CN102925384B - High-activity strain capable of realizing malolactic conversion - Google Patents
High-activity strain capable of realizing malolactic conversion Download PDFInfo
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Abstract
The invention provides a high-activity strain capable of realizing malolactic conversion. The high-activity strain is Lactobacillus plantarum subsp. Plantarum RF17 which is preserved in China Center for Type Culture Collection in Wuhan University on Mar.16, 2012, with the preservation number of CCTCC NO: M 2012085. The high-activity strain capable of realizing malolactic conversion, provided by the invention has the advantages that by utilizing the strain, namely the Lactobacillus plantarum subsp. Plantarum RF17, the content of malic acid in fruit wine and fruit juice can be reduced more efficiently, content of lactic acid, volatile ester and the like in the fruit wine and the fruit juice can be increased, so that the fruit wine and the fruit juice is more gentle and coordinated in mouthfeel, and is sweet-scented in flavor, a solid foundation is laid to improve quality of the fruit wine and the fruit juice, and therefore a novel strain source is provided for the research on the biological acid-degradation and lactic acid fermentation of the fruit wine.
Description
[technical field]
The present invention relates to a kind of microorganism, relate in particular to a kind of malolactic acid and transform high dynamic strain, this bacterial strain be plant lactobacillus plant subspecies (
Lactobacillus plantarum subsp.plantarum) RF17.
[background technology]
In fruit wine, the oxysuccinic acid of high-content can cause that the wine body is sour and astringent, harsh feeling is strong, and malo-lactic fermentation is milk-acid bacteria take the L MALIC ACID of two carboxyls as substrate, is transformed into mono carboxylic Pfansteihl and CO under oxysuccinic acid-lactalase catalysis
2Process, this process can make the wine body soften, and plays the local flavor modification, is one of important biomolecule acid descending process of brewageing in high-quality fruit wine.But the sulfurous gas of high-content and ethanol concn in fruit wine, low pH value, reach lower leavening temperature etc. and all suppressed the growth of milk-acid bacteria, thereby greatly reduce the deacidification effect of milk-acid bacteria.
In order to address the above problem, the applicant is that CN102358888A, name are called " a lactobacillus plantarum R23 " Chinese patent in the publication number of application on 09 20th, 2011; " Chinese agronomy circular (supplementary issue) ", in August, 2010,26:204-208; And " Fujian Journal of Agricultural Sciench " in December, 2009,24(6): all having disclosed the bacterial strain that a strain filters out from the loquat distiller's wort on the documents such as 570-574 is plant lactobacillus R23, this plant lactobacillus R23 has stronger malolactic fermentation ability and resistance, at fruit wine biological acid reductions such as loquat wine, Vitis davidi wine, red hayberry wines, and obtain applications well in the lactic fermenting beverage production of acid garden spgarden stuff; Its main mechanism is, the oxysuccinic acid in fruit wine is converted into lactic acid under the catalysis of the synthetic malolactic acid enzyme of thalline, thereby wine body acidity is descended, and mouthfeel is soft, and this process is the gordian technique of producing high-quality fruit wine; And in the garden spgarden stuff lactic fermentation, this bacterial strain can adapt to the above sour environment of pH3.0 fast, and breeds take organic acid as main carbon source, breaks through lactic acid fermented technical bottleneck, gives simultaneously garden spgarden stuff the happy fermentation perfume of coordinating; The size of malolactic acid conversion vigor has reflected the size of malolactic acid enzyme activity indirectly, and directly affect the degree that malolactic fermentation carries out, it is the primary standard of selecting good malolactic fermentation (malolactic fementation, MLF) bacterial strain.
Although above-mentioned plant lactobacillus R23 can play good deacidification effect to fruit wine, the practitioner wishes to obtain malolactic acid and transforms the higher bacterial strain of vigor, thereby provides new bacterium source for the biological acid reduction of fruit wine and the research of lactic fermentation functional drinks etc.
[summary of the invention]
Technical problem to be solved by this invention is to provide a kind of malolactic acid to transform high dynamic strain, this bacterial strain be plant lactobacillus plant subspecies (
Lactobacillus plantarum subsp.plantarum) RF17, and these plant lactobacillus plant subspecies RF17 was preserved on 03 16th, 2012 the Chinese Typical Representative culture collection center that is positioned at Wuhan City Wuhan University, and deposit number is CCTCC NO:M2012085.
The present invention solves the problems of the technologies described above by the following technical programs:
The applicant is to plant lactobacillus R23(
Lactobacillus plantarum) carry out the bacterial strain that ultraviolet nitrosoguanidine complex mutation obtains, by this bacterial strain is carried out morphology, Physiology and biochemistry and molecular biology identification, identify that finally it is a bacterial strain of plant lactobacillus plant subspecies.
Further, this bacterial strain can be in having the wine body of following at least one condition normal growth:
Beneficial effect of the present invention is: provide a kind of plant lactobacillus plant subspecies (
Lactobacillus plantarum subsp.plantarum) RF17, this bacterial strain not only has stronger malolactic acid and transforms vigor, probiotic properties, and resistance is high, can carry out malolactic fermentation under the katalysis of malolactic acid enzyme, can more effectively reduce the content of oxysuccinic acid, increase lactic acid and volatilization ester etc. in fruit wine, fruit juice, thereby make fruit wine, fruit juice mouthfeel softer, coordinate, fragrant odour is strong, for improve fruit wine, the fruit juice quality lays the foundation, and then provides new bacterium source for the biological acid reduction of fruit wine and the research of lactic fermentation functional drinks.
[description of drawings]
The invention will be further described in conjunction with the embodiments with reference to the accompanying drawings.
Fig. 1 is the gramstaining schematic diagram of bacterial strain RF17 in the present invention.
Fig. 2 is the scanning electron microscope schematic diagram of bacterial strain RF17 in the present invention.
Fig. 3 is the transmission electron microscope schematic diagram of bacterial strain RF17 in the present invention.
Fig. 4 is that in the present invention, bacterial strain RF17 grows the tree schematic diagram to the 16SrDNA sequential system of relevant kind.
Fig. 5 is that in the present invention, bacterial strain RF17 grows the tree schematic diagram to the pheS sequential system of relevant kind.
Fig. 6 is the aggegation characteristic schematic diagram of bacterial strain RF17 and plant lactobacillus R23 in the present invention.
Fig. 7 is the resistance of oxidation schematic diagram of bacterial strain RF17 and control strain in the present invention.
[embodiment]
Plant lactobacillus plant subspecies of the present invention (
Lactobacillus plantarum subsp.plantarum) RF17 is to plant lactobacillus R23(
Lactobacillus plantarum) carry out that ultraviolet nitrosoguanidine complex mutation obtains.
One, the acquisition of this bacterial strain RF17
Be 2.0～3.0 * 10 with 2mL concentration
7The bacteria suspension of cfu/mL plant lactobacillus R23 carries out centrifugal collection thalline, and add nitrosoguanidine to form nitroso guanidine solution in the thalline of collecting, and in this nitroso guanidine solution, the concentration of nitrosoguanidine is 2mg/mL, put afterwards 28 ℃ of shaking table 150r/min oscillation treatment 60min, vibration adds 25% hypo solution termination reaction by 2.5 times of nitroso guanidine solution volumes after finishing, follow centrifugal 10min under 12000rpm, abandoning supernatant is used 5mLPBS solution washing thalline three times; Then add 2mLPBS solution in the thalline after washing and get bacterium liquid with the mixing thalline, getting this bacterium liquid 0.1mL coats on the counting flat board, to count again flat board and be placed in apart from shining 20s under 15w ultraviolet lamp 20cm, shine that complete rear to wrap counting with black cloth dull and stereotyped and be placed under 28 ℃ and cultivate 3d; The bacterial strain that morphological specificity is identical is rejected, and remains bacterial strain in improvement TJA slant medium with the transfering loop picking, and numbering, cultivates 3d for 28 ℃; The bacterial strain equivalent that will improve afterwards in the TJA slant medium is inoculated in test media, after 28 ℃ of cultivation 18h, measure respectively the total acid in the test media of inoculating different strains, and take plant lactobacillus R23 as the contrast bacterium, filter out the strongest bacterial strain of deacidification ability, and to name this bacterial strain be bacterial strain RF17.
Wherein, the collecting cells method of plant lactobacillus R23 is: plant lactobacillus R23 is inoculated in the LH18 substratum cultivates, take the logarithm vegetative period bacterium liquid 5mL in the aseptic centrifuge tube of 10mL, and should be placed in centrifugal 10min under 12000r/min by aseptic centrifuge tube, abandoning supernatant with aseptic PBS solution 5mL washed twice, then adds the aseptic PBS solution of 5mL mixing, with 100 times of this bacterium liquid dilutions, it is 2.0～3.0 * 10 that the thallus suspension liquid of gained is concentration at last
7The bacteria suspension of cfu/mL, stand-by;
The preparation of PBS solution: the ratio according to 0.2mol/L Sodium phosphate dibasic 26.5mL:0.2mol/L SODIUM PHOSPHATE, MONOBASIC 73.5mL is mixed, and the pH to 6.4 of regulator solution namely gets required PBS solution;
The dull and stereotyped component with improvement TJA slant medium of counting is: tomato juice 50mL, yeast extract 5g, extractum carnis 10, lactose 20g, glucose 2g, dipotassium hydrogen phosphate 2g, tween 80 .1g, sodium acetate 5g, adding distil water is to 1L, and 20min sterilizes under pH6.8 ± 0.2,121 ℃;
The component of test media is: tomato juice 10%, yeast extract paste 0.2%, extractum carnis 0.4%, MgSO
40.02%, sodium acetate 0.5%, Tween-800.1%, ammonium citrate 0.2%, MnSO
40.005%, Tryptones 1%, oxysuccinic acid 0.5%, pH4.8 ± 0.2,1 * 10
520min sterilizes under Pa;
The component of LH18 substratum (lower same) is: tomato juice 100mL, yeast extract paste 7.4g, extractum carnis 10g, glucose 30g, MgSO
40.36g, sodium malate 20g, tween 1g, Tryptones 15g, ammonium citrate 2g, MnSO
40.05g, add water and supply 1L.
Two, the evaluation of this bacterial strain RF17
1. preliminary evaluation---morphology and Physiology and biochemistry are identified
The bacterial strain RF17 of screening gained is carried out the researchs such as form and physicochemical property, and the Main Morphology and the cultural characteristic that obtain this bacterial strain RF17 are as follows: colony diameter 1.0～1.8mm, and the bacterium colony oyster white, circle, smooth surface, projection, moistening, opaque, neat in edge; To bacterial strain RF17 carry out gramstaining (schematic diagram as shown in Figure 1) and bacterial strain RF17 is placed in scanning electron microscope and transmission electron microscope observe respectively (wherein the scanning electron microscope schematic diagram as shown in Figure 2, the transmission electron microscope schematic diagram as shown in Figure 3) learn, thalline is shaft-like, 0.5 μ m * 1.33～1.67 μ m, single, in pairs or be the short chain shape and arrange, do not give birth to spore, Gram-positive; Liquid culture sterile film,, layering muddy without bubble, bacterium liquid are obviously, thalline is difficult for centrifugal and bottom adularescent precipitation.Its concrete Physiology and biochemistry identification of indicator in following table 1:
The Physiology and biochemistry identification of indicator of table 1 bacterial strain RF17
Annotate: "+" expression is positive; "-" expression is negative; "+W " expression is weak positive.
2. further identify---molecular biology identification:
16SrDNA sequence and pheS sequence to bacterial strain RF17 are measured respectively, the 16SrDNA sequence that records as shown in SEQ ID NO.1, the pheS sequence is as shown in SEQ ID NO.2; And adopt MEGA4.1 software, ortho position connection method phylogenetic tree construction respectively, carry out the similarity of 1000 times and calculate.As shown in Figure 4, the 16SrDNA sequential analysis shows, the bacterial strain homology that bacterial strain RF17 and plant lactobacillus belong to reaches more than 90%; As shown in Figure 5, the pheS sequential analysis shows, genetic evolution distance and the plant lactobacillus of bacterial strain RF17 belong to recently, and be in the branch of a minimum with plant lactobacillus plant subspecies (Lactobacillus plantarum subsp.plantarum), with type strain Lactobacillus plantarum subsp.plantarum LMG6907T(AM087714) homology reach 100%, illustrate that this bacterial strain RF17 belongs to plant lactobacillus plant subspecies on the Molecular Phylogeny taxonomy.
Principle of classification according to homology in phylogeny and combining form, physiological and biochemical property, with reference to " common bacteria system identification handbook, finally determine bacterial strain RF17 be plant lactobacillus plant subspecies (
Lactobacillus plantarum subsp.plantarum).
Three, the characteristic research of this bacterial strain RF17
1. the malolactic acid of bacterial strain RF17 transforms vigor and deacidification ability
Under equal conditions plant lactobacillus R23 and bacterial strain RF17 are carried out respectively inoculation culture, measure its malolactic acid and transform vigor and deacidification amount, measurement result is as shown in table 2, the conversion vigor of plant lactobacillus R23 and bacterial strain RF17 is respectively 119.5u and 137.9u, two bacterial strain differences are extremely remarkable, and the malolactic acid of bacterial strain RF17 transforms vigor and improved 15.4% than plant lactobacillus R23; The deacidification amount of plant lactobacillus R23 and bacterial strain RF17 is respectively 0.55mg/mL and 0.98mg/mL, and two bacterial strain differences are remarkable, and the acid molar ratio plant lactobacillus R23 that falls of bacterial strain RF17 has improved 78.2%.
The malolactic acid of table 2 bacterial strain RF17 and plant lactobacillus R23 transforms vigor and deacidification ability
2. the tolerance of bacterial strain RF17
Respectively to bacterial strain RF17 and plant lactobacillus R23 tolerance SO
2The ability of concentration, alcoholic strength, pH value and low temperature is tested, and test-results sees Table 3, and as can be known, R23 compares with plant lactobacillus, and bacterial strain RF17 is except relatively relatively poor to the tolerance of 13% alcohol, to SO
2, pH and temperature tolerance be respectively 1.7,18.4,1.1 times of plant lactobacillus R23, namely acid-fast ability has had and has significantly improved, and is the good mutagenic strain of a strain.
Table 3 bacterial strain RF17 and the plant lactobacillus R23 tolerance to environmental factor
Annotate: 1) isolated strains to the tolerance of each environmental factors with (A) b(as (10.20 ± 1.30) +++) form represents, wherein: A represents that the bacterium amount increases multiple: A＜1, expression bacterium amount is negative growth; A 〉=1, expression bacterium amount is positive growth; B represents that the bacterium magnitude is other: " ++ ++ " represent that plate count bacterium amount reaches the 109CFU/mL level; " +++" represent that plate count bacterium amount reaches the 108CFU/mL level; " ++ " expression plate count bacterium amount reaches the 107CFU/mL level; "+" expression plate count bacterium amount reaches the 106CFU/mL level.
3. the probiotic properties of bacterial strain RF17
(1) acid and bile tolerance ability
Be that plant lactobacillus R23, lactobacillus delbruockii subspecies bulgaricus 6045(are available from the commercial strain of Chinese industrial microbial strains preservation administrative center with bacterial strain RF17 and the contrast bacterium of collecting) respectively with after stroke-physiological saline solution washing 2 times, and make bacteria suspension with 1/10th of original volume, inoculum size take 1% be inoculated in respectively the pH value as 3.5,3.0,2.5,2.0,1.5 and gallbladder salinity in 0%, 0.1%, 0.3%, 0.5% LH18 substratum, be placed in 25 ℃ of constant temperature culture 4h, and live bacterial count is carried out in every 2h sampling, averages for parallel 3 times.Each bacterial strain cultivates in different pH values and gallbladder salinity environment that bacterium quantitative changeization after 4h sees Table respectively 4, table 5.
The acid-fast ability of table 4 bacterial strain RF17 (lgCFU/mL,
)
Annotate: ND: expression does not detect, and is lower same.
Table 5 bacterial strain RF17 to the tolerance of cholate (lgCFU/mL,
)
By table 4 and table 5 as can be known: after cultivating 4h under the environment of pH value 2.0, plant lactobacillus R23 and lactobacillus delbruockii subspecies bulgaricus 6045 can't be grown, and bacterial strain RF17 bacterium amount is still near 10
6Cfu/mL, its acid-fast ability is the strongest; After cultivating 2h under 0.5% cholate environment, lactobacillus delbruockii subspecies bulgaricus 6045 exists without viable cell; The equal dead of bacterial strain RF17 after cultivation 4h, and the biomass of plant lactobacillus R23 still can reach 10
7CFU/mL.Result shows, bacterial strain RF17 acid-fast ability is the strongest, and the ability of tolerance cholate is inferior to plant lactobacillus R23.
In addition, described in bacterial strain acid and bile tolerance capability study process, the concrete preparation process of bacteria suspension is: bacterial strain RF17, plant lactobacillus R23, lactobacillus delbruockii subspecies bulgaricus 6045 are accessed respectively in the LH18 substratum, and in 28 ℃ of constant temperature culture until the thalline biomass reaches 10
8More than CFU/mL, afterwards the fermented liquid of constant temperature culture gained be placed under 3000r/min, 4 ℃ centrifugal 10min and collect bacterium mud, standby; Get intestinal bacteria (indicator) 25 ℃ of constant temperature culture to thalline biomasss in bacteria culture medium and reach 10
7More than CFU/mL, then with nutrient solution centrifugal 10min and collect bacterium mud under 3000r/min, 4 ℃; Respectively with pH7.0 phosphate buffered saline buffer washing 2 times, and the absorbance that is formed in wavelength 600nm place is that 0.4 ± 0.1 suspension bacteria liquid is bacteria suspension with each bacterial classification bacterium mud of collecting.
(2) stick ability
Measure respectively the surface hydrophobicity of bacterial strain RF17 and contrast bacterium plant lactobacillus R23, measurement result as shown in Figure 6, as seen from Figure 6, bacterial strain RF17 reaches respectively 32.67% and 14.96% from aggegation rate and his aggegation rate, though a little less than plant lactobacillus R23, adhesive capacity is still stronger.
(3) resistance of oxidation
Be that plant lactobacillus R23, lactobacillus delbruockii subspecies bulgaricus 6045 carry out respectively determination oxidative to bacterial strain RF17 and contrast bacterium, measurement result as shown in Figure 7, as shown in Figure 7, the centrifugal bacterium liquid of each bacterial strain all has certain removing ability to superoxide anion and hydroxy radical qiao, born of the same parents' extra-metabolite that each bacterial strain namely is described all has resistance of oxidation, especially removes the ability of ultra-oxygen anion free radical; Wherein the ability of bacterial strain RF17 removing free radical is the strongest, fermented liquid after fermentation 24h reaches respectively 78.2% and 81.0% to the clearance rate of ultra-oxygen anion free radical and hydroxy radical qiao, and is higher 37.1 and 8.2 percentage points than contrast bacterium lactobacillus delbruockii subspecies bulgaricus 6045 respectively.
By above-mentioned research, bacterial strain RF17 of the present invention contains following characteristic as can be known: 1. stronger resistance: can tolerate the pH value more than or equal to 1.5 acidity, can tolerate the gallbladder salinity up to 0.3%, 2. stronger resistance of oxidation: born of the same parents' extra-metabolite that bacterial strain RF17 cultivates after 24h reaches respectively 78.2% and 81.0% to the clearance rate of ultra-oxygen anion free radical and hydroxy radical qiao, 3. stronger adhesive capacity: reach respectively 32.67% and 14.96% from aggegation rate and his aggegation rate.
And this bacterial strain RF17 is normal growth in having the wine body of following at least one condition:
Four, the application of this bacterial strain RF17
1. the application of bacterial strain RF17 in producing fermented type aseptic " No. 1, Portugal, osmanthus " grape juice beverage
With bacterial strain RF17 in the seed fermentation substratum be in the LH18 substratum after activation to obtain to spread cultivation bacterium liquid, afterwards this bacterium liquid that spreads cultivation with 5.7 * 10
8The cfu/ml inoculum size is linked in aseptic fermentor tank, the Sucus Vitis viniferae of processing 2min through 95～100 ℃ is housed in this tank, 30 ℃ ± 2 ℃ of manual shift leavening temperatures, aerobic fermentation obtain appropriate lactic acid fermented Sucus Vitis viniferae lactic acid fermentation liquid after 48 hours, carry out Beverage Service after filtration, make content at 20% Cloudy lactic fermenting beverage, then the can sterilization gets final product.
In " No. 1, Portugal, osmanthus " Sucus Vitis viniferae fermenting process, the variation of leading indicator is as shown in table 6; Through fermentation in 48 hours, oxysuccinic acid almost disappeared, and lactic acid generates in a large number; Bacterial strain RF17 preferentially carries out during the fermentation MLF and produces lactic acid take oxysuccinic acid as main carbon source, total acid descends; Carry out heterolactic fermentation take carbohydrate metabolism as main after MLF finishes, product is partly lactic acid, and total acid increases thereupon; Fermentation 48h aromatic series tyrosine and proline(Pro) increase suddenly, have even surpassed unfermented juice, and by the flavor evaluation mark, this moment, the fermented juice best flavor formed the phase just.
The variation of leading indicator in table 6 " No. 1, Portugal, osmanthus " Sucus Vitis viniferae fermenting process
2. the application of bacterial strain RF17 in " liberation clock " loquat wine is brewageed
With bacterial strain RF17 in the seed fermentation substratum be in the LH18 substratum activation after, with 5.7 * 10
8Cfu/mL inoculum size access pH is 3.45, total sulphur concentration is that 120mg/L, alcoholic strength are in " liberation clock " loquat wine of 11.5%, and in 25 ± 1 ℃ of constant temperature culture, and the carrying out with paper chromatography monitoring MLF, monitoring finds that in the paper chromatography of cultivation after 3 days, oxysuccinic acid disappears, and represents that MLF finishes to stop course of fermentation.
" liberation clock " main Oranoleptic indicator's of loquat wine variation sees Table 7, and larger variation has all occured for the total acid, pH value, the volatilization ester that use bacterial strain RF17 to carry out " liberation clock " loquat wine of MLF reaction; Use a large amount of minimizings of oxysuccinic acid and the total acid of the loquat wine of bacterial strain RF17 to descend and the rising of pH value, the deacidification rate reaches 47%, has reached the effect of biological acid reduction; The volatilization ester is one of main component that affects loquat wine fragrance and local flavor, the volatilization ester of proper content can make aroma strong, be conducive to the formation of the mellow local flavor of wine body and complicacy, wine sample volatilization ester total amount after using bacterial strain RF17 through the MLF effect slightly increases, and shows that bacterial strain RF17 can increase fragrance and the local flavor of loquat wine; Soft index is to estimate an important indicator of coordinating in wine making, test is quoted in grape wine this important evaluation index the effect that bacterial strain RF17 improves the fruit wine local flavor is carried out quantization assessment, result shows that bacterial strain RF17 can improve the soft index of loquat wine, the loquat wine mouthfeel is softer, coordination thereby improvement fruit wine quality makes, and fragrant odour is strong.
Table 7 " liberation clock " the main Oranoleptic indicator's of loquat wine variation
In summary, plant lactobacillus plant subspecies of the present invention (
Lactobacillus plantarum subsp.plantarum) RF17 not only has stronger malolactic acid and transform vigor, probiotic properties, and resistance is high, can carry out malolactic fermentation under the katalysis of malolactic acid enzyme, can more effectively reduce the content of oxysuccinic acid, increase lactic acid and volatilization ester etc. in fruit wine, fruit juice, thereby make fruit wine, fruit juice mouthfeel softer, coordinate, fragrant odour is strong, for raising fruit wine, fruit juice quality lay the foundation.
Claims (1)
1. a malolactic acid transforms high dynamic strain, it is characterized in that: described bacterial strain be plant lactobacillus plant subspecies (
Lactobacillus plantarum subsp.Plantarum) RF17, being preserved on 03 16th, 2012 the Chinese Typical Representative culture collection center that is positioned at Wuhan City Wuhan University, deposit number is CCTCC NO:M2012085.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108546654B (en) * | 2018-03-23 | 2020-06-02 | 广东省农业科学院蚕业与农产品加工研究所 | Acid-reducing lactobacillus fermentum and application thereof |
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