CN105733996B - A kind of extracellular polysaccharide and the lactobacillus plantarum CZ401 bacterial strain of beta galactosidase and application thereof - Google Patents

A kind of extracellular polysaccharide and the lactobacillus plantarum CZ401 bacterial strain of beta galactosidase and application thereof Download PDF

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CN105733996B
CN105733996B CN201610271772.8A CN201610271772A CN105733996B CN 105733996 B CN105733996 B CN 105733996B CN 201610271772 A CN201610271772 A CN 201610271772A CN 105733996 B CN105733996 B CN 105733996B
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bacterial strain
lactobacillus plantarum
extracellular polysaccharide
beta galactosidase
milk
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CN105733996A (en
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程国军
马成杰
殷杰
田梦洋
张欢欢
何冬兰
李晓华
彭杨
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South Central Minzu University
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/225—Lactobacillus
    • C12R2001/25—Lactobacillus plantarum
    • 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
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    • A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
    • A23C9/123—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt
    • A23C9/1234—Fermented milk preparations; Treatment using microorganisms or enzymes using only microorganisms of the genus lactobacteriaceae; Yoghurt characterised by using a Lactobacillus sp. other than Lactobacillus Bulgaricus, including Bificlobacterium sp.
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    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2468—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1) acting on beta-galactose-glycoside bonds, e.g. carrageenases (3.2.1.83; 3.2.1.157); beta-agarase (3.2.1.81)
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    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01023—Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase

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Abstract

The present invention provides lactobacillus plantarum (L.plantarum) CZ401 bacterial strains of a kind of extracellular polysaccharide and beta galactosidase, the bacterial strain is preserved in China typical culture collection center on March 7th, 2016, address are as follows: Wuhan City, Hubei Province Wuchang District Wuhan University, deposit number is CCTCC NO:M2016092, and the 16S rDNA sequence of the bacterial strain is as shown in SEQ ID NO.1.Observation bacterium colony is in light yellow on MRS solid medium, shows smooth, rounded, center projections；It is Gram-positive, rod-shaped, atrichia, no gemma that microscopy, which observes bacterial strain,.Above-mentioned CZ401 bacterial strain provided by the invention has higher extracellular polysaccharide ability and betagalactosidase activity, and the cow's milk that can ferment generates lactic acid drink, is with a wide range of applications.

Description

A kind of lactobacillus plantarum CZ401 bacterial strain of extracellular polysaccharide and beta galactosidase and Its purposes
Technical field
The present invention relates to a kind of Bacillus acidi lactici new strains, and in particular to a plant height imitates extracellular polysaccharide and beta galactosidase Bacillus acidi lactici CZ401 and its application.
Background technique
Lactic acid bacteria be recognized be a kind of aliment security level microorganism.The thermophilus of composition common sour milk leavening at present Bacterium and lactobacillus bulgaricus etc., due to not stomach juice-resistant and bile, are largely destroyed, survival rate when by human body alimentary canal Less than 0.1%, thus the level for restoring colony balance in human body intestinal canal is very restricted.Lactobacillus plantarum then can be effective Ground is resistant to gastral enzyme, the gastric juice of pH value and bile acid etc., into after people's enteron aisle, is adjustable improvement gastrointestinal tract, restores enteron aisle Interior colony balance.Blood pressure blood fat is adjusted, cholesterol is reduced, adjusts blood lipid, enhancing immunity of organisms in addition, lactobacillus plantarum has With the functions such as resistance, antitumor and pre- anti-cancer.Therefore, suitable lactobacillus plantarum fermentation milk beverage is developed, for changing Kind consumer health's situation is of great significance.
Exopolysaccharides Produced by Lactic Acid Bacteria (extracellular polysaccharide, EPS) is lactic acid bacteria in growth metabolism mistake It is secreted into outside cell wall in journey, often seeps in one of culture medium pod membrane or glue polysaccharide.Exopolysaccharides Produced by Lactic Acid Bacteria has following 5 The function of a aspect.1. improving dairy products state, whey being prevented to be precipitated；2. improving lactic acid bacteria to the adsorption capacity of enteron aisle；3. keeping Moisture absorbs metal ion, prevents bacteriophage from corroding, thus effective protection thallus；4. cell factor is generated by stimulation human body, And the modes such as lymphopoiesis is promoted to enhance human immunological competence；5. effectively improving the anti-cancer ability of human body.
Beta galactosidase (EC3.2.1.23) scientific name is β-D- galactoside galactohydrolase, is lactic acid lactose metabolism Key enzyme in the process, it can be hydrolyzed lactose into as galactolipin and glucose, it may have the transferance of galactoside.Mesh Before, there is the people especially Asian of significant proportion with lactose intolerance in the world, and beta galactosidase can effectively drop Lactose content in low dairy products, alleviates lactose intolerance to a certain extent.On the other hand, the beta galactosidase in lactic acid bacteria It is endocellular enzyme, after reaching small intestine, the effect through cholate is discharged into enteron aisle, and then absorption of the enteron aisle to lactose from now on.
Therefore, if one plant of separation screening of energy has higher extracellular polysaccharide ability and betagalactosidase activity, and energy The cow's milk that ferments generates the lactobacillus plantarum of lactic acid drink, and the exploitation for lactobacillus plantarum fermentation milk beverage will have extensively Application value.
Summary of the invention
The present invention solves the problems in background technique, and providing one kind has higher extracellular polysaccharide ability and β-half Gal activity, but the cow's milk that can ferment generates the lactobacillus plantarum CZ401 bacterial strain of lactic acid drink, and the bacterial strain is for plant cream The exploitation of bacillus fermentation milk beverage, is with a wide range of applications.
The lactobacillus plantarum (L.plantarum) of a kind of extracellular polysaccharide and beta galactosidase provided by the invention CZ401 bacterial strain, the deposit number of the bacterial strain are as follows: CCTCC NO:M2016092, the 16S rDNA sequence such as SEQ ID of the bacterial strain Shown in NO.1.
The bacterial strain has efficient extracellular polysaccharide and beta galactosidase ability, and there is fermentation cow's milk to produce the latent of Yoghourt Energy.
Detailed description of the invention
Fig. 1 is CZ401 bacterial strain in the embodiment of the present invention to the acidity curve graph after Cow Milk Fermentation.
Specific embodiment
Detailed specific description done to the present invention combined with specific embodiments below, but protection scope of the present invention not office It is limited to following embodiment.
The separation and identification of bacterial strain
Isolated enteric microorganism in cheese is made by oneself from from Inner Mongol prairie herdsman in the present embodiment, is named as CZ401, the form and physiological and biochemical property of the bacterial strain are as follows: observation bacterium colony is in light yellow on MRS solid medium, shows light It is sliding, rounded, center projections；It is Gram-positive, rod-shaped, atrichia, no gemma that microscopy, which observes bacterial strain,.
By CZ401 strain inoculated to 5ml MRS culture solution (casein peptone 10.0g；Beef leaches object 10.0g；Yeast extracts Liquid 5.0g；Glucose 5.0g；Sodium acetate 5.0g；Lemon acid diamine 2.0g；Tween 80 1.0g；Dipotassium hydrogen phosphate 2.0g；Seven water sulphur Sour magnesium 0.2g；Seven water manganese sulfate 0.05g；Calcium carbonate 20.0g；Distilled water 1000ml), 37 DEG C, stationary culture 24 hours, 10000rpm is centrifuged 1min, collects thallus in 1.5ml centrifuge tube, extracts bacterial genomes.Use 16S rDNA universal primer (upstream primer: 5 '-AGAGTTTGATCCTGGCTCAG-3 '；Downstream primer: 5 '-AAGGAGGTGATCCAGCCGCA-3 ') it carries out PCR amplification, its 16S rDNA sequence is as shown in SEQ ID NO.1 after being sequenced for amplified production, with the listed 16S of GenBank RDNA sequence carries out nucleotide sequence homology and compares analysis, the 16S rDNA with Lactobacillus paraplantarum Sequence similarity reaches 99%, is accredited as this category.The CZ401 bacterial strain is preserved in Chinese Typical Representative training on March 7th, 2016 Support object collection, address are as follows: Wuhan City, Hubei Province Wuchang District Wuhan University, deposit number are CCTCC NO:M2016092.
The performance study of bacterial strain
Under provide some performances of lactobacillus plantarum CZ401 of the invention and grind the result that makes internal disorder or usurp:
1, influence of the pepsin to lactobacillus plantarum CZ401 Strain survival
The MRS medium treatment group of the pepsin containing 5g/L is tested, and it is pair that the MRS culture medium of pepsin, which is not added, According to every group of 3 repetitions.The lactobacillus plantarum CZ401 bacterial strain of activation is inoculated in processing group and control group by 10% inoculum concentration In, 37 DEG C of stationary culture 2h.The bacterium solution of simulate the gastric juice before and after the processing is spread evenly across respectively on MRS solid plate, 37 DEG C of trainings 48h is supported, plate count is carried out.The bacterium number of lactobacillus plantarum CZ401 control group is (9.66 ± 0.19) X109Cfu/ml, warp After pepsin, the bacterium number of processing group becomes (8.87 ± 0.24) X109cfu ml-1, become.Compared with the control group, it handles Group bacterium number reduces 8.2%, shows that bacterial strain CZ401 has very strong tolerance to pepsin.
2, resistance of the lactobacillus plantarum CZ401 bacterial strain to common antibiotics
The lactobacillus plantarum CZ401 bacterium solution of activation is linked into the MRS containing various concentration antibiotic by 2% inoculum concentration Culture solution in 37 DEG C of biochemical cultivation cases after stationary culture 3 days, measures OD600nmIt is worth (OD600nm< 0.1 indicates not grow, OD600nm > 0.3 indicates growth), whether research thallus grows or growing state.As a result such as table 1, bacterial strain CZ401 to the antibiotic of selection all There are 15 μ g mL-1Above resistance.
Resistance of the table 1CZ401 bacterial strain to different antibiotic
3, the extracellular polysaccharide acid producing ability of lactobacillus plantarum CZ401 bacterial strain
The lactobacillus plantarum CZ401 bacterium solution of activation is linked into MRS culture solution, 37 DEG C of stationary cultures by 5% inoculum concentration 48h.10mL bacterium solution is taken, 2000rpm is centrifuged 10min and collects supernatant, adds 80% trichloroacetic acid to final concentration 4%, 4 DEG C of standings 2h.8000rpm is centrifuged 20min, and supernatant is transferred to bag filter, to flowing water dialysis 2d, during which repeatedly changes water.After dialysis Exocellular polysaccharide sample is dissolved in distilled water, fixed molten.Make standard curve with glucose, by the exocellular polysaccharide sample sulfuric acid-of acquisition Phynol method measurement excludes background interference using blank cultures as control.Exocellular polysaccharide content is 183.47 in CZ401 bacterium solution ±6.58mg L-1, show that CZ401 bacterial strain has very strong extracellular polysaccharide ability.
4, lactobacillus plantarum CZ401 bacterial strain produces beta galactosidase ability
The lactobacillus plantarum CZ401 bacterium solution of activation is linked into MRS culture solution, 37 DEG C of stationary cultures by 5% inoculum concentration 48h.10mL bacterium solution is taken, 4 DEG C, 12000rpm, 10min is centrifuged, collects thallus.Thallus is washed with the phosphate buffer of pH 7.0 Twice, 4 DEG C, 12000rpm, it is centrifuged 10min, abandons supernatant.Thallus is dissolved in phosphate buffer, and Ultrasonic Cell Disruptor is crushed 3min. 4 DEG C, 12000rpm, it is centrifuged 10min, takes supernatant.
Betagalactosidase activity is measured with ortho-nitrophenyl β-D- synthesis (ONPG) method, uses o-nitrophenol (ONP) make standard curve, according to ONP standard curve, calculate ONP total amount in reaction, i.e. beta galactosidase catalysis ONPG is raw At the amount of ONP.Enzyme-activity unit calculation method: with unit mass thallus (g), ONPG is decomposed within the unit time (min) and is generated The amount of 1mmol ONP is as a beta galactosidase unit of activity U (mmol L-1min-1g-1).Specific steps are as follows: cell is broken 20mmol L is added in broken supernatant samples 37 DEG C of water-bath 5min in water-bath-1Ortho-nitrophenyl β-D- synthesis (ONPG) phosphate buffer, 37 DEG C of water-bath 10min.It is added immediately 0.5M NaCO3 solution, reaction is terminated, at 420nm Light absorption value is measured, using phosphate buffer as blank control when measurement.CZ401 bacterial strain produce betagalactosidase activity be 0.46 ± 0.04U shows that CZ401 bacterial strain has very strong production beta galactosidase ability.
5, application of the lactobacillus plantarum CZ401 bacterial strain in fermentation cow's milk
The lactobacillus plantarum CZ401 bacterium solution of activation is linked into 12% skimmed milk by 10% inoculum concentration and is fermented, 42 DEG C of stationary cultures.Since accessing strain, the acidity of acidified milk is measured, and observes the structural state of cow's milk.Cream is taken every 2h Sample adds 0.5% phenolphthalein as indicator, is titrated with 0.1M NaOH standard solution to milk sample, when sample in pale pink and is protected Hold 30s it is colour-fast when, that is, reach titration end-point, read the amount of the NaOH solution of consumption, calculate gill Nie Erdu, i.e., often 100ml fermentation liquid consumption 1ml 0.1M Na0H is denoted as 10T.And results of regular determination milk sample structural state.
Acidity curve as shown in Figure 1, begin to produce acid after inoculation CZ401 bacterium solution for 4 hours；After fermentation 10 hours, cow's milk Acidity is 930T；Production acid peaks within 12 hours, is 1080T, hereafter acidity does not change substantially.CZ401 strain fermentation cow's milk Can complete curdled milk, institutional framework is good, without whey be precipitated, have the special fragrance of Yoghourt.Show that lactobacillus plantarum CZ401 produces sour feelings Condition and structural state reach the requirement of dairy fermentation, can be used as dairy starter.

Claims (3)

1. lactobacillus plantarum (L.plantarum) CZ401 bacterial strain of a kind of extracellular polysaccharide and beta galactosidase, the bacterial strain Deposit number are as follows: CCTCC NO:M2016092, for the 16S rDNA sequence of the bacterial strain as shown in SEQ ID NO.1, the bacterial strain is same When have dairy fermentation produce acid function.
2. the purposes of lactobacillus plantarum CZ401 bacterial strain described in claim 1, it is characterised in that: for produce exocellular polysaccharide and Beta galactosidase.
3. the purposes of lactobacillus plantarum CZ401 bacterial strain described in claim 1, it is characterised in that: for fermenting, cow's milk produces Yoghourt.
CN201610271772.8A 2016-04-27 2016-04-27 A kind of extracellular polysaccharide and the lactobacillus plantarum CZ401 bacterial strain of beta galactosidase and application thereof Expired - Fee Related CN105733996B (en)

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CN109679864B (en) * 2018-12-13 2023-11-10 石河子大学 Strain for producing transglycosylation active beta-galactosidase and method for producing galactooligosaccharide by using same
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