction kit is purchased from Shanghai haze growth Science and Technology Ltd..
BLG integrated enzyme reaction kit is purchased from Shanghai haze growth Science and Technology Ltd..
The screening technique of 1 bacillus cereus of embodiment (CCTCC NO:M 2016530)
It weighs 1.0g mixing soil sample to mix in 10mL centrifuge tube with 5mL sterile water, after concussion mixes, respectively using sterile It is coated with skimmed milk power plating medium after 10,100,1000 times of water dilutions, then in 30 DEG C of stationary cultures in constant incubator 24h。
It selects the bacterium colony that hydrolysis circle generates and is transferred to new skimmed milk power plating medium, then in constant incubator 30 DEG C of stationary cultures are for 24 hours.Then observation hydrolysis circle size and colonial morphology will screen the maximum single colonie S1 of hydrolysis circle and turn It is connected to after being activated 8 hours in LB culture medium, then transfers in the LB culture medium containing 20% glycerol, be preserved in -80 DEG C.
The 16S rRNA sequence such as SEQ ID NO.1 of bacterial strain S1, bacterial strain qualification result are shown to be bacillus cereus.
The α of embodiment 2 bacillus cereus S1 (CCTCC NO:M 2016530) fermentation liquidS1Casein removal efficiency
By the starting strain bacillus cereus being preserved in -80 DEG C of glycerol tubes (CCTCC NO:M 2016530) with 2% Inoculum concentration access LB culture medium, at 30 DEG C, 220rpm, cultivate 12h.
Activated bacillus cereus is taken to be transferred in fresh LB culture medium respectively with 2% inoculum concentration, at 30 DEG C, 220rpm collects fermentation liquid after cultivating 12h, and fermented liquid supernatant is collected by centrifugation in 8000rpm.
By fermented liquid supernatant and αS11h is reacted after casein standard items are by 1:1 mixing, then at 30 DEG C.With αS1Casein Standard items are control, use CSN1S1 integrated enzyme reaction kit, the α of difference test sampleS1Casein content.
50 μ l samples will be added on ELISA Plate hole bottom respectively, hole wall is not touched as far as possible, shake gently mixing.With sealing plate film 37 DEG C of sealing plate postposition incubate 30 minutes.Will be spare after 30 times of concentrated cleaning solutions, 30 times of distilled water dilutions, carefully take sealing plate film off, Liquid is discarded, is dried, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, is so repeated 5 times, pats dry.Enzyme marking reagent is added in every hole 50 μ l, except blank well.It is incubated 30 minutes with 37 DEG C of postposition of sealing plate film sealing plate.It carefully takes sealing plate film off, discards liquid, dry, Cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, is so repeated 5 times, pats dry.Color developing agent A50 μ l is first added in every hole, adds aobvious Toner B50 μ l, gently concussion mixes, and 37 DEG C are protected from light colour developing 15 minutes.Every hole adds 50 μ l of terminate liquid, and it is (blue at this time to terminate reaction It is vertical to turn yellow).With blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole.Measurement is 15 after adding terminate liquid It is carried out within minute.
The α of table 1. bacillus cereus S1 (CCTCC NO:M 2016530) fermentation liquidS1Casein removal efficiency
The beta lactoglobulin removal efficiency of embodiment 3 bacillus cereus S1 (CCTCC NO:M 2016530) fermentation liquid
By the starting strain bacillus cereus being preserved in -80 DEG C of glycerol tubes (CCTCC NO:M 2016530) with 2% Inoculum concentration access LB culture medium, at 30 DEG C, 220rpm, cultivate 12h.
Activated bacillus cereus is taken to be transferred in fresh LB culture medium respectively with 2% inoculum concentration, at 30 DEG C, 220rpm collects fermentation liquid after cultivating 12h, and fermented liquid supernatant is collected by centrifugation in 8000rpm.
1h is reacted after fermented liquid supernatant is mixed with beta lactoglobulin standard items by 1:1, then at 30 DEG C.With beta lactoglobulin Standard items are control, use CSN1S1 integrated enzyme reaction kit, the α of difference test sampleS1Casein content.
50 μ l samples will be added on ELISA Plate hole bottom respectively, hole wall is not touched as far as possible, shake gently mixing.With sealing plate film 37 DEG C of sealing plate postposition incubate 30 minutes.Will be spare after 30 times of concentrated cleaning solutions, 30 times of distilled water dilutions, carefully take sealing plate film off, Liquid is discarded, is dried, cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, is so repeated 5 times, pats dry.Enzyme marking reagent is added in every hole 50 μ l, except blank well.It is incubated 30 minutes with 37 DEG C of postposition of sealing plate film sealing plate.It carefully takes sealing plate film off, discards liquid, dry, Cleaning solution is filled it up in every hole, is discarded after standing 30 seconds, is so repeated 5 times, pats dry.Color developing agent A50 μ l is first added in every hole, adds aobvious Toner B50 μ l, gently concussion mixes, and 37 DEG C are protected from light colour developing 15 minutes.Every hole adds 50 μ l of terminate liquid, and it is (blue at this time to terminate reaction It is vertical to turn yellow).With blank air-conditioning zero, 450nm wavelength sequentially measures the absorbance (OD value) in each hole.Measurement is 15 after adding terminate liquid It is carried out within minute.
The beta lactoglobulin removal efficiency of 2. bacillus cereus of table (CCTCC NO:M 2016530) fermentation liquid
Beta lactoglobulin standard items Reaction solution Removal efficiency
Beta lactoglobulin concentration (ng/mL) 4.032 3.406 15.510%
SEQUENCE LISTING
<110>Southern Yangtze University
<120>one plants of bacillus cereus and its applications
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1451
<212> DNA
<213>bacillus cereus
<400> 1
ggggtggcgc agctataatg cagtcgagcg atggattaag agcttgctct tatgaagtta 60
gcggcggacg ggtgagtaac acgtgggtaa cctgcccata agactgggat aactccggga 120
aaccggggct aataccggat aacattttga actgcatggt tcgaaattga aaggcggctt 180
cggctgtcac ttatggatgg acccgcgtcg cattagctag ttggtgaggt aacggctcac 240
caaggcaacg atgcgtagcc gacctgagag ggtgatcggc cacactggga ctgagacacg 300
gcccagactc ctacgggagg cagcagtagg gaatcttccg caatggacga aagtctgacg 360
gagcaacgcc gcgtgagtga tgaaggcttt cgggtcgtaa aactctgttg ttagggaaga 420
acaagtgcta gttgaataag ctggcacctt gacggtacct aaccagaaag ccacggctaa 480
ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg ttatccggaa ttattgggcg 540
taaagcgcgc gcaggtggtt tcttaagtct gatgtgaaag cccacggctc aaccgtggag 600
ggtcattgga aactgggaga cttgagtgca gaagaggaaa gtggaattcc atgtgtagcg 660
gtgaaatgcg tagagatatg gaggaacacc agtggcgaag gcgactttct ggtctgtaac 720
tgacactgag gcgcgaaagc gtggggagca aacaggatta gataccctgg tagtccacgc 780
cgtaaacgat gagtgctaag tgttagaggg tttccgccct ttagtgctga agttaacgca 840
ttaagcactc cgcctgggga gtacggccgc aaggctgaaa ctcaaaggaa ttgacggggg 900
cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960
cttgacatcc tctgacaacc ctagagatag ggcttctcct tcgggagcag agtgacaggt 1020
ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg caacgagcgc 1080
aacccttgat cttagttgcc atcatttagt tgggcactct aaggtgactg ccggtgacaa 1140
accggaggaa ggtggggatg acgtcaaatc atcatgcccc ttatgacctg ggctacacac 1200
gtgctacaat ggacggtaca aagagctgca agaccgcgag gtggagctaa tctcataaaa 1260
ccgttctcag ttcggattgt aggctgcaac tcgcctacat gaagctggaa tcgctagtaa 1320
tcgcggatca gcatgccgcg gtgaatacgt tcccgggcct tgtacacacc gcccgtcaca 1380
ccacgagagt ttgtaacacc cgaagtcggt ggggtaacct tttggagcca gccgctaatg 1440
tgatcaggtt t 1451

Claims (7)

1. one plant of bacillus cereus (Bacillus cereus) S1, which is characterized in that in being preserved on September 27th, 2016 State's Type Tissue Collection, deposit number are CCTCC NO:M 2016530, and preservation address is Wuhan, China, and Wuhan is big It learns.
2. application of bacillus cereus (Bacillus cereus) S1 in milk powder producing described in claim 1.
3. application of bacillus cereus (Bacillus cereus) S1 described in claim 1 in production protease.
4. bacillus cereus (Bacillus cereus) S1 described in claim 1 is removed in vitro in milk powder anaphylactogen Using.
5. bacillus cereus (Bacillus cereus) S1 described in claim 1 removes α in vitroS1Answering in casein With.
6. bacillus cereus (Bacillus cereus) S1 described in claim 1 is removed in beta lactoglobulin in vitro Using.
7. application of bacillus cereus (Bacillus cereus) S1 described in claim 1 in production dairy produce.
CN201611004340.7A 2016-11-15 2016-11-15 One plant of bacillus cereus and its application Active CN106754464B (en)

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Publication number Priority date Publication date Assignee Title
CN109837219B (en) * 2017-11-24 2020-09-04 江南大学 Separation and purification of hydrolyzed cow milk allergen beta-lactoglobulin protease and application thereof

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CN104745504B (en) * 2015-03-06 2018-04-13 华南理工大学 The Bacillus cercus in one plant of deep-sea source and its application in neutral proteinase is produced
CN105950497A (en) * 2016-04-27 2016-09-21 广东温氏大华农生物科技有限公司 Keratinase producing bacillus cereus and application thereof

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