CN106520620A - High-diacetyl-yield Lactobacillus plantarum strain and application thereof - Google Patents
High-diacetyl-yield Lactobacillus plantarum strain and application thereof Download PDFInfo
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Abstract
The invention discloses a high-diacetyl-yield Lactobacillus plantarum strain of which the collection number of the strain is CCTCC M2016513. The invention also discloses a working ferment which contains the Lactobacillus plantarum strain. The invention also discloses application of the high-diacetyl-yield Lactobacillus plantarum strain in fermented food. The detection and organoleptic evaluation on the diacetyl content in the Lactobacillus-plantarum-containing fermented milk indicate that the Lactobacillus plantarum has higher diacetyl yield and higher organoleptic scores, improves the fragrance quality of the fermented milk and promotes the enhancement of the fermented milk quality.
Description
Technical field
The invention belongs to microbiological art, is related to a kind of Lactobacillus plantarum, a kind of specifically high yield biacetyl
Lactobacillus plantarum (Lactobacillus plantarum) and its application.
Background technology
Lactic acid bacteria (Lactic acid bacteria, LAB) is that a class can produce a large amount of lactic acid using fermentable sugar
Antibacterial general name, belongs to at least 23 on taxonomy on this kind of antibacterial that nature has found at present.In food, medicine and other fields
Mainly there are Lactobacillus, Streptococcus, Enterococcus, Lactococcus, Pediococcuses and leukonid using more lactic acid bacteria
Category etc..Lactic acid bacteria is the topmost source of probiotic bacteria, and many lactic acid bacterias are the intrinsic probiotic bacterias of human body intestinal canal, with improving people
Body intestinal microbial population, adjusts immunity of organisms, and inhibiting cancer, reduce serum cholesterol adjust the important physiologically actives such as blood pressure.
Local flavor is one of key factor of fermentation milk quality responses, and affects key of the consumer to fermentation milk degree of recognition
Index.Biacetyl (Diacetyl) is butter, Yoghourt, cheese and a kind of flavor substances in the non-milk product for much needing creamy taste
Matter [list of references (Yang Lijie, Wang Junhu. the regulation and control [J] of biacetyl metabolism branch road in lactococcus lactis. China Dairy Industry,
2004,32 (5)：24-29.)], have when being diluted to 1mg/L butter fragrance [list of references (in Tian Li, Jiao Weina, celebrate ring.
Lactococcus lactis subsp.lactis produce the optimizing research [J] of diacetyl. food science and technology, 2011,36 (2)：238-242.)/(Wang Hong
Leaf, Li Lihua, Lu Chun. the impact [J] of acetaldehyde, diacetyl to fermentation milk local flavor. China Dairy Industry, 2010,38 (10)：32-
34.)].In flavour and the fragrance of fermented dairy product, biacetyl is considered as one of important compound for working, even if depositing
In micro biacetyl, also can largely improve the local flavor of Yoghourt.Current America and Europe occurs producing main flavor
The ketone odor type Yoghourt of biacetyl, China also have ketone odor type Yoghourt of the research main flavor material for biacetyl, for improving tradition
The aldehyde odor type local flavor of Yoghourt.Therefore the ability that lactic acid bacteria produces biacetyl is improved, the local flavor of yogurt can be improved from source, be improved and send out
Kefir milk flavouring essence quality, the production for dairy industry provide certain directive significance.
The content of the invention
For above-mentioned technical problem of the prior art, the invention provides a kind of Lactobacillus plantarum of high yield biacetyl and
Its application, the Lactobacillus plantarum of described this high yield biacetyl and its application will solve lactic acid bacteria of the prior art and produce double second
The technical problem of the scarce capacity of acyl.
The invention provides a kind of Lactobacillus plantarum of high yield biacetyl, its deposit number is CCTCC M 2016513
Present invention also offers a kind of working stock culture, above-mentioned Lactobacillus plantarum strain is inoculated in sterilized milk, culture
To curdled milk, in continuous culture two generations of activation, are used as mother culture；Mother culture is inoculated in into addition milk surum by percent by volume 3-5%
Cultivate to curdled milk in the sterilized milk of albumen, obtain final product working stock culture.
Further, during culture to curdled milk, 10-12h is cultivated under the conditions of 35-37 DEG C.
Present invention also offers above-mentioned Lactobacillus plantarum strain is inoculated in fluid medium by a kind of working stock culture
In, in two generations of continuous activation, then activation culture thing is inoculated in fluid medium by percent by volume 3-5% and is cultivated, solid-liquid
Isolated cell precipitation, precipitation is suspended with aseptic breast, working stock culture is obtained final product, wherein, in described leaven, viable bacteria
Number is 109More than cfu/mL.
Further, during activating in liquid medium within, 10-12h is cultivated under the conditions of 35-37 DEG C.
Present invention also offers a kind of purposes of the Lactobacillus plantarum of above-mentioned high yield biacetyl in fermented food.
Further, described fermented food is lactic acid bacteria milk beverage or fermentation milk.
Present invention also offers a kind of preparation method of above-mentioned lactobacillus beverage, cools down after raw milk is sterilized, then
Above-mentioned working stock culture mix homogeneously is added, makes above-mentioned Lactobacillus plantarum concentration reach 106More than cfu/mL, that is, contained
The lactic acid bacteria milk beverage of above-mentioned Lactobacillus plantarum.
Present invention also offers a kind of preparation method of above-mentioned fermentation milk, cools down after raw milk is sterilized, adds
The working stock culture stated and customary commercial leaven, it is 3-5% that described working stock culture accounts for the volume of fermentation milk, and described is normal
It is 3-5% that rule commodity leaven accounts for the volume of fermentation milk, is fermented, to titratable acidity 70- after mixing under conditions of 35-37 DEG C
80 ° of T, that is, obtain the fermentation milk of above-mentioned Lactobacillus plantarum.
Further, described customary commercial leaven is Lactobacillus bulgaricus, bacillus bifiduss, streptococcus thermophiluss or thermophilic
Any one in Lactobacillus lactiss or two or more combinations.
Wherein, described sterilizing methods are conventional raw milk sterilizing methods, preferably such as instantaneous ultrahigh-temperature sterilization, more preferably
Such as at 95 DEG C heating sterilization 20min.Described Lactobacillus plantarum working stock culture, cooling are added after breast cooling subject to sterilization
Conventional Temperature, is preferably cooled to 37-40 DEG C.Fermentation temperature routine, preferably 37 DEG C.
The assay method of biacetyl is that conventional headspace solid-phase microextraction combines gas chromatography combined with mass spectrometry (HS-SPME-
GC-MS method) is detected, preferably includes for the fermentation milk of Lactobacillus plantarum CCTCC M 2016513 being placed in head space admittedly
It is to be measured in phase extraction bottle.
Wherein, the condition determination of headspace solid-phase microextraction is：The above-mentioned fermentation milks of 5g-10g are placed in 20ml ml headspace bottles, and 55
30-40min is extracted under DEG C -60 DEG C of waters bath with thermostatic control, extracting head is placed on GC-MS and parses 5min.
Wherein, GC-MS condition determinations are：60m × 0.25mm × 0.25 μm HP-INNOWX capillary chromatographs (U.S. J＆W
Company)；50/30 μm of DVB/CAR/PDMS solid phase micro-extracting head (Supelco companies of the U.S.)；60 DEG C of initial temperature, holding 3min, 5
DEG C/min is warming up to 230 DEG C, keeps 10min.
Lactobacillus plantarum (Lactobacillus plantarum) bacterial strain that the present invention is provided, its biolvgical name are referred to as：
Lactobacillus plantarum Z01, belong to Lactobacillus, Lactobacillus plantarum kind.In September in 2016 is preserved on the 22nd
State's Type Tissue Collection, preservation address：Luojiashan, Wuchang, Wuhan City, Hubei Province Wuhan University collection.Postcode：
430072, its deposit number is CCTCC M 2016513.
The present invention is compared with prior art, and its technological progress is significant.The plant breast bar of the high yield biacetyl of the present invention
Bacterium CCTCC M2016513, by being detected to the diacetyl content in its fermentation milk and subjective appreciation, as a result show this
Bright Lactobacillus plantarum biacetyl yield is higher, and sensory evaluation scores are higher, improves the flavouring essence quality of fermentation milk, promotes fermented milk products
The raising of matter, has broad prospects in fermentation milk field.
Description of the drawings
In conjunction with the following drawings, the feature and beneficial effect of the present invention are illustrated.
Fig. 1 shows the colonial morphology of Lactobacillus plantarum CCTCC M of the present invention 2016513.
Fig. 2 shows the cellular morphology (× 1000) of Lactobacillus plantarum CCTCC M of the present invention 2016513.
Fig. 3 shows the growth curve of Lactobacillus plantarum CCTCC M of the present invention 2016513.
Fig. 4 shows the optimum growth temperature of Lactobacillus plantarum CCTCC M of the present invention 2016513.
Fig. 5 shows the fermentation milk GC-MS total ion current figures of Lactobacillus plantarum CCTCC M of the present invention 2016513.
Fig. 6 shows that the fermentation yogurt of Lactobacillus plantarum CCTCC M of the present invention 2016513 and the sense organ of blank yogurt are obtained
Divide radar map.
Specific embodiment
The present invention gathers sample from lactic acid bacteria habitat, screens the lactobacilli strain of high yield biacetyl, studies which and produces double second
The ability of acyl, develops new probiotic bacteria.Raw material or reagent used by the present invention is in addition to special instruction, commercially available.
The present invention filters out a strains of lactic acid bacteria 1-33 from lactic acid bacteria habitat, using morphological characteristic, cultural colony and physiology
The Microbiological Characteristics such as biochemical character and its inherited character 16s rDNA are accredited as Lactobacillus plantarum to lactic acid bacteria 1-33
(Lactobacillus plantarum), the bacterial strain are preserved in China typical culture collection center on 22nd in September in 2016
(abbreviation CCTCC), its deposit number are CCTCC M 2016513.
The morphological feature of Lactobacillus plantarum CCTCC M of the present invention 2016513：
Colony characteristicses：Bacterial strain is separated in the flat lining outs of MRS, and 37 DEG C of culture 48h, strain growth are good, its colonial morphology
As shown in Figure 1.Bacterium colony size 0.2-2mm, bacterium colony are circular, neat in edge, front microprotrusion, and the milky white middle band point of color is slightly yellow,
Opaque, surface wettability is smooth.
Thalline feature：In shaft-like (Fig. 2), thalline size is generally 1.5 μm of 0.6 μ m to thalline, does not produce spore, gram dye
Color is positive.
The cultural characteristic of Lactobacillus plantarum CCTCC M of the present invention 2016513：Lactobacillus plantarum CCTCC M's 2016513
Minimum growth temperature is 15 DEG C, and maximum growth temperature is 45 DEG C, optimal in 30-40 DEG C of growth temperature；Bacterial strain CCTCC M
2016513 period of delay is relatively short, and 2h enters exponential phase, and 11h reaches stable phase.
The Lactobacillus plantarum CCTCC M 2016513 of the present invention derive from traditional fermented food, belong to generally recognized as safe
(Generally Recognized As Safe, GRAS) strain, can be used in lactobacillus food.
Therefore, the invention further relates to purposes of the described Lactobacillus plantarum CCTCC M 2016513 in fermented food.Institute
Stating the fermented food containing Lactobacillus plantarum CCTCC M 2016513 includes lactic acid bacteria milk beverage and fermentation milk.
The present invention also provides 2016513 working stock cultures of Lactobacillus plantarum CCTCC M.
What working stock culture of the present invention was preferably prepared using following preparation methoies：By Lactobacillus plantarum CCTCC M
2016513 strains are inoculated in the skimmed milk of 12% (w/v), cultivate 10-12h to curdled milk under the conditions of 37 DEG C, and continuous culture is lived
Changed for two generations, use as mother culture；Mother culture is inoculated in above-mentioned sterilized milk by 3-5% (v/v), 10-12h is cultivated
To curdled milk, now in curdled milk viable count about 109Cfu/mL, obtains described working stock culture；Or by Lactobacillus plantarum
2016513 strains of CCTCC M are inoculated in MRS fluid mediums, are cultivated 10-12h and are activated, continuously under the conditions of 37 DEG C
Activated for two generations, then activation culture thing is inoculated in MRS culture medium by 3-5% (v/v), cultivate 10-12h, under the conditions of 4 DEG C
6000r/min is centrifuged 10min, removes supernatant, obtains cell precipitation, precipitation is suspended with a certain amount of sterile absorbent breast, is obtained
To described working stock culture.
In the present invention, preferred described lactic acid bacteria milk beverage is prepared as steps described below：Raw milk is at 95 DEG C
Lower heating sterilization 20min, is then cooled to 40 DEG C, adds the described work fermentations of Lactobacillus plantarum CCTCC M 2016513
Agent so as to which concentration reaches 106More than cfu/mL, obtains containing Lactobacillus plantarum CCTCC M 2016513 in 4 DEG C of stored refrigerated
Lactic acid bacteria milk beverage.
In the present invention, preferred described fermentation milk is prepared as steps described below：Raw milk is heated at 95 DEG C
Sterilization 20min or at 140 DEG C high temperature thermal sterilization 2s, be then cooled to 37 DEG C, add the plant according still further to 3-5% (v/v)
Lactobacilluss CCTCC M 2016513, add 3-5% (v/v) fermentation milk commodity leaven, after mixing at 37 DEG C mixed fungus fermentation
To 70-80 ° of T of titratable acidity, it is subsequently cooled to 40 DEG C, then carries out stored refrigerated obtain containing Lactobacillus plantarum CCTCC M
2016513 fermentation milk.
Below with embodiment further illustrating the present invention, but the present invention is not intended to be limited thereto.Do not note in the following example
The experimental technique of bright actual conditions, generally according to normal condition, or according to the condition proposed by manufacturer.Described in embodiment
" room temperature " refer to the temperature of the operation room tested, generally 25 DEG C.
The collection of 1 Lactobacillus plantarum CCTCC M 2016513 of embodiment, separation
(1), sample pretreatment
3g tibet koumiss several pieces are taken respectively, are respectively put in the 50ml conical flasks equipped with 15ml sterilized water, it is after concussion, quiet
20min is put, it is standby.
(2), lactobacilli strain is separated
Using sterilized water by volume, according to 1：10 pairs of above-mentioned samples carry out serial dilution, take 0.1mL in each dilution factor
Dilute sample, is respectively coated MRS agar plates and M17 agar plates, and constant temperature culture 24-48h under 37 DEG C of anaerobic conditions, with nothing
Bacterium toothpick picking is of different sizes, raised, micro white, moistening, neat in edge, single bacterium colony of the bacterium colony back side for yellow.Then in phase
On the agar plate answered, line point is pure, obtains pure single bacterium colony, carries out Gram’s staining, catalase experiment.Purification bacterial strain preservation
In corresponding isolation medium, used as protective agent, -20 DEG C frozen for the glycerol of addition 30%.
Culture medium prescription used in which is as follows：
MRS culture medium (Bifidobacterium selective culture medium, Beijing overpass technical concern company limited buys).
M17 culture medium (Lactococcus selective medium, Beijing overpass technical concern company limited buys).
Different batches sample isolates 90 plants of bacterium on MRS agar culture mediums and M17 agar culture mediums.These bacterial strains exist
Separate and on flat board, show projection, micro white, moistening, neat in edge, the bacterium colony back side are yellow.
(3) different strains produce biacetyl ability and determine
The separation strains obtained from flat board are inoculated in MRS fluid mediums and cultivate 12h, then by the inoculation of 5% (v/v)
Amount is inoculated into re-activation in fluid medium containing MRS, and ferment 10h at 37 DEG C.By fermentation liquid at 4 DEG C under 6000rpm/min
Centrifugation 10min, takes thalline.With aseptic water washing 3 times, sterilized water is added to mix, it is standby.
Raw milk heats sterilization 20min or the high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 40 DEG C, then
Add bacteria suspension, add 5% (v/v) fermentation milk commodity leaven, after mixing at 37 DEG C mixed fungus fermentation to titratable acidity 70-
80 ° of T, are subsequently cooled to room temperature, then carry out 4 DEG C of stored refrigerated.
The fermentation milk of above-mentioned cold preservation is weighed into 5g, is placed in headspace solid-phase microextraction bottle (20ml).Ml headspace bottle is placed in
In 55 DEG C of thermostat water bath, the prior aging good extracting head (50/30 μm of DVB/CAR/PDMS) of insertion, headspace absorption
40min, extraction terminate, and extracting head is transferred at GC-MS injection ports, parse 5min, complete sample introduction.
Wherein, the fermentation milk of described Lactobacillus plantarum is conventional fermentation milk, preferably 37 DEG C condition bottom fermentation 10h and
Obtain fermentation milk.
Wherein, the condition determination of headspace solid-phase microextraction is：The above-mentioned fermentation milks of 5g are placed in 20ml ml headspace bottles, 55 DEG C of constant temperature
40min is extracted under water-bath, and extracting head is placed on GC-MS and parses 5min.
GC-MS is kept 3min, 5 DEG C/min to be warming up to 230 DEG C, is kept the ramp of 10min, completed with 60 DEG C of initial temperature
The detection of fermentation milk volatile compound, so that determine the content for drawing biacetyl in fermentation milk.Experimental result is listed in Table 1 below.From
As can be seen that the biacetyl yield of bacterial strain 1-33 is of a relatively high in table, this plant of bacterium is chosen, CCTCC M 2016513 are named as.
Table 1. produces the initial gross separation of biacetyl lactic acid bacteria
The identification of 2 Lactobacillus plantarum CCTCC M 2016513 of embodiment
(1) physiological and biochemical test
Bacterial strain CCTCC M 2016513 are Gram-positive, peroxidase is negative, no motion of bacillus, at 15 DEG C and 45
DEG C can grow.
(2) the 16s rDNA sequence analysis of bacterial strain CCTCC M 2016513
2016513 genome DNA extracting methods of bacterial strain CCTCC M：2016513 single bacterium colonies of CCTCC M of picking purification
It is inoculated in 10mL MRS fluid mediums, bacterium solution is centrifuged into (5000g, 10min) collects thalline after 37 DEG C of culture 8h.Using base
Because group DNA extraction agent boxes (Sangon Biotech (Shanghai) Co., Ltd.) is extracted.PCR amplifications are using the logical of two kinds of synthesis
Use primer
16s 27F:GAGAGTTTGATCCTGGCTCAG
16s 1492R：CGGCTACCTTGTTACGACTT
PCR primer is reclaimed using pillar PCR primer purification kit (Sangon Biotech (Shanghai) Co., Ltd.),
Sangon Biotech (Shanghai) Co., Ltd. is sent to be sequenced after purification.The 16s DNA cores of obtained strains CCTCC M 2016513
Nucleotide sequence is 1448bp (the SEQ ID NO in sequence table：1) GenBank (GenBank accetion number, are sent：
GQ359860 Blast analyses) are done.2016513 homology highest bacterial strains of bacterial strain CCTCC M are L.plantarum qz-227
(Sequence ID：AB904768.1), homology is 99%.
The G+C (mol%)≤10%～12% of the DNA according to described in Goodfellow and O ' Donnell and 16S rRNA
The kind of sequence homology >=95% can be classified as a category, and Embley and Stackebrangdt thought when 16S rRNA's
A kind is may be considered during sequence homology >=97%.It is possible thereby to infer：Bacterial strain CCTCC M 2016513 with
L.plantarum qz-227 belong to same kind.Bacterial strain CCTCC M 2016513 are accredited as Lactobacillus plantarum.
According to the Microbiological Characteristics such as morphological characteristic, physiological and biochemical property and its inherited character 16s rDNA to lactic acid bacteria
CCTCC M 2016513 are accredited as Lactobacillus plantarum (Lactobacillus plantarum), and the bacterial strain is in September, 2016
China typical culture collection center (abbreviation CCTCC) is preserved within 22nd, its deposit number is CCTCC M 2016513.
The growth characteristics of 2016513 bacterial strains of embodiment 3CCTCC M
(1) drafting of 2016513 strain growth curves of CCTCC M
The Lactobacillus plantarum CCTCC M 2016513 for having activated are accessed into MRS fluid mediums by 5% (v/v) inoculum concentration
In, 37 DEG C of constant temperature culture 16h, every the OD values that 1-2h determines culture fluid in 600nm, are plotted against time with OD values and obtain bacterial strain
Growth curves of the CCTCC M 2016513 in MRS, its result (Fig. 3) show：Lactobacillus plantarum CCTCC M 2016513 exist
Grow rapidly in MRS culture medium, logarithmic (log) phase, 11h or so is entered in 2h or so and enter stable phase.
(2) 2016513 bacterial strain optimum growth temperatures of CCTCC M are determined
The Lactobacillus plantarum CCTCC M 2016513 for having activated are connected to into 10mL MRS liquid respectively by 5% (V/V) inoculum concentration
In body culture medium, constant temperature culture 8h under the conditions of being respectively placed in 15 DEG C, 25 DEG C, 32 DEG C, 37 DEG C, 40 DEG C and 45 DEG C, with nonvaccinated
MRS fluid mediums are compared, and determine the OD values of the culture fluid cultivated under different temperatures in 600nm, and the size according to OD values is true
Determine optimum growth temperature.As a result show：(Fig. 4) growth temperature range of Lactobacillus plantarum CCTCC M 2016513 is wider, from 15
DEG C all grow to 45 DEG C, in 30 DEG C of -40 DEG C of well-growns, optimum growth temperature is 37 DEG C.
4 Lactobacillus plantarum CCTCC M of embodiment 2016513 produce biacetyl ability and determine
(1) actication of culture：2016513 strains of Lactobacillus plantarum CCTCC M are inoculated in MRS fluid mediums, 37
Cultivate 10h to be activated under the conditions of DEG C, two generations of continuous activation.
(2) preparation of working stock culture：Activation culture thing is inoculated in MRS culture medium by 5% (v/v), 10h is cultivated,
Under the conditions of 4 DEG C 6000r/min centrifugation 10min, remove supernatant, obtain cell precipitation, will precipitation with it is a certain amount of it is aseptic take off
Fat breast suspends, and obtains described working stock culture.
(3) prepared by fermentation milk：Raw milk heats sterilization 20min or the high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, then
40 DEG C are cooled to, working stock culture and the volume hundred of 5% Lactobacillus plantarum CCTCC M 2016513 of percent by volume is subsequently adding
Divide 5% commodity leaven of ratio, after fermentation is mixed to 70-80 ° of T of titratable acidity, that is, obtain the fermentation containing the Lactobacillus plantarum
The fermentation milk of newborn CCTCC M 2016513.
(4) diacetyl content is determined：The fermentation milk of above-mentioned cold preservation is weighed into 5g, headspace solid-phase microextraction bottle is placed in
(20ml) in.Ml headspace bottle is placed in 55 DEG C of thermostat water bath, prior aging good extracting head (the 50/30 μm of DVB/ of insertion
CAR/PDMS), headspace absorption 40min, extraction terminate, and extracting head is transferred at GC-MS injection ports, parse 5min, complete into
Sample.GC-MS is kept 3min, 5 DEG C/min to be warming up to 230 DEG C, is kept the ramp of 10min, complete fermentation milk with 60 DEG C of initial temperature
The detection of volatile compound, so as to determine the content for drawing biacetyl in fermentation milk, total ion current figure is shown in Fig. 5.
1 Lactobacillus plantarum CCTCC M of Application Example, 2016513 working stock cultures
2016513 strains of Lactobacillus plantarum CCTCC M are inoculated in into 12% (w/v) of 1% lactalbumin of addition (WPC)
In 115 DEG C of skimmed milks for sterilizing 15min, 10h is cultivated under the conditions of 37 DEG C to curdled milk, continuous culture two generations of activation, used as mother
Leaven is used；Mother culture is inoculated in above-mentioned sterilized milk by 5% (v/v), 10h is to curdled milk for culture, now in curdled milk
Viable count is about 109Cfu/mL, obtains the working stock culture (1) of the present invention.
2016513 strains of Lactobacillus plantarum CCTCC M are inoculated in MRS fluid mediums, are cultivated under the conditions of 37 DEG C
10h is activated, and in two generations of continuous activation, is then inoculated in MRS fluid mediums activation culture thing by 5% (v/v), is cultivated
10h, under the conditions of 4 DEG C, 6000r/min centrifugations 10min, removes supernatant, obtains cell precipitation, and precipitation is used a certain amount of nothing
Bacterium skimmed milk suspends, and obtains the working stock culture (2) of the present invention.
Lactic acid bacteria milk beverage of the Application Example 2 containing Lactobacillus plantarum CCTCC M 2016513
Raw milk heats sterilization 20min or the high temperature thermal sterilization 2s at 140 DEG C at 95 DEG C, is then cooled to 40 DEG C, with
The amount of 5% (v/v) adds 2016513 working stock cultures of Lactobacillus plantarum CCTCC M obtained by Application Example 1【Work fermentation
Agent (1) or (2)】So as to concentration reaches 106More than cfu/mL, obtains containing Lactobacillus plantarum in 4 DEG C of stored refrigerated
The lactic acid bacteria milk beverage of CCTCC M 2016513.
Fermentation milk of the Application Example 3 containing Lactobacillus plantarum CCTCC M 2016513
By raw milk fresh milk in 95 DEG C of heat sterilization 20min or at 140 DEG C high temperature thermal sterilization 2s, be cooled to 40 DEG C,
2016513 working stock cultures of Lactobacillus plantarum CCTCC M obtained by Application Example 1 are added with the amount of 5% (v/v)【Work is sent out
Ferment agent (1) or (2)】, and commercial fermentation agent Lactobacillus bulgaricus, it is 70 ° that the mixed bacterium is fermented at 37 DEG C to titratable acidity
T, cold preservation is to 4 DEG C and stored refrigerated obtains the fermentation milk containing Lactobacillus plantarum CCTCC M 2016513.
The preparation of 1 blank run of comparative example breast
By raw milk fresh milk after 95 DEG C of heat sterilization 20min, 37 DEG C are cooled to, business is added with the amount of 5% (v/v)
Leaven Lactobacillus bulgaricus, it is 70 ° of T to ferment at 37 DEG C to titratable acidity, and cold preservation is to 4 DEG C and stored refrigerated obtains blank
Fermentation milk.
Fermentation milk of the effect example 1 containing Lactobacillus plantarum CCTCC M 2016513 and blank run breast biacetyl contain
Fermentation milk in above-mentioned Application Example 3 and comparative example 1 is carried out double second according to the method in embodiment 4 by amount contrast respectively
Acyl assay, as a result shows that the diacetyl content of the fermentation milk containing Lactobacillus plantarum CCTCC M 2016513 is
32.14mg/L, and in comparative example 1 blank run breast in diacetyl content be 14.58mg/L, diacetyl content improve
Twice or so, it was demonstrated that Lactobacillus plantarum CCTCC M 2016513 have the ability of higher product biacetyl.
Fermentation milk of the effect example 2 containing Lactobacillus plantarum CCTCC M 2016513 and blank run breast sensory evaluation
Contrast.
Sensory evaluation is carried out by 8 valuation officers (3 is male, 5 female), needs, to their professional trainings, to make them complete before evaluation
Understand organoleptic attribute, know the evaluation criterion of fermentation milk.According to [list of references (Chen Jie. mesophilic digestion yogurt product Quality Research
[D]. Southern Yangtze University, 2008.)/(Kang Huan. the pre-test [D] of flavor substance and enzyme activity and organoleptic attribute relation in Yoghourt. northeast agriculture
Sparetime university is learned, 2013.)], 9 organoleptic properties that may have significant contribution to the acceptability of product of initial option.Sensory evaluation
Definition is shown in Table 2 to term accordingly.
2 sensory evaluation term of table and standards of grading
First, the sample (sample in Application Example 2 and comparative example 1) of mark random code is taken out from refrigerator,
Valuation officer is presented at random.After they open lid, be gently agitated for yogurt with spoon first, by smelling determine odor types and
Other properties are evaluated by intensity successively.Using 9 points of scaling laws.All of evaluation procedure is all having white light and controllable environment
Sensory evaluation laboratory in carry out.In evaluation procedure, prepare one glass of pure water and gargle.Each sample is in triplicate.As a result see
Fig. 6.
We can see that the fermentation milk of Lactobacillus plantarum CCTCC M 2016513 in typical Yoghourt taste, creamy taste from Fig. 6
With in overall acceptance higher than blank run breast, this with Lactobacillus plantarum CCTCC M 2016513 in effect example 1 with compared with
The ability of high product biacetyl is consistent, and biacetyl has the fragrance of whipping cream, and it is whole that its presence can significantly improve fermentation milk
The flavor characteristic of body, brings the sensory experience for more pleasing to consumer, can also draw this conclusion from sensory evaluation figure.
It should be understood that after the above for having read the present invention, those skilled in the art can make various to the present invention
The present invention is made various changes or modifications, these equivalent form of values equally fall within the model limited by the application appended claims
Enclose.
Sequence table
<110>Shanghai application technology university
<120>A kind of Lactobacillus plantarum of high yield biacetyl and its application
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cgattactag cgattccgac ttcatgtagg cgagttgcag cctacaatcc gaactgagaa 180
tggctttaag agattagctt actctcgcga gttcgcaact cgttgtacca tccattgtag 240
cacgtgtgta gcccaggtca taaggggcat gatgatttga cgtcatcccc accttcctcc 300
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aagacctggt aaggttcttc gcgtagcttc gaattaaacc acatgctcca ccgcttgtgc 540
gggcccccgt caattccttt gagtttcagc cttgcggccg tactccccag gcggaatgct 600
taatgcgtta gctgcagcac tgaagggcgg aaaccctcca acacttagca ttcatcgttt 660
acggtatgga ctaccagggt atctaatcct gtttgctacc catactttcg agcctcagcg 720
tcagttacag accagacagc cgccttcgcc actggtgttc ttccatatat ctacgcattt 780
caccgctaca catggagttc cactgtcctc ttctgcactc aagtttccca gtttccgatg 840
cacttcttcg gttgagccga aggctttcac atcagactta aaaaaccgcc tgcgctcgct 900
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Claims (10)
1. a plant height produces the Lactobacillus plantarum of biacetyl, it is characterised in that its deposit number CCTCC M 2016513.
2. a kind of working stock culture, it is characterised in that：Lactobacillus plantarum strain described in claim 1 is inoculated in into sterilized milk
In, to cultivate to curdled milk, in continuous culture two generations of activation, are used as mother culture；Mother culture is inoculated in by percent by volume 3-5% and is added
Plus cultivate to curdled milk in the sterilized milk of lactalbumin, obtain final product working stock culture.
3. a kind of working stock culture according to claim 2, it is characterised in that：During culture to curdled milk, in 35-
10-12h is cultivated under the conditions of 37 DEG C.
4. a kind of working stock culture, it is characterised in that：Lactobacillus plantarum strain described in claim 1 is inoculated in into liquid culture
In base, in two generations of continuous activation, then activation culture thing is inoculated in fluid medium by percent by volume 3-5% and is cultivated, solid-liquid
Isolated cell precipitation, precipitation is suspended with aseptic breast, working stock culture is obtained final product, and in described leaven, viable count is
109More than cfu/mL.
5. a kind of working stock culture according to claim 4, it is characterised in that：The process activated in liquid medium within
In, 10-12h is cultivated under the conditions of 35-37 DEG C.
6. purposes of the Lactobacillus plantarum of a kind of high yield biacetyl described in claim 1 in fermented food.
7. the purposes according to claim 4, it is characterised in that：Described fermented food is lactic acid bacteria milk beverage or sends out
Kefir milk.
8. a kind of preparation method of lactobacillus beverage, it is characterised in that：Cool down after raw milk is sterilized, be subsequently adding claim
Working stock culture mix homogeneously described in 2 or 4 so as in Lactobacillus plantarum concentration reach 106More than cfu/mL, obtains final product
To the lactic acid bacteria milk beverage containing the Lactobacillus plantarum described in claim 1.
9. a kind of preparation method of fermentation milk, it is characterised in that：After raw milk is sterilized cool down, add claim 2 or
Working stock culture and customary commercial leaven described in person 4, it is 3-5% that described working stock culture accounts for the volume of fermentation milk, described
Customary commercial leaven account for fermentation milk volume be 3-5%, after mixing under conditions of 35-37 DEG C ferment, to titratable acidity
70-80 T, that is, obtain the fermentation milk containing Lactobacillus plantarum described in claim 1.
10. the preparation method of a kind of fermentation milk according to claim 9, it is characterised in that：Described commodity leaven is
In conventional Lactobacillus bulgaricus, bacillus bifiduss, streptococcus thermophiluss or bacillus acidophilus any one or it is two or more
Combination.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674852A (en) * | 2017-11-16 | 2018-02-09 | 扬州市扬大康源乳业有限公司 | A kind of Lactobacillus plantarum of high yield diacetyl and its application |
| CN112458003A (en) * | 2020-10-28 | 2021-03-09 | 四川农业大学 | Diacetyl-producing lactobacillus plantarum and application thereof in pickled vegetables |
| CN115088767A (en) * | 2022-05-30 | 2022-09-23 | 锡林郭勒职业学院 | High-fat type milk tofu and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN102453689A (en) * | 2011-12-06 | 2012-05-16 | 光明乳业股份有限公司 | Lactobacillus plantarum strain producing extracellular polysaccharide, and application thereof |
| CN106085901A (en) * | 2016-06-08 | 2016-11-09 | 江南大学 | A kind of Lactobacillus plantarum with biacetyl synthesis capability and application thereof |
-
2016
- 2016-11-28 CN CN201611064174.XA patent/CN106520620A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101144065A (en) * | 2007-09-03 | 2008-03-19 | 江南大学 | Oxidation resistant Lactobacillus casei capable of resisting hydrogen peroxide and eliminating free radical, and use thereof |
| CN102453689A (en) * | 2011-12-06 | 2012-05-16 | 光明乳业股份有限公司 | Lactobacillus plantarum strain producing extracellular polysaccharide, and application thereof |
| CN106085901A (en) * | 2016-06-08 | 2016-11-09 | 江南大学 | A kind of Lactobacillus plantarum with biacetyl synthesis capability and application thereof |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107674852A (en) * | 2017-11-16 | 2018-02-09 | 扬州市扬大康源乳业有限公司 | A kind of Lactobacillus plantarum of high yield diacetyl and its application |
| WO2019095274A1 (en) * | 2017-11-16 | 2019-05-23 | 扬州市扬大康源乳业有限公司 | Lactobacillus plantarum with high butanedione yield and use thereof |
| CN107674852B (en) * | 2017-11-16 | 2021-02-02 | 扬州市扬大康源乳业有限公司 | Lactobacillus plantarum for producing butanedione and application thereof |
| CN112458003A (en) * | 2020-10-28 | 2021-03-09 | 四川农业大学 | Diacetyl-producing lactobacillus plantarum and application thereof in pickled vegetables |
| CN115088767A (en) * | 2022-05-30 | 2022-09-23 | 锡林郭勒职业学院 | High-fat type milk tofu and preparation method thereof |
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