CN103060243A - Sub-lactobacillus casei and sub-cheese subspecies strain - Google Patents
Sub-lactobacillus casei and sub-cheese subspecies strain Download PDFInfo
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Abstract
The invention provides an auxiliary lactobacillus casei and auxiliary cheese subspecies strain, and the strain is auxiliary lactobacillus casei and auxiliary cheese subspecies R37(Lactobacillus paracasei subsp.paracasei R37), which is preserved in China Typical Culture Preservation Center of Wuhan University in Wuhan on Aug. 17th, 2012, and the preservation number is CCTCC NO: M2012311. The strain disclosed by the invention has the advantages that: the strain, namely the Sub-lactobacillus casei and sub-cheese subspecies R37, not only has high malic acid to lactic acid transformation activity, stress resistance and easy growth characteristics, but also can be quickly propagated in grape juice, generates unique fermentation flavor accordant to special flavor of grape juice, and gives the grape juice more functional nutrient factors, and increases the antioxidant function of fermented juice, thereby providing a new zymophyte source for the research and preparation of lactic acid fermented grape juice beverage.
Description
[technical field]
The present invention relates to microbial technology field, relate in particular to the secondary cheese subspecies of strain lactobacillus paraceasi bacterial strain.
[background technology]
Development along with Food science; lactic fermentation also is used in the processing of juice drinks; lactobacillus fermented fruits and vegetables juice not only can provide happy lactic fermentation flavour substances; can also eliminate and give birth to blue or green flavor, protection color, promote mineral absorption, produce the materials such as the necessary multivitamin of human body, lactobacillin, digestive ferment, thereby give the whole intestines of nectar, stomach invigorating, aid digestion and improve the effect of body immunity.But the fermentation that milk-acid bacteria is applied to garden spgarden stuff is processed, and faces lot of challenges.Because garden spgarden stuff lacks the important promotion factor lactose of milk-acid bacteria propagation, be used for the lactic acid fermented milk-acid bacteria of garden spgarden stuff and just must be able to utilize other monose and the polyoses such as the contained organic acid of garden spgarden stuff, glucose, obtaining energy breeds rapidly, and the fermentation of generation and garden spgarden stuff flavor coordination is fragrant, improves nutrition and the function of product.And the garden spgarden stuff zymotechnique of lactic acid of existing report adopts tradition to be applied to the milk-acid bacteria of Yoghourt fermentation more, employing is added a certain amount of milk powder or is contained newborn substratum as seed liquor in raw material, or directly add milk-product and ferment, be unfavorable for that the pure and fresh straightforward local flavor of fruit juice keeps.
In order to address the above problem: the applicant is CN200910304966.3 in the application number of application on 07 29th, 2009, name is called on the Chinese patent of " a kind of lactic acid fermentation broth of loquat juice and preparation method thereof " and has disclosed the lactic acid fermentation broth of loquat juice that does not add milk powder and utilize milk-acid bacteria direct fermentation, the fruit juice fruital that solves loquat product in the prior art is not enough, the dull defective of local flavor, nutritive ingredient can not be the shortcomings such as the abundant absorption of human body institute, it need not to add milk powder, kept the intrinsic nutritive ingredient of loquat, the distinctive nutrition of lactobacillus-fermented Sucus Eriobotryae and local flavor have been given again, be a kind of high-quality novel loquat juice fermented drink, enriched the kind of lactic fermenting beverage; The applicant is CN201110281566.2 in the application number of application on 09 20th, 2011, name is called on the Chinese patent of " a lactobacillus plantarum R23 " and has disclosed plant lactobacillus R23, and the application number of applying on 05 17th, 2010 is CN201010174093.1, name is called the lactobacterium casei that has disclosed the lactobacillus of called after R35 on the Chinese patent of " a kind of Novel fruit wine biological-deacidification bacterial strain and application thereof ", and this two bacterial strain all is disclosed on the document of " tropical crops journal " the 32nd volume the 3rd phase 550-553 page or leaf in 2011, this two bacterial strain all can adapt to the above sour environment of pH3.2 fast, and breed take organic acid as main carbon source, break through lactic acid fermented technical bottleneck, it is fragrant to produce simultaneously the happiness fermentation of coordinating mutually with Sucus Eriobotryae, and gives the distinctive nutrition of Sucus Eriobotryae lactic fermentation and the beneficial function of giving birth to.
The practitioner continues further investigation, the lactic fermentation technique extension is applied in the grape juice, and wishes to select the lactic acid fermented suitable bacterial strain of grape, thereby provide new zymophyte source for the development of lactic fermentation grape juice beverage.
[description]
Technical problem to be solved by this invention is to provide the secondary cheese subspecies of strain lactobacillus paraceasi bacterial strain, this bacterial strain is the secondary cheese subspecies of lactobacillus paraceasi R37(Lactobacillus paracasei subsp.paracasei R37), and the secondary cheese subspecies of this lactobacillus paraceasi R37 is preserved in the Chinese Typical Representative culture collection center that is positioned at Wuhan City Wuhan University on August 17th, 2012, and deposit number is CCTCC NO:M2012311.
The present invention solves the problems of the technologies described above by the following technical programs:
The milk-acid bacteria that the applicant separates from the loquat wine of spontaneous fermentation and preliminary screening goes out to have the malolactic fermentation ability, and the gained milk-acid bacteria screened to obtain the wherein the strongest bacterial strain of a strain malolactic fermentation ability further, by this bacterial strain being carried out morphology, Physiology and biochemistry and 16S rDNA sequencing and homology analysis, identify that finally it is a bacterial strain of the secondary cheese subspecies of lactobacillus paraceasi.
This obtained strains can be used in producing grape juice lactic fermentation aseptic beverage, particularly: and the grape juice of in aseptic fermentor tank, packing into and processing 10min through 95～100 ℃, and obtained strains is with 10
7The inoculum size of cfu/mL is connected in this aseptic fermentor tank, and leavening temperature is 30 ℃ ± 5 ℃, ferment and get appropriate lactic acid fermented active fruit juice lactic acid fermentation liquid after 48 ± 24 hours, carry out Beverage Service after the active fruit juice lactic acid fermentation liquid of gained filtered and namely get described grape juice lactic fermentation aseptic beverage to make the lactic fermenting beverage of active fruit juice lactic acid fermentation liquid mass percent as 20%, at last the can of grape juice lactic fermentation aseptic beverage is got final product.
Beneficial effect of the present invention is: the secondary cheese subspecies of strain lactobacillus paraceasi R37(Lactobacillus paracasei subsp.paracasei R37 is provided), this bacterial strain not only has stronger malolactic acid and transforms vigor, resistance and probiotic properties, and can be in grape juice Fast-propagation, it is fragrant to produce the uniqueness fermentation of coordinating mutually with the grape juice local flavor, and give grape juice the more function nutrition factor, improve the anti-oxidant function of fermented juice, thereby provide new zymophyte source for the development of lactic fermentation grape juice beverage.
[description of drawings]
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is that bacterial strain R37 grows evolutionary tree with relevant kind 16SrDNA sequential system among the present invention.
Fig. 2 is the colonial morphology figure of bacterial strain R37 among the present invention.
Fig. 3 is the cellular form figure (scanning electron microscope) of bacterial strain R37 among the present invention.
Fig. 4 is the gramstaining synoptic diagram of bacterial strain R37 among the present invention.
Fig. 5 is the adhesive capacity synoptic diagram of bacterial strain R37 among the present invention.
Fig. 6 is the resistance of oxidation synoptic diagram of the interior extra-metabolite of bacterial strain R37 born of the same parents among the present invention.
Fig. 7 is the canonical plotting of Determination of Cholesterol Content among the present invention.
Fig. 8 is the canonical plotting of GABA assay among the present invention.
Fig. 9 is the growth curve of bacterial strain R37 in " No. 1, Portugal, osmanthus " grape juice among the present invention.
[embodiment]
The secondary cheese subspecies of lactobacillus paraceasi of the present invention R37(Lactobacillus paracasei subsp.paracasei R37) be that separation screening is out from the loquat wine of spontaneous fermentation.
One, the acquisition of bacterial strain R37
Loquat is divided in loquat juice in the 5L fermentor tank and in 20-25 ℃ of lower spontaneous fermentation afterwards through stoning, fragmentation, making beating; And adopt malolactic acid content in the paper chromatography qualitative detection loquat distiller's wort, select to occur malolactic fermentation and SO
2Concentration be the loquat distiller's wort of 40-100mg/L as separation source, for subsequent use; Under aseptic technique, separation source is carried out 10 times of gradient dilutions, and to choose extent of dilution be 10
-2Diluent, draw the selected diluent of 0.1mL and coat the ATB isolation medium, put 28 ℃ and cultivate 6d, single bacterium colony that the picking circle is slightly flat is repeatedly rule at the MRS flat board and to be gone down to posterity until obtain pure bacterial strain; Obtained strains is separated, each bacterial strain is all through the turn sour test of ability of gramstaining, catalase reaction, indole test, sugar fermentating test and lactic acid, preliminary screening goes out to have the milk-acid bacteria of malolactic fermentation ability, afterwards the gained milk-acid bacteria is screened to obtain the wherein the strongest bacterial strain of malolactic fermentation ability further, and to name this bacterial strain be bacterial strain R37.
Two, the evaluation of bacterial strain R37
1. the morphology of bacterial strain R37 and Physiology and biochemistry are learned feature
The form of obtained strains R37 and physicochemical property etc. are studied, and the Main Morphology and the cultural characteristic that obtain this bacterial strain R37 are as follows: as shown in Figure 2, colony diameter 0.7～1.3mm, rounded, moistening, smooth surface, projection, oyster white, opaque, neat in edge; Bacterial strain R37 placed under the scanning electron microscope observe (the scanning electron microscope synoptic diagram as shown in Figure 3) and bacterial strain R37 is carried out gramstaining (synoptic diagram as shown in Figure 4) as can be known, cell is shaft-like, the thalline size be 0.5 μ m * 1.0～1.67 μ m(long * wide), single, paired or short chain shape arrangement that cell is is without gemma; Liquid culture bacterium liquid is muddy, sterile film, and without bubble, layering is obvious, bottom adularescent precipitation; Following table 1 is that physiologic character, the table 2 of bacterial strain R37 is carbohydrate metabolism test-results of bacterial strain R37:
The physiologic character of table 1 bacterial strain R37
The carbohydrate metabolism test-results of table 2 bacterial strain R37
Annotate: in table 1 and the table 2, "+" expression is positive; "-" expression is negative; "+W " expression is weak positive.
Can find out that by above-mentioned table 1, table 2 bacterial strain R37 has following physio-biochemical characteristics: bacterial strain can be 10 ℃, 40 ℃ lower growths, and the polychrom hydrolysis experiment is positive, amphimicrobian, and catalase test, oxidase test, urease test etc. are all negative; Sugar fermentating test is the result show, bacterial strain R37 is 21 kinds of carbon sources such as energy metabolism ribitol, L-fucose, inositol, rhamnosyl, D-R alcohol, 5-ketone group-gluconate, D-wood sugar, galactitol, starch, L-arabinose alcohol, A-methyl D-mannoside, L-wood sugar, D-R, tetrahydroxybutane, B-methyl D-xyloside, D-Fucose, glycerine, D-lyxose, L-arabinose, 2-ketone group-gluconate, Xylitol not, and other 25 kinds of carbon sources all can be utilized in the table 2.
2. 16S rDNA sequencing and the homology analysis of bacterial strain R37
Adopting the aimed strain of the bacterial genomes DNA extraction test kit extraction pure culture of TIANGEN company is the genomic dna of bacterial strain R37.Entrust biotechnology (Shanghai) Co., Ltd. to utilize the synthetic primer of dna synthesizer: F968(5 '-GAG TTT GAT CCT GGC TCA G-3 ') and L1401(5 '-AGAAAG GAG GTGATC CAG CC-3 ').
Take the genomic dna that extracts the bacterial strain R37 obtained as template, and take above-mentioned primer (F968/L1401) as primer to its 16S rDNA gene of pcr amplification V6～V8 variable region:
PCR reaction composition (50 μ L): ddH
2O34.6 μ L, 10 * PCR reaction buffered soln, 5.0 μ L, dNTP damping fluid 4.0 μ L, primer (F968) 2.0 μ L, primer (L1401) 2.0 μ L, template DNA 2.0 μ L, Taq TM archaeal dna polymerase 0.4 μ L;
The pcr amplification program is: 94 ℃ of denaturation 5min; Then 94 ℃ of sex change 30s, 55 ℃ of annealing 1min, 72 ℃ are extended 1min, totally 35 circulations; Last 72 ℃ are fully extended 10min, 4 ℃ of preservations.
The PCR product detects: the PCR product of getting 5 μ L, the sample-loading buffer of 2 μ L, gel electrophoresis separates in 1.0% agarose, at the EB dyestuff 10min that dyes, analyze with gel imaging system, the product (being the 16S rDNA of bacterial strain R37) that will contain target fragment checks order, and records the 16S rDNA sequence of bacterial strain R37 shown in SEQ ID NO.1.The 16S rDNA sequence input Genebank of bacterial strain R37 is carried out sequence homology relatively with Blast software, and utilize MEGA4.0 software building phylogenetic evolution tree, carry out sibship and Phylogenetic Analysis.As shown in Figure 1, the 16SrDNA sequential analysis of bacterial strain R37 shows, bacterial strain R37 and L.casei, L.paracasei subsp.paracasei, L.paracasei subsp.tolerans and L.rhamnosus type strain gather in a nearest branch, sequence homology is more than 99.1%, the Bootstrap supporting rate is 100%, illustrates that this bacterial strain R37 belongs to the secondary cheese subspecies of lactobacillus paraceasi at the Molecular Phylogeny taxonomy.
According to principle of classification and the combining form, physiological and biochemical property of homology in phylogeny, determine that finally bacterial strain R37 is the secondary cheese subspecies (Lactobacillus paracasei subsp.Paracasei R37) of lactobacillus paraceasi.
Three, the seed selection of bacterial strain R37
Will be available from the yoghurt production commercial strain lactobacillus delbruockii subspecies bulgaricus 6045(numbering CICC6045 of Chinese industrial microbial strains preservation administrative center; Below all be called for short bacterial strains 6045), lactobacterium casei 20986(numbers CICC20986; Below all be called for short bacterial strains 20986), thermophilus streptococcus 20379(numbers CICC20379; Below all be called for short bacterial strains 20379), Sucus Eriobotryae lactic fermentation optimum bacterial strain lactobacterium casei R35(deposit number CGMCC NO:3635; Below all be called for short bacterial strain R35), and the secondary cheese subspecies of the lactobacillus paraceasi R37 activation of the application's gained after proceed as follows respectively: the inoculation after activating in liquid nutrient medium LH16, behind 25 ± 1 ℃ of cultivation 24h, adopt 3000r/min, 4 ℃ of centrifugal 10min also to collect bacterium mud, then get bacterium mud and add pretreated Sucus Vitis viniferae vibration and suspend and reach 10 to form the nectar degree
9The seed liquor that cfu/mL is above, afterwards with this seed liquor with 10
7In the inoculum size access Sucus Vitis viniferae of cfu/mL, 25 ± 1 ℃ of temperature controls are cultivated, and sense organ is passed judgment on the flavor variations of fermentation 1-3d with the suitable bacterial strain of screening lactic fermentation; And adopt three kinds of grape juices to carry out parallel test; Test-results is as shown in table 3 below.
The flavor evaluation of each bacterial strain of table 3 in grape juice
As shown in Table 3, although 3 kinds of grape juices all can obtain excellent flavor in 2 days at the participation bottom fermentation to the of each bacterial strain, ferment and reached the best on the 3rd day, it is aromatic strongly fragrant and coordinate with former fruital to ferment, be that each bacterial strain all is milk-acid bacterias of suitable grape juice physical property, but show that by table 3 fermented flavour of bacterial strain R37 obviously is better than other a few strain bacterial strains.
The detection method of this test flavor evaluation is: fermented sample is divided into 5 grades by its flavor characteristic.One-level is for there being the grape fruital, and it is aromatic strongly fragrant to ferment, and flavour is good, and 90-100 divides; Secondary is for there being the grape fruital, and fermentation perfume (or spice) is stronger, and flavour is good, and 80-89 divides; Three grades for there being the grape fruital, and fermentation is fragrant general, and flavour is better, and 70-79 divides; Level Four is that the grape fruital is light, and fermentation is fragrant not obvious, and 60-69 divides; Pyatyi is that fragrance and flavour are all unhappy, and score value was less than 60 minutes.Please 12 professional persons through training carry out independent marking, the statistical study of averaging.
Four, the characteristic research of bacterial strain R37
1. the probiotic properties of bacterial strain R37
(1) acid and bile tolerance ability
The acid-fast ability of each bacterial strain of table 4 (lgCFU/mL,
Annotate: ND: expression does not detect, and is lower same.
Each bacterial strain of table 5 to the tolerance of cholate (lgCFU/mL,
By table 4 and table 5 as can be known: be that the bacterium amount of bacterial strain R37 is still near 10 after cultivating 4h under 2.5 the environment in the pH value
6Cfu/mL, and bacterial strain R35 and bacterial strain 6045 can't have been grown, namely the acid-fast ability of bacterial strain R37 is the strongest; After cultivating 4h under the 0.3% cholate environment, the bacterium amount of three bacterial strains still reaches 10
6Cfu/mL, the equal dead of three bacterial strains behind the cultivation 2h in 0.5% cholate environment.The result shows that bacterial strain R37 has stronger resistance, can tolerate the pH value more than or equal to 3.0 acidity, and can tolerate the gallbladder salinity up to 0.3%.
(2) stick ability
Investigate bacterial strain 6045, bacterial strain R35, bacterial strain R37 from aggegation rate and his aggegation rate, the results are shown in Figure 5.As seen from Figure 5, bacterial strain R37's reaches respectively 36.28% and 23.29% from aggegation rate and his aggegation rate; The result shows, although bacterial strain R37 from aggegation rate and his aggegation rate a little less than bacterial strain R35, its adhesive capacity is still stronger.
In addition, bacterial strain from the measuring method of aggegation rate and his aggegation rate is described in the bacterial strain acid and bile tolerance capability study process: bacterial strain R37, bacterial strain R35, bacterial strain 6045 are accessed respectively in the LH18 substratum, and in 25 ℃ of constant temperature culture until the thalline biomass reaches 10
8More than the CFU/mL, the fermented liquid with the constant temperature culture gained places 3000r/min, 4 ℃ of lower centrifugal 10min and collects bacterium mud afterwards, and is for subsequent use; Get intestinal bacteria (indicator) 25 ℃ of constant temperature culture to thalline biomasss in bacteria culture medium and reach 10
7More than the CFU/mL, then with nutrient solution in 3000r/min, 4 ℃ of lower centrifugal 10min and collect bacterium mud; The bacterium mud of each bacterial classification of collecting is washed 2 times with the phosphate buffered saline buffer of pH value 7.0 respectively, and the absorbance that is formed in wavelength 600nm place is 0.4 ± 0.1(A
0) suspension bacteria liquid be bacteria suspension, leave standstill 24h and measure absorbance A24, be (A from the formula of aggegation rate (%)
0-A
24)/A
0Milk-acid bacteria and colibacillary plastc ring absorbance are adjusted to 0.6 ± 0.1(A
0), leave standstill 24h and measure absorbance A
24, the formula of his aggegation rate (%) is (A
0-A
24)/A
0
(3) resistance of oxidation
Bacterial strain R35, bacterial strain R37 are cultivated 24h in the LH18 substratum, measure to its cell-free extract and without the resistance of oxidation of fermented liquid, measurement result as shown in Figure 6.As shown in Figure 6, each bacterial strain cell-free extract and superoxide anion and hydroxy radical qiao are all had certain removing ability without fermented liquid,
All reach extremely significantly (P＜0.01) with the difference of substratum, wherein, bacterial strain R37 cell-free extract and the ultra-oxygen anion free radical clearance rate is respectively 71.03% and 52.13% without fermented liquid has improved 69.82 and 50.92 per-cents than substratum respectively.The result shows that bacterial strain R37 has stronger resistance of oxidation.
The detection method of ultra-oxygen anion free radical clearance rate is as follows: adopt the pyrogallol Autoxidation Method to measure fermented liquid to the clearance rate of superoxide radical.According to obtain solution A00, blank solution A0, sample blank solution A 0 ' and sample determination solution A x in the table 6, in 25 ℃ of water-baths, keep 5min, then add rapidly the HCl stopped reaction of 1mL8mol/L, measure light absorption value.Take the substratum LH18 solution that do not add milk-acid bacteria as contrast.Ultra-oxygen anion free radical clearance rate (%)=[A0-(Ax-A0 ')]/A0 * 100%.
Table 6 ultra-oxygen anion free radical clearance rate is analyzed application of sample table (mL)
(4) decreasing cholesterol ability
Cholesterol solution is added in the LH18 substratum to form new substratum with the addition of 100mg/L, and the pH value of regulating this new substratum is to identical with the LH18 substratum; Then with each strains of lactic acid bacteria seed liquor with 10
8The CFU/mL inoculum size accesses in the new substratum, and 25 ℃ of temperature controls detect cholesterol level in the new substratum after cultivating 48h, calculate removal rate of cholesterol; Investigate the ability of bacterial strain 6045, bacterial strain R35, bacterial strain R37 removing cholesterol, the results are shown in Table 7.As can be seen from Table 7, bacterial strain R37 decreasing cholesterol ability is the strongest, can remove 7.8% cholesterol, secondly be bacterial strain R35, and bacterial strain 6045 does not have the decreasing cholesterol ability substantially, only has 0.15%.The result shows that bacterial strain R37 has stronger decreasing cholesterol ability.
The decreasing cholesterol ability of each bacterial strain of table 7
And wherein the method for cholesterol determination adopts o-phthalaldehyde method, and concrete operations are as follows:
A, standard curve making
Get 6 test tubes, the 1mg/mL cholesterol mark liquid that adds respectively 0 μ L, 10 μ L, 30 μ L, 50 μ L, 70 μ L, 90 μ L, and complement to 400 μ L with the analytical pure glacial acetic acid, in every pipe, add afterwards 0.1mg/mL o-phthalaldehyde(OPA) solution 1.5mL, add vitriol oil 1.0mL while vibrating, abundant mixing, room temperature is surveyed absorbance after leaving standstill 10min under 550nm; Take optical density(OD) (OD) value as X-coordinate, cholesterol level is ordinate zou drawing standard curve, the gained typical curve as shown in Figure 7, regression curve equation is: y=181.44x-11.623, coefficient R
2Be 0.9971, show that the equation of gained meets the requirements, can be used for Determination of Cholesterol Content in the fermented liquid.
Determination of Cholesterol Content in b, the sample
In the LH18 substratum, add the 100mg/L cholesterol solution to form new substratum, each inoculation will be fermented to new substratum, and it is extremely identical with the LH18 substratum to regulate the pH value of this new substratum; Measure remaining cholesterol level M in the new substratum behind the fermentation 48h
48, with the new substratum that do not access bacterial strain M in contrast
0, each sample is got supernatant liquor 100 μ L, ice acetic acid 300 μ L, o-phthalaldehyde(OPA) 1.5mL, vitriol oil 1.0mL behind 9000r/min, 4 ℃ of centrifugal 10min.Fully vibration, mixing, room temperature is surveyed absorbance after leaving standstill 10min under 550nm.Respectively do 2 parallel, bring mean value into typical curve and draw corresponding cholesterol level, calculate each bacterial strain decreasing cholesterol rate.Decreasing cholesterol rate %=(M
0-M
48)/M
0
(5) bacterial strain R37 produces γ-aminobutyric acid (GABA) ability
With bacterial strain 6045, bacterial strain R35, bacterial strain R37 with 10
7In the inoculum size access LH18 liquid nutrient medium of cfu/mL, behind 30 ℃ of constant temperature culture 48h, measure the content of GABA in the nutrient solution, the results are shown in Table 8.The result shows that the amount of bacterial strain GABA that R37 produces is the highest, reaches 899mg/L, be bacterial strain R35 secondly, and bacterial strain 6045 produces GABA hardly, and namely bacterial strain R37 has stronger product GABA ability.
Each bacterial strain of table 8 produces the GABA ability
Wherein, the measuring method of GABA adopts colorimetry, and concrete operations are as follows:
A, GABA standard curve making
Get GABA reference liquid (1mg/mL) 0mL that configures, 0.2mL, then 0.4mL, 0.6mL, 0.8mL, 1.0mL add respectively distilled water and complement to lmL in 6 colorimetric cylinders in each pipe; Follow the 0.01mol/mL sodium tetraborate that adds 1.0mL in every pipe, 6% phenol of 1.0mL, mixing, add the 1.0mL chlorine bleach liquor, leave standstill 7min in the darkroom behind the mixing, boiling water bath 10min, ice bath 5min immediately adds 60% ethanol of 2.0mL, left standstill behind the mixing 3 minutes, and measured its absorbancy at 640nm; Take optical density(OD) (OD) value as X-coordinate, GABA content is ordinate zou drawing standard curve.Test obtains the γ-aminobutyric acid typical curve as shown in Figure 8, and regression curve equation is: y=0.7166x+0.1276, coefficient R
2Be 0.9977, show that the equation of gained meets the requirements, can be used for the GABA assay in the fermented liquid.
B, GABA assay
The bacterium liquid of each bacterial strain placed respectively under 4 ℃, 9000r/min respectively get the 1.0mL supernatant liquor behind the centrifugal 10min; In the every 0.01mol/mL sodium tetraborate that adds 1.0mL in the supernatant liquor, 6% phenol of 1.0mL got, mixing, add afterwards the 1.0mL chlorine bleach liquor, leave standstill 7min in the darkroom behind the mixing, boiling water bath 10min, ice bath 5min immediately adds 60% ethanol of 2.0mL, left standstill behind the mixing 3 minutes, and measured its absorbancy at 640nm.Respectively do 2 parallel, after bring mean value into typical curve and draw corresponding GABA content.
2. the malolactic acid of bacterial strain R37 transforms (MLF) vigor
Under equal conditions bacterial strain R35 and bacterial strain R37 are carried out respectively inoculation culture, the malolactic acid of measuring separately transforms vigor, and measurement result is as shown in table 9.As shown in Table 9, bacterial strain R35 and bacterial strain R37 all have stronger MLF ability, and its MLF conversion vigor is respectively 140.5U and 133.8U, and two bacterial strain differences are extremely remarkable.
The malolactic acid of each bacterial strain of table 9 transforms vigor
Five, the application of bacterial strain R37 in grape juice
1. the application of bacterial strain R37 in " No. 1, Portugal, osmanthus " grape juice
(1) growth curve of bacterial strain R37 in " No. 1, Portugal, osmanthus " grape juice
With bacterial strain R37 with 10
7In inoculum size access " No. 1, Portugal, osmanthus " grape juice of cfu/mL, 25 ℃ of temperature controlled fermentations get its growing state as shown in Figure 9.As shown in Figure 9, the adaptability of bacterial strain R37 in " No. 1, Portugal, osmanthus " grape juice is good, without obvious lag phase, enters fast increased logarithmic phase after the inoculation, and degradation period does not appear in fermentation 120h yet, and the growth curve of its increased logarithmic phase meets respectively equation:
Y=7.2027+0.031651*X-0.000119*X
2(F value 354.58, P＜0.01).
(2) acid metabolic of bacterial strain R37
With bacterial strain R37 with 10
7In inoculum size access " No. 1, Portugal, osmanthus " grape juice of cfu/mL, 25 ℃ of temperature controlled fermentations, its total acid metabolic situation sees Table 10.The result shows that bacterial strain R37 mainly carries out malolactic fermentation (MLF) at earlier fermentation, the oxysuccinic acid in the fermented substrate is converted into lactic acid and total acid is descended, and the later stage is mainly carried out glycolysis-rises total acid.
The acid metabolic of table 10 bacterial strain R37
(3) carbohydrate metabolism of bacterial strain R37
With bacterial strain R37 with 10
7In inoculum size access " No. 1, Portugal, osmanthus " grape juice of cfu/mL, 25 ℃ of temperature controlled fermentations, total carbohydrate metabolism situation sees Table 11 in the grape juice.As can be seen from Table 11, the Total Soluble Sugar content of grape juice is on a declining curve all the time during the fermentation; Total sugar content descended rapidly after bacterial strain R37 rigidly connected grape juice, had descended 3.51% in the 12h, and then total sugar content keeps the trend of slow decreasing.
The carbohydrate metabolism of table 11 bacterial strain R37
(4) bacterial strain R37 is to amino acid whose metabolism
With bacterial strain R37 with 10
7In inoculum size access " No. 1, Portugal, osmanthus " grape juice of cfu/mL, 25 ℃ of temperature controlled fermentation 4d, it sees Table 12 to amino acid whose metabolism situation.As known from Table 12, bacterial strain R37 can utilize other amino acid except Gelucystine, methionine(Met), and the amino acid composition of its consumption is mainly arginine, L-glutamic acid and Threonine, wherein arginic content decrease 0.3081g/L, rate of consumption reaches 94.08%.
The amino acid metabolism (g/L) of table 12 bacterial strain R37
(5) bacterial strain R37 is on the impact of fermented flavour
With bacterial strain R37 with inoculum size 10
7In cfu/mL access " No. 1, Portugal, osmanthus " grape juice, at 25 ℃ of bottom fermentation 3d, adopt the GC-MS analytical procedure to investigate bacterial strain to the contribution of grape juice fermented flavour, the results are shown in Table 13.As shown in Table 13, behind bacterial strain R37 fermentation 3d, the relative content of aromatic hydrocarbon, ester class, ketone and alcohols volatile flavor substance all is significantly increased (P＜0.01) in the grape juice, wherein, the relative content of Ester increases at most, than having increased by 3.23% before the fermentation.The result shows that bacterial strain R37 can improve local flavor and form, and increases flavor complexity.
The variation of table 13 milk-acid bacteria R37 fermenting must volatile flavor substance
(6) bacterial strain R37 is on the impact of grape juice anti-oxidant function
With bacterial strain R37 with 10
7In inoculum size access " No. 1, Portugal, osmanthus " grape juice of cfu/mL, the situation of superoxide-dismutase (SOD) vigor is as shown in table 14 in 25 ℃ of temperature controlled fermentations, fermented liquid (being bacterial strain R37 grape lactic acid fermentation fruit juice).As shown in Table 14, the variation of SOD vigor is the rear downtrending of rising first in the fermented liquid.The result shows, bacterial strain R37 has the ability that metabolism produces the outer SOD of born of the same parents, and during fermentation 3d, the SOD vigor reaches maximum value 101.18U/mL in the bacterial strain R37 grape lactic acid fermentation fruit juice, has increased by 15.7% than former grape juice.
The SOD vigor of table 14 bacterial strain R37 fermented juice changes
2. the application of bacterial strain R37 in " huge peak " grape juice
Bacterial strain R37 carries out lactic fermentation the impact of " huge peak " grape juice local flavor is seen Table 15 under 25 ℃.As can be seen from Table 15, bacterial strain R37 is the rear downward trend that rises first at the lactic fermentation local flavor of " huge peak " grape juice: fermentation to the 2nd day grape juice can obtain excellent flavor; The 3-4 days local flavors that ferment reach best, and it is aromatic strongly fragrant to ferment; Obvious bad flavor appears in fermentation to the bacterial strain R37 fermentation in 6 days.
The flavor evaluation of table 15 bacterial strain R37 fermentation " huge peak " grape juice
3. the application of bacterial strain R37 in Vitis davidi fruit juice
Bacterial strain R37 carries out lactic fermentation the impact of Vitis davidi good flavor is seen Table 16 under 25 ℃.As can be seen from Table 16, bacterial strain R37 is the rear downward trend that rises first at the lactic fermentation local flavor of Vitis davidi fruit juice: fermentation to the 2nd day grape juice can obtain excellent flavor; The 3-5 days R37 that ferment can keep excellent flavor, and the 6th day grape juice local flavor that ferment slightly descends.
The flavor evaluation of table 16 milk-acid bacteria R37 fermentation Vitis davidi fruit juice
By the characteristic research of above-mentioned bacterial strains R37 and the application in grape juice thereof as can be known bacterial strain R37 have that following benefit is given birth to and functional performance: a, stronger resistance: can tolerate the pH value more than or equal to 3.0 acidity, can tolerate the gallbladder salinity up to 0.3%; B, stronger resistance of oxidation: after bacterial strain R37 cultivates 24h, its cell-free extract and the ultra-oxygen anion free radical clearance rate is respectively 71.03% and 52.13% without fermented liquid; C, stronger adhesive capacity: it reaches respectively 36.28% and 23.29% from aggegation rate and his aggegation rate; D, stronger decreasing cholesterol ability: can remove in the substratum 7.8% cholesterol; E, have product γ-aminobutyric acid (GABA) ability: fermentation 2d can ferment and produce the GABA of 899mg/L in substratum; F, have the ability that metabolism produces the outer SOD of born of the same parents: during fermentation 3d, the SOD vigor has increased by 15.7% than original fruit juice in the bacterial strain R37 grape lactic acid fermentation fruit juice.
And the characteristic research by above-mentioned bacterial strain R37 and the application in grape juice thereof as can be known bacterial strain R37 can be used in producing grape juice lactic fermentation aseptic beverage, particularly: the grape juice of in aseptic fermentor tank, packing into and processing 10min through 95～100 ℃, and obtained strains is with 10
7The inoculum size of cfu/mL is connected in this aseptic fermentor tank, and leavening temperature is 30 ℃ ± 5 ℃, ferment and get appropriate lactic acid fermented active fruit juice lactic acid fermentation liquid after 48 ± 24 hours, carry out Beverage Service after the active fruit juice lactic acid fermentation liquid of gained filtered and namely get described grape juice lactic fermentation aseptic beverage to make the lactic fermenting beverage of active fruit juice lactic acid fermentation liquid mass percent as 20%, at last the can of grape juice lactic fermentation aseptic beverage is got final product.
To sum up, the invention provides the secondary cheese subspecies of strain lactobacillus paraceasi R37(Lactobacillus paracasei subsp.paracasei R37), this bacterial strain not only has stronger malolactic acid and transforms vigor, resistance and probiotic properties, and can be in grape juice Fast-propagation, it is fragrant to produce the uniqueness fermentation of coordinating mutually with the grape juice local flavor, and give grape juice the more function nutrition factor, improve the anti-oxidant function of fermented juice, thereby provide new zymophyte source for the development of lactic fermentation grape juice beverage.
In addition, related test method among the present invention, and each nutrient media components as follows:
1. total number of bacterial colony: with reference to milk-acid bacteria check in GB/T4789.35-2003(microbiological test of food hygiene-lactobacillus drink), the method for plate culture count.
2. flavour substances relative content
2.1 pre-treatment: get the 200mL sample, respectively with 100mL, 50mL and 30mL heavily steam 2 times dichloromethane extraction 3 times, merge organic phase, dry with anhydrous sodium sulfate dehydration, be concentrated into 5mL, Rotary Evaporators is concentrated into 1mL ,-40 ℃ of preservations;
2.2 instrument condition: adopt U.S. Agilent7980A/5975C GC/MSD gas chromatography mass spectrometry system, chromatographic column HP-FFAP30m * 0.25m * 0.25mm.Chromatographic condition: carrier gas He gas, 250 ℃ of injector temperatures, column temperature adopts temperature programming, and 6O ℃ keeps 5min, rises to 24O ℃ with 5 ℃/min, keeps 10min constant current (1mL/min), Splitless injecting samples; Mass spectrum condition: ion source temperature: 230 ℃; Level Four bar temperature: 150 ℃, ionization mode: EI source.
3. amino acid composition: with reference to amino acid whose mensuration in the GB/T5009.124-2003 food.
4.SOD measuring method: the pyrogallol Autoxidation Method, press table 17 preparation reaction solution, in 25 ℃ of water-baths, keep 1min behind 10 times of the diluted samples, then add rapidly the 8mol/L HCl stopped reaction of 1mL, measure light absorption value.Inhibiting rate (%)=[A
0-(A
x-A
0')]/A
0* 100.Enzyme amount when the SOD vigor suppresses pyrogallol autoxidation rate 50% with per minute in the 1mL reaction solution is decided to be a unit of activity, that is: sample enzyme (U/mL)=inhibiting rate alive/50% * V
(reaction solution)* extension rate/V
(sample volume)
Table 17 ultra-oxygen anion free radical clearance rate is analyzed application of sample table (mL)
5.ATB the component of isolation medium: peptone 1%, yeast extract 0.5%, glucose 1%, sal epsom 0.02%, manganous sulfate 0.005%, cysteine hydrochloride 0.05%, tomato juice 25%, agar 1.2% is transferred pH to 4.8 with 1mol/L HCl or NaOH.
6.MRS dull and stereotyped component: yeast extract paste 0.5%, extractum carnis 1%, Tryptones 1.5%, sodium acetate 0.5%, ammonium citrate 0.2%, manganous sulfate 0.005%, sal epsom 0.02%, glucose 2%, tween-80 0.1%, pH4.8.
7. liquid nutrient medium LH16 component: be tomato juice 100mL, yeast extract paste 7.4g, extractum carnis 10g, glucose 30g, sal epsom 0.36g, sodium malate 20g(57ml), tween 1g, Tryptones 15g, ammonium citrate 2g.PH transfers to 4.8 ± 0.2.
8.LH18 nutrient media components: tomato juice 100mL, yeast extract paste 7.4g, extractum carnis 10g, glucose 30g, sal epsom 0.36g, sodium malate 20g, tween 1g, Tryptones 15g, ammonium citrate 2g, the 0.05g manganous sulfate adds water and supplies 1L, and regulating pH is 4.8 ± 0.2.
9. bacteria culture medium component: the 10g peptone, the 3g extractum carnis, 5g NaCl, 20g agar, adding distil water is supplied 1L.
Claims (1)
1. the secondary cheese subspecies of strain lactobacillus paraceasi bacterial strain, it is characterized in that: this bacterial strain is the secondary cheese subspecies of lactobacillus paraceasi R37(Lactobacillus paracasei subsp.paracasei R37), be preserved in the Chinese Typical Representative culture collection center that is positioned at Wuhan City Wuhan University on August 17th, 2012, deposit number is CCTCC NO:M2012311.
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| CN105400727A (en) * | 2015-12-21 | 2016-03-16 | 南昌大学 | Lactobacillus paracasei with antioxidant activity and application thereof |
| CN108330082A (en) * | 2017-12-20 | 2018-07-27 | 云南皇氏来思尔乳业有限公司 | One plant of Lactobacillus paracasei and its application |
| WO2020140319A1 (en) * | 2019-01-04 | 2020-07-09 | 中国食品发酵工业研究院有限公司 | Lactobacillus paracasei and use thereof |
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| CN110656061A (en) * | 2019-08-30 | 2020-01-07 | 福建省农业科学院农业工程技术研究所 | Lactobacillus paracasei subspecies paracasei RP38 |
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| CN113892651A (en) * | 2021-10-08 | 2022-01-07 | 河北一然生物科技股份有限公司 | Novel application of composite probiotic preparation in antioxidant field |
| CN114752525A (en) * | 2022-04-21 | 2022-07-15 | 广西壮族自治区水牛研究所 | Lactobacillus casei with blood pressure lowering effect and application thereof |
| CN114752525B (en) * | 2022-04-21 | 2022-09-09 | 广西壮族自治区水牛研究所 | Lactobacillus casei with blood pressure lowering effect and application thereof |
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