CN106834435A - A kind of special throw type leaven orientation preparation method of hot pickled mustard tube based on 16S rDNA sequencings - Google Patents
A kind of special throw type leaven orientation preparation method of hot pickled mustard tube based on 16S rDNA sequencings Download PDFInfo
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Abstract
The present invention relates to fruits and vegetables and technical field of food biotechnology, and in particular to a kind of special throw type leaven orientation preparation method of hot pickled mustard tube based on 16S rDNA sequencings, including：Sampled in hot pickled mustard tube salt marsh pond；Extract sample DNA and be PCR, amplification 16S rDNA after doing agarose gel electrophoresis detection；The PCR primer of acquisition carries out high-flux sequence and the analysis that compares, and determines dominant microflora species and proportion of composing；The directed screening of dominant strain；The proliferation of high-density culture of directed screening bacterial strain；The bacterial strain freeze dried powder of each directed screening and high density increment culture is obtained through centrifugation and freeze-drying through the bacterium solution of high density increment culture, the dominant microflora species determined according to 16S rDNA sequencings mixes with proportion of composing, and the special throw type leaven of hot pickled mustard tube is obtained.The present invention can effectively solve traditional direct vat inoculation and there are problems that fermented product local flavor is single, ferment local-flavor not, with product stabilization, content of nitrite is low the characteristics of.
Description
Technical field
The present invention relates to fruits and vegetables and technical field of food biotechnology, and in particular to a kind of squeezing based on 16S rDNA sequencings
The special throw type leaven orientation preparation method of dish.
Background technology
Hot pickled mustard tube is special product of China, belongs to one of big pickles in the world three.Hot pickled mustard tube is wide in the cultivated area of China, and yield is high, is
Main pickles one of vegetable for processing.More than traditional hot pickled mustard tube salt marsh using high salt concentration, based on spontaneous fermentation.Traditional natural hair
Ferment is present in a series of applications such as work consuming is time-consuming, fermentation period is long, miscellaneous bacteria easily pollutes, product special flavour is impure, edible safety is poor
The drawbacks of.The modernization of hot pickled mustard tube salt marsh, is given birth to the controllability of manufacturing process and standardization, standardization and anniversary stable equilibrium
A kind of production technology pattern for representative is produced, wherein be exactly one of important step using pure tungus inoculation, i.e., by people's work point
The main bacteria seeds such as Leuconostoc mesenteroides, Lactobacillus plantarum, lactobacillus lactis in salted vegetable bittern, after freeze-dried, compounding
Throw type leaven is fabricated to for hot pickled mustard tube salt marsh.Because throw type leaven has viable bacteria content high by (109~1012cfu/g)、
Security is good, long shelf-life, it is easy to use, the features such as avoid spawn degeneration, and can suppress beneficial to standardized production and effectively
The advantages of miscellaneous bacteria, shortening fermentation period, reduction content of nitrite, many deficiencies of hot pickled mustard tube salt marsh spontaneous fermentation are compensate for, can
To solve above-mentioned drawback, had broad application prospects in traditional zymotic vegetables industry.
The salting process of hot pickled mustard tube is the fermentation process of microorganism.Exactly using the principle of fermentation of microorganism, hot pickled mustard tube is in nature
In fermentation process, abundant microorganism species play very big work to the preservation of hot pickled mustard tube product and each unique local flavor of formation
With high concentration mineral matter, lactic acid and the bioactivator that lactic acid bacteria metabolism is produced in hot pickled mustard tube salting process are that composition pickles are only
The major reason of special local flavor.Lactic fermentation is the topmost fermentation of various fermentation kinds in hot pickled mustard tube salting process.It is main
The lactic acid bacteria wanted includes lactobacillus, Pediococcus, bright string strain Pseudomonas, streptococcus, lactococcus.To a certain extent, salt
In many degree of quality and local flavor of stain hot pickled mustard tube determined by the lactic acid bacteria flora in fermentation process.
But due to the not Culturability or poor repeatability of some floras, cause traditional mode that is separately cultured to be difficult to entirely
Bacterial community and change in face, objectively reflection hot pickled mustard tube salting process, therefore be separately cultured according to tradition at present obtained
Leavening production hot pickled mustard tube be difficult to really reflect its local flavor and quality, occur in that the problems such as local flavor is single, fermented flavour is not mellow.Separately
Outward, the method for traditional microbiological Identification of Species is typically identified using phenotypic characteristic, but this method has complex operation step, workload
Greatly, the problems such as cycle is long, some testing results are unstable.Therefore, according to the fermentation character of different vegetable, find it is more excellent,
Effective leavening preparation method is significant for traditional fruits and vegetables mode.
With developing rapidly for biotechnology, traditional microorganism identification method is often difficult to the numerous habit of identification
Complicated microorganism, thus the Molecular Identification based on genome sequence receives significant attention.In bacterial genomes, 16S is encoded
The rDNA genes of rRNA have good evolutionary conservatism, the length (about 1540bp) of fit analysis, and and evolutionary distance
The good variability for matching, so the standard logo sequence as bacteria molecule identification.One of 16S rDNA sequence analyses
Distinguishing feature is in time to identify the inert bacterium of slow-growing or biochemical reaction.
The content of the invention
The present invention has that fermented product local flavor is single for traditional direct vat inoculation, ferment local-flavor not, carry
A kind of special throw type leaven orientation preparation method of hot pickled mustard tube based on 16S rDNA sequencings is gone out, the method is orientable to be filtered out
Leading hot pickled mustard tube salt marsh and hot pickled mustard tube local flavor, the different genus lactubacillus of quality, specificity and goal orientation are strong, and the degree of accuracy is high, it is not necessary to
Purify repeatedly.
To realize goal of the invention of the invention, inventor provides following technical scheme：
The special throw type leaven orientation preparation method of a kind of hot pickled mustard tube based on 16S rDNA sequencings, at least including following step
Suddenly：
(1) sampled in hot pickled mustard tube salt marsh pond, sample is placed in sterile cryo pipe, be immediately placed in preservation in liquid nitrogen, then put
In ultra low temperature freezer,
(2) extract sample DNA and be PCR, amplification 16S rDNA, described PCR expansions after doing agarose gel electrophoresis detection
The sense primer 515F of the specific primer of increasing have as shown in SEQ ID NO.1 nucleotide sequence (5 '-
GTGCCAGCMGCCGCGG-3 '), the anti-sense primer 907R of specific primer has the nucleotides sequence as shown in SEQ ID NO.2
Row (5 '-CCGTCAATTCMTTTRAGTTT-3 '), PCR amplification regions are the V4-V5 Variable Areas of microorganism 16S rDNA, are surveyed
Need to carry out recovery purifying to the PCR primer before sequence,
(3) PCR primer that step (2) is obtained is carried out into high-flux sequence and the analysis that compares, determines dominant microflora species
With proportion of composing,
(4) directed screening of dominant strain,
(5) the proliferation of high-density culture of directed screening bacterial strain,
(6) centrifugation and freeze-drying,
Step (5) is added into skimmed milk power through the bacterium solution of high density increment culture, is centrifuged after shaking up, remove supernatant culture
Liquid, collects test tube bottom thalline；The thalline of collection does freeze-drying after adding freeze drying protectant, wherein：Thalline and frozen-dried protective
Agent mass fraction proportioning is 1:3-1:2.5,
(7) the bacterial strain freeze dried powder of each directed screening and high density the increment culture for obtaining step (6) is according to 16S
The dominant microflora species that rDNA sequencings determine mixes with proportion of composing, and the special throw type leaven of hot pickled mustard tube is obtained.
Preferably, it is special directly putting type fermented according to a kind of hot pickled mustard tube based on 16S rDNA sequencings of the present invention
Agent orients preparation method, wherein, sampling uses 3 point samplings in described step (1), and collection point is located in salt marsh pond respectively
The heart, 2 points of salt marsh pond diagonal, 3 samples of often place's collection, mix after collection.
Preferably, it is special directly putting type fermented according to a kind of hot pickled mustard tube based on 16S rDNA sequencings of the present invention
Agent orients preparation method, wherein, the directed screening of described step (4) dominant strain is operated as following：In the step (1)
In samples taken, take 1ml liquid carries out 10 times of gradient dilution in physiological saline, is uniformly coated on post-directed training base, so
Train 36~72h for a certain area in 35~37 DEG C of insulating boxs afterwards, observe bacterium colony；Then the bacterial strain of post-directed training is carried out into Physiology and biochemistry
Characterization is tested；According to qualification result, the bacterial strain after directed screening is carried out into inclined-plane culture.
Preferably, it is special directly putting type fermented according to a kind of hot pickled mustard tube based on 16S rDNA sequencings of the present invention
Agent orients preparation method, wherein, the proliferation of high-density culture of described step (5) directed screening bacterial strain is operated as follows：
Above-mentioned steps (4) directed screening and the bacterial strain of purifying culture are carried out into high density increment culture, high density increment culture medium is MRS
Agar medium, condition of culture is 48~72h of culture in 35~37 DEG C of insulating boxs；Described MRS agar medium formulas are：Egg
White 10.0 grams of peptone, 5.0 grams of beef extract, 4.0 grams of dusty yeast, 20.0 grams of glucose, 1.0 milliliters of Tween 80, dipotassium hydrogen phosphate 2.0
Gram, 5.0 grams of sodium acetate, 2.0 grams of triammonium citrate, 0.2 gram of magnesium sulfate, 0.05 gram of manganese sulfate, 15.0 grams of agar powder and distilled water 1
Rise, after heating for dissolving, correct pH=6.2~7.2,121 DEG C of autoclavings 15~20 minutes.
Preferably, it is special directly putting type fermented according to a kind of hot pickled mustard tube based on 16S rDNA sequencings of the present invention
Agent orients preparation method, wherein, step (5) is cultivated through high density increment bacterium solution in described step (6), addition 2.7~
3.5% through high-temperature sterilization skimmed milk power, 25-30 points is centrifuged under the conditions of -5~10 DEG C after shaking up with 4000-5000rpm rotating speeds
Clock.
Preferably, it is special directly putting type fermented according to a kind of hot pickled mustard tube based on 16S rDNA sequencings of the present invention
Agent orients preparation method, wherein, frozen-dried protective agent prescription is in described step (6)：25-30 grams of skimmed milk, lactose 10-15
Gram, 5-12 grams of glucose sugar, 5-6.5 grams of vitamin C, 5-7.2 grams of D-glucitol, 5-6 grams of Pidolidone and distilled water 250mL；Freeze
After dry protective agent heating for dissolving, 121 DEG C standby after autoclaving 15-20 minutes.
Preferably, it is special directly putting type fermented according to a kind of hot pickled mustard tube based on 16S rDNA sequencings of the present invention
Agent orients preparation method, wherein, the main technologic parameters of freeze-drying are in described step (6)：- 50~-70 DEG C of pre-freeze 12-
After 24h, -60~80 DEG C of lyophilized 24-72h.
Compared with prior art, the beneficial effects of the invention are as follows：
(1) compared with the traditional zymotic agent for being separately cultured preparation using tradition, the present invention identifies micro- by high-flux sequence
The 16SrDNA of biological flora, can accurately obtain detailed biological community structure and proportion of composing after hot pickled mustard tube spontaneous fermentation, and really
Fixed main lactic acid bacteria flora, so as to accurately judge different microorganisms flora to hot pickled mustard tube spontaneous fermentation process and product property
Influence.The target strain for being used has diversity, specificity, it is to avoid traditional zymotic agent strain is single, flora is very different,
Non-specific the shortcomings of.
(2) the hot pickled mustard tube special leaven that present invention orientation is prepared has specificity and goal orientation feature.According to
The different process and quality, local flavor demand of different places hot pickled mustard tube processing, raising fermentation efficiency is constituted by matching strain, and product is steady
Fixed, content of nitrite significantly reduces (Fig. 3 and Fig. 4), it is to avoid the ferment local-flavor resulted in traditional zymotic agent fermentation process
Impure shortcoming.
(3) compared with leavening prepared by conventional screening methods, one kind is special for hot pickled mustard tube based on 16S rDNA sequencings beam system
With the method for throw type leaven in leavening preparation process, the leavening dry powder that strain selectivity is strong, activity is high, obtained
It is easy to storage with transport.
Brief description of the drawings
Fig. 1 hot pickled mustard tube sample 16S rRNA gene PCR amplified production agarose gel electrophoretograms.
Distribution of the bacterium in different classifications level in Fig. 2 hot pickled mustard tubes.
Fig. 3 spontaneous fermentation hot pickled mustard tube nitrite column diagrams.
The hot pickled mustard tube nitrite column diagram of Fig. 4 hot pickled mustard tube special leavens of the present invention.
Specific embodiment
With reference to embodiment, present disclosure is further illustrated.It should be appreciated that implementation of the invention is not limited to
In the following examples, any formal accommodation and/or change made to the present invention fall within the scope of the present invention.
In the present invention, if not refering in particular to, all of part, percentage are unit of weight, and all of equipment and raw material etc. are
It is commercially available or the industry is conventional.If without specializing, the method that embodiment is used is technology generally in the art.
Embodiment 1
A kind of special throw type leaven orientation preparation method of hot pickled mustard tube based on 16S rDNA sequencings, enters according to the following steps
OK：
(1) sampled in hot pickled mustard tube salt marsh pond, using 3 point samplings, collection point is located at salt marsh pond center, salt marsh pond pair respectively
2 points of linea angulata, 3 samples of often place's collection.Mixed after collection, be placed in the sterile cryo pipe of 15ml, be immediately placed in guarantor in liquid nitrogen
Deposit, be subsequently placed in -70 DEG C of ultra low temperature freezers.
(2) sample for gathering step (1) DNA extraction kit extracts DNA (the English contracting of microorganism
Write DNA), the DNA extraction steps of all samples are with reference to DNA extraction kit specification.To the sample gene group DNA for extracting
Carry out 3% agarose gel electrophoresis detection (Fig. 1) (3) and the DNA specific primers that step (2) is extracted are entered into performing PCR, expand micro-
Biological 16SrDNA, the sense primer 515F of specific primer have as shown in SEQ ID NO.1 nucleotide sequence (5 '-
GTGCCAGCMGCCGCGG-3 '), anti-sense primer 907R have as shown in SEQ ID NO.2 nucleotide sequence (5 '-
CCGTCAATTCMTTTRAGTTT-3 '), PCR (i.e. PCR) amplification region is the V4-V5 of microorganism 16S rDNA
Variable Area, recovery purifying is carried out before sequencing to the PCR primer.
(4) PCR primer that step (3) is obtained is carried out into high-flux sequence.Screening sequencing the data obtained and and data with existing
Storehouse information carries out sequence alignment, obtains the operable taxon (OTUs) of flora；And with existing 16S V4-V5 databases ratio
It is right.According to sequence label and reads, it is 72.7% that the pickled rear lactobacillus (Lactobacillus) of hot pickled mustard tube occupies ratio, respectively
For：Lactobacillus alimentarius (Lactobacillus alimentarius) (nucleotide sequence is as shown in SEQ ID NO.3),
Lactobacillus versmoldensis (nucleotide sequence is as shown in SEQ ID NO.4), Leuconostoc mesenteroides
(Leuconostoc mesenteroides) (nucleotide sequence is as shown in SEQ ID NO.5), Lactobacillus plantarum
(Lactobacillus plantarum) (nucleotide sequence is as shown in SEQ ID NO.6), Lactobacillus brevis (Lactobacillus
Brevis) (nucleotide sequence is as shown in SEQ ID NO.7), ratio is respectively：22.7%th, 37%, 8%, 2.6%, 2.4%
(Fig. 2).
(5) Lactobacillus alimentarius (Lactobacillus alimentarius), Lactobacillus
Versmoldensis, Leuconostoc mesenteroides (Leuconostoc mesenteroides), Lactobacillus plantarum
(Lactobacillus plantarum), the directed screening of Lactobacillus brevis (Lactobacillus brevis)：
(5.1) directed screening of Lactobacillus alimentarius (Lactobacillus alimentarius)：In above-mentioned steps (1)
In samples taken, take 1ml liquid carries out 10 times of gradient dilution in physiological saline, is uniformly coated on MRS culture mediums, Ran Hou
48~72h is cultivated in 35~37 DEG C of insulating boxs, bacterium colony is observed.Pick out be creamy white, opaque, circular smooth single bacterium colony,
Carry out 48~72h of inclined-plane culture；Access 48~60h of liquid MRS medium cultures.MRS agar medium formulas are：Peptone
10.0 grams, 5.0 grams of beef extract, 4.0 grams of dusty yeast, 20.0 grams of glucose, 1.0 milliliters of Tween 80,2.0 grams of dipotassium hydrogen phosphate, second
5.0 grams of sour sodium, 2.0 grams of triammonium citrate, 0.2 gram of magnesium sulfate；1 liter of 0.05 gram of manganese sulfate, 15.0 grams of agar powder and distilled water；Plus
After heat of solution, correction pH is 6.2,121 DEG C of autoclavings 15~20 minutes.
The bacterial strain of screening is carried out into Physiology and biochemistry identification experiment, meets following result：
(5.2) directed screening of Lactobacillus versmoldensis：In the samples taken of above-mentioned steps (1),
Take 1ml liquid carries out 10 times of gradient dilution in physiological saline, is uniformly coated on MRS culture mediums, then in 36~37 DEG C of perseverances
48~72h is cultivated in incubator, bacterium colony is observed.Pick out be creamy white, opaque, circular smooth single bacterium colony, carry out inclined-plane training
Support 48~72h；Access 48~72h of liquid MRS medium cultures.MRS agar medium formulas are：10.0 grams of peptone, beef
5.0 grams of cream, 4.0 grams of dusty yeast, 20.0 grams of glucose, 1.0 milliliters of Tween 80,2.0 grams of dipotassium hydrogen phosphate, 5.0 grams of sodium acetate, lemon
2.0 grams of triamine of lemon acid, 0.2 gram of magnesium sulfate；1 liter of 0.05 gram of manganese sulfate, 15.0 grams of agar powder and distilled water；After heating for dissolving, school
Positive pH is 6.2,121 DEG C of autoclavings 15~20 minutes.
By the bacterial strain after activation, by 3% inoculum concentration be inoculated in cholate mass concentration be respectively 0,0.3, the MRS liquid of 0.6g/l
In body culture medium, 37 DEG C of Anaerobic culturel 16h.Then screening carries out Physiology and biochemistry identification experiment, meets following result：
(5.3) directed screening of Leuconostoc mesenteroides (Leuconostoc mesenteroides)：Take 1ml in step (1)
Liquid carries out 10 times of gradient dilution in physiological saline, is uniformly coated on YGPB culture mediums, then picking colonies typical in
Rule on YGPB culture medium upper flat plates, be placed in Anaerobic culturel 2~3 days in 25 DEG C of incubators, observe colony growth situation.YGPB
Culture medium prescription is：Glucose 10g, peptone 10g, beef extract 8g, yeast extract 3g, NaCl 5g, potassium dihydrogen phosphate
2.5g, dipotassium hydrogen phosphate 2.5g, 0.2 gram of magnesium sulfate；1 liter of 0.05 gram of manganese sulfate, pH6.8,15.0 grams of agar powder and distilled water；Plus
After heat of solution, correction pH is 6.2,121 DEG C of autoclavings 15~20 minutes.
The bacterial strain of screening is carried out into bio-chemical characteristics, meets following result：
(5.4) directed screening of Lactobacillus plantarum (Lactobacillus plantarum)：In the institute of above-mentioned steps (1)
Take in sample, take 1ml liquid carries out 10 times of gradient dilution in physiological saline, be uniformly coated on orientation MRS culture mediums, in
48~72h is cultivated in 35~37 DEG C of insulating boxs, bacterium colony is observed, picked out molten calcium circle, be creamy white, it is opaque, circular smooth
Single bacterium colony, in 48~72h of the flat lining out cultures of MRS；After inclined-plane culture, access Liquid Culture MRS and cultivate 48~72h.MRS
Agar medium formula is：10.0 grams of peptone, 5.0 grams of beef extract, 4.0 grams of dusty yeast, 20.0 grams of glucose, Tween 80 1.0
Milliliter, 2.0 grams of dipotassium hydrogen phosphate, 5.0 grams of sodium acetate, 2.0 grams of triammonium citrate, 0.2 gram of magnesium sulfate；0.05 gram of manganese sulfate, 1%
1 liter of calcium carbonate, 0.05 ‰ natamycin, 15.0 grams of agar powder and distilled water；After heating for dissolving, correction pH is 6.2,121 DEG C
Autoclaving 15~20 minutes.
The bacterial strain of screening is carried out into bio-chemical characteristics, meets following result：
(5.5) directed screening of Lactobacillus brevis (Lactobacillus brevis)：In the samples taken of above-mentioned steps (1)
In, take 1ml liquid carries out 10 times of gradient dilution in physiological saline, MRS improved culture mediums is uniformly coated on, then at 37 DEG C
Cultivate 48h in insulating box, observe bacterium colony, pick out be creamy white, opaque, circular smooth single bacterium colony, improved in MRS and cultivated
The flat lining out culture 48h of base.After inclined-plane culture, 72h is cultivated in access liquid MRS improved culture mediums.MRS improved culture mediums are matched somebody with somebody
Fang Wei：Glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g, diammonium hydrogen citrate 2g and distilled water 1000ml.Dissolving
Afterwards, correction pH is 6.5,121 DEG C of autoclavings 15~20 minutes.
The bacterial strain of screening is carried out into bio-chemical characteristics, meets following result：
(6) the proliferation of high-density culture of directed screening bacterial strain.By step (5) directed screening and the newborn bar of the food of purifying culture
Bacterium, Lactobacillus versmoldensis, Leuconostoc mesenteroides, Lactobacillus plantarum and Lactobacillus brevis carry out high density increasing
It is worth culture, high density increment culture medium is MRS agar mediums, and condition of culture is 64~72h of culture in 35~37 DEG C of insulating boxs.
MRS agar medium formulas are：10.0 grams of peptone, 5.0 grams of beef extract, 4.0 grams of dusty yeast, 20.0 grams of glucose, tween
801.0 milliliters, 2.0 grams of dipotassium hydrogen phosphate, 5.0 grams of sodium acetate, 2.0 grams of triammonium citrate, 0.2 gram of magnesium sulfate, manganese sulfate 0.05
Gram, 1 liter of 15.0 grams of agar powder and distilled water；After heating for dissolving, correction pH is 6.2,121 DEG C of autoclavings 15~20 minutes.
(7) collection of directed screening bacterial strain：The bacterium solution through high density increment culture that step (6) is obtained, adds 3.5%
Through the skimmed milk power of high-temperature sterilization, it is centrifuged under the conditions of -5 DEG C 30 minutes with 5000rpm rotating speeds after shaking up, removes supernatant culture
Liquid, collects test tube bottom thalline.
(8) freeze-drying of throw type leaven.The thalline that step (7) is collected adds freeze drying protectant, frozen-dried protective
Agent prescription is：30 grams of skimmed milk, 15 grams of lactose, 12 grams of glucose sugar, 6.5 grams of vitamin C, 7.2 grams of D-glucitol, Pidolidone 6
Gram and distilled water 250mL.After freeze drying protectant heating for dissolving, 121 DEG C of autoclavings 20 minutes.Thalline and freeze drying protectant quality
Fraction proportioning is 1:3.Freeze-drying condition is：After -70 DEG C of pre-freeze 24h, -80 DEG C of lyophilized 24h.
(9) preparation of the special throw type leaven of hot pickled mustard tube.The group of the lactic acid bacteria confirmed according to 16S rDNA sequencing results
Into by above-mentioned lyophilized Lactobacillus alimentarius (Lactobacillus alimentarius), Lactobacillus
Versmoldensis, Leuconostoc mesenteroides (Leuconostoc mesenteroides), Lactobacillus plantarum
(Lactobacillus plantarum), Lactobacillus brevis (Lactobacillus brevis) are according to 22.7%, 37%, 8%,
2.6%th, 2.4% ratio mixing, is obtained the special throw type leaven of hot pickled mustard tube.After testing, viable bacteria content is 109~1012cfu/g。
The special throw type leaven of hot pickled mustard tube obtained in the present invention is used for hot pickled mustard tube salt marsh, is compared with the hot pickled mustard tube of spontaneous fermentation,
Local flavor is consistent, and content of nitrite is substantially reduced；Hot pickled mustard tube is used for using the special throw type leaven of hot pickled mustard tube of the invention simultaneously
Salt marsh, the characteristics of with product stabilization, it is to avoid the impure shortcoming of ferment local-flavor resulted in traditional zymotic agent fermentation process.
Hot pickled mustard tube obtained in the special throw type leaven of hot pickled mustard tube of the present invention compares with spontaneous fermentation hot pickled mustard tube nitrite, as a result as schemed
Shown in 3 and Fig. 4.As can be seen that the special throw type leaven of hot pickled mustard tube of the invention can significantly reduce containing for hot pickled mustard tube nitrite
Amount, content of nitrite almost only has the half of spontaneous fermentation.This is the hardly possible in short-term because spontaneous fermentation flora is complex
Directly affect slowly and harmful microbe is suppressed to form dominant microflora and acid production speed；The special direct putting type of hot pickled mustard tube of the invention
Leavening then can in the short term quickly form dominant microflora and quickly produce acid and harmful microorganism is effectively suppressed, therefore
Harmful microorganism is greatly weakened the reduction ability of nitrate, suppress and reduce the generation of nitrite.
Embodiment 2
Other are with embodiment 1, difference：
Step (7)：The bacterium solution through high density increment culture that step (6) is obtained, adds 2.7% through the de- of high-temperature sterilization
Fat milk powder, is centrifuged 25 minutes with 4000rpm rotating speeds after shaking up under the conditions of 10 DEG C, removes supernatant nutrient solution, collects test tube bottom
Thalline.
The freeze-drying of step (8) throw type leaven.The thalline that step (7) is collected adds freeze drying protectant, freezes
Protection agent prescription be：25 grams of skimmed milk, 10 grams of lactose, 5 grams of glucose sugar, 5 grams of vitamin C, 5 grams of D-glucitol, 5 grams of Pidolidone
With distilled water 250mL；After freeze drying protectant heating for dissolving, 121 DEG C of autoclavings are standby after 15 minutes.Thalline and freeze drying protectant
Mass fraction proportioning is 1:2.5.Freeze-drying condition is：After -50 DEG C of pre-freeze 12h, -60 DEG C of lyophilized 72h.
After testing, the present embodiment reaches similar technology effect with embodiment 1, repeats no more.
SEQUENCE LISTING
<110>Zhejiang Academy of Agricultural Science
<120>A kind of special throw type leaven orientation preparation method of hot pickled mustard tube based on 16S rDNA sequencings
<130> Z164859
<160> 7
<170> PatentIn version 3.3
<210> 1
<211> 16
<212> DNA
<213>Artificial sequence
<400> 1
gtgccagcmg ccgcgg 16
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<400> 2
ccgtcaattc mtttragttt 20
<210> 3
<211> 450
<212> DNA
<213> Lactobacillus alimentarius
<400> 3
gtagggaatc ttccacaatg gacgaaagtc tgatggagca atgccgcgtg agtgaagaag 60
gttttcggat cgtaaaactc tgttgttgaa gaagaacata tgtgagagta actgttcacg 120
tactgacggt attcaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 180
gtaggtggca agcgttgtcc ggatttattg ggcgtaaaga gaatgtaggc ggttcattaa 240
gtttgaagtg aaagccctcg gctcaaccga ggaagtgctt cgaaaactgg tgaacttgag 300
tgcagaagag gaaagtggaa ctccatgtgt agcggtggaa tgcgtagata tatggaagaa 360
caccagtggc gaaggcggct ttctggtctg taactgacgc tgagattcga aagcatgggt 420
agcaaacagg attagaaacc cgagtagtcc 450
<210> 4
<211> 451
<212> DNA
<213> Lactobacillus versmoldensis
<400> 4
gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag 60
gttttcggat cgtaaaactc tgttgttgaa gaagaacatg cgtgagagta actgttcatg 120
tattgacggt attcaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 180
gtaggtggca agcgttatcc ggatttattg ggcgtaaagc gagtgcaggc ggtttattag 240
gtctgatgtg aaagccctcg gctcaaccga ggaagtgcat cggaaaccgg taaacttgag 300
tgcagaagag gagagtggaa ctccatgtgt agcggtggaa tgcgtagata tatggaagaa 360
caccagtggc gaaggcggct ctctggtctg taactgacgc tgaggctcga aagcgtgggt 420
agcaaacagg attagaaacc cgagtagtcc g 451
<210> 5
<211> 450
<212> DNA
<213> Leuconostoc mesenteroides
<400> 5
gtagggaatc ttccacaatg ggcgaaagcc tgatggagca acgccgcgtg tgtgatgaag 60
gctttcgggt cgtaaagcac tgttgtatgg gaagaaaagt taaggtaggg aatgacctta 120
gcctgacggt accataccag aaagggacgg ctaaatacgt gccagcagcc gcggtaatac 180
gtatgtcccg agcgttatcc ggatttattg ggcgtaaagc gagcgcagac ggttggttaa 240
gtctgatgtg aaagcccgga gctcaactcc ggaaaggcat tggaaactgg ttaacttgag 300
tgcagtagag gtaagtggaa ctccatgtgt agcggtggaa tgcgtagata tatggaagaa 360
caccagcggc gaaggcggct tactggactg taactgacgt tgaggctcga aagtgtgggt 420
agcaaacagg attagatacc ctagtagtcc 450
<210> 6
<211> 450
<212> DNA
<213> Lactobacillus plantarum
<400> 6
gtagggaatc ttccacaatg gacgaaagtc tgatggagca acgccgcgtg agtgaagaag 60
gttttcggat cgtaaaactc tgttgttgga gaagaatgta tctgatagta actgatcagg 120
tagtgacggt atccaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 180
gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggtttcttaa 240
gtctgatgtg aaagccttcg gctcaaccga agaagtgcat cggaaactgg gaaacttgag 300
tgcagaagag gacagtggaa ctccatgtgt agcggtgaaa tgcgtagata tatggaagaa 360
caccagtggc gaaggcggct gtctggtctg taactgacgc tgaggctcga aagcatgggt 420
agcaaacagg attagaaacc ctagtagtcc 450
<210> 7
<211> 450
<212> DNA
<213> Lactobacillus brevis
<400> 7
gtagggaatc ttccacaatg gacgaaagtc tgatggagca atgccgcgtg agtgaagaag 60
ggtttcggct cgtaaaactc tgttgttaaa gaagaacact cttgagagta actgttcagg 120
agttgacggt atttaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac 180
gtaggtggca agcgttgtcc ggatttattg ggcgtaaagc gagcgcaggc ggtttcttaa 240
gtctgatgtg aaagccttcg gcttaaccgg agaagtgcat cggaaactgg gagacttgag 300
tgcagaagag gacagtggaa ctccatgtgt agcggtggaa tgcgtagata tatggaagaa 360
caccagtggc gaaggcggct gtctagtctg taactgacgc tgaggctcga aagcatgggt 420
agcaaacagg attagatacc cttgtagtcc 450
Claims (7)
1. a kind of special throw type leaven of hot pickled mustard tube based on 16S rDNA sequencings orients preparation method, it is characterised in that at least wrap
Include following steps：
(1) sampled in hot pickled mustard tube salt marsh pond, sample is placed in sterile cryo pipe, be immediately placed in preservation in liquid nitrogen, be subsequently placed in super
In low temperature refrigerator,
(2) extract sample DNA and be PCR, amplification 16S rDNA after doing agarose gel electrophoresis detection, what described PCR was expanded
The sense primer 515F of specific primer has the nucleotide sequence as shown in SEQ ID NO.1, and the downstream of specific primer is drawn
Thing 907R has the nucleotide sequence as shown in SEQ ID NO.2, and PCR amplification regions can for the V4-V5 of microorganism 16S rDNA
Become region, need to carry out recovery purifying to the PCR primer before sequencing,
(3) PCR primer that step (2) is obtained is carried out into high-flux sequence and the analysis that compares, determines dominant microflora species and group
It is proportional,
(4) directed screening of dominant strain,
(5) the proliferation of high-density culture of directed screening bacterial strain,
(6) centrifugation and freeze-drying,
Step (5) is added into skimmed milk power through the bacterium solution of high density increment culture, is centrifuged after shaking up, remove supernatant nutrient solution, received
Collection test tube bottom thalline；The thalline of collection does freeze-drying after adding freeze drying protectant, wherein：Thalline and freeze drying protectant quality
Fraction proportioning is 1:3-1:2.5,
(7) the bacterial strain freeze dried powder of each directed screening and high density the increment culture for obtaining step (6) is according to 16S rDNA
The dominant microflora species for determining is sequenced to mix with proportion of composing, the special throw type leaven of hot pickled mustard tube is obtained.
2. a kind of special throw type leaven beam system of hot pickled mustard tube based on 16S rDNA sequencings as claimed in claim 1 is standby square
Method, it is characterised in that sampling uses 3 point samplings in described step (1), and collection point is located at salt marsh pond center, salt marsh respectively
2 points of pond diagonal, 3 samples of often place's collection, mixes after collection.
3. a kind of special throw type leaven beam system of hot pickled mustard tube based on 16S rDNA sequencings as claimed in claim 1 is standby square
Method, it is characterised in that the directed screening of described step (4) dominant strain is by following operation：In being sampled for the step (1)
In product, take 1ml liquid carries out 10 times of gradient dilution in physiological saline, is uniformly coated on post-directed training base, then 35
Train 36~72h in~37 DEG C of insulating boxs for a certain area, observe bacterium colony；Then the bacterial strain of post-directed training is carried out into physiological and biochemical property mirror
Fixed experiment；According to qualification result, the bacterial strain after directed screening is carried out into inclined-plane culture.
4. a kind of special throw type leaven beam system of hot pickled mustard tube based on 16S rDNA sequencings as claimed in claim 1 is standby square
Method, it is characterised in that the proliferation of high-density culture of described step (5) directed screening bacterial strain is operated as follows：Will be above-mentioned
Step (4) directed screening carries out high density increment culture with the bacterial strain of purifying culture, and high density rises in value culture medium for MRS agar is trained
Base is supported, condition of culture is 48~72h of culture in 35~37 DEG C of insulating boxs；Described MRS agar medium formulas are：Peptone
10.0 grams, 5.0 grams of beef extract, 4.0 grams of dusty yeast, 20.0 grams of glucose, 1.0 milliliters of Tween 80,2.0 grams of dipotassium hydrogen phosphate, second
1 liter of 5.0 grams of sour sodium, 2.0 grams of triammonium citrate, 0.2 gram of magnesium sulfate, 0.05 gram of manganese sulfate, 15.0 grams of agar powder and distilled water, plus
After heat of solution, pH=6.2~7.2,121 DEG C of autoclavings 15~20 minutes are corrected.
5. a kind of special throw type leaven beam system of hot pickled mustard tube based on 16S rDNA sequencings as claimed in claim 1 is standby square
Method, it is characterised in that add 2.7~3.5% to pass through through the bacterium solution of high density increment culture step (5) in described step (6)
The skimmed milk power of high-temperature sterilization, is centrifuged 25-30 minutes with 4000-5000rpm rotating speeds after shaking up under the conditions of -5~10 DEG C.
6. a kind of special throw type leaven beam system of hot pickled mustard tube based on 16S rDNA sequencings as claimed in claim 1 is standby square
Method, it is characterised in that frozen-dried protective agent prescription is in described step (6)：25-30 grams of skimmed milk, 10-15 grams of lactose, glucose
Sugared 5-12 grams, 5-6.5 grams of vitamin C, 5-7.2 grams of D-glucitol, 5-6 grams of Pidolidone and distilled water 250mL；Freeze drying protectant
After heating for dissolving, 121 DEG C standby after autoclaving 15-20 minutes.
7. a kind of special throw type leaven beam system of hot pickled mustard tube based on 16S rDNA sequencings as claimed in claim 1 is standby square
Method, it is characterised in that the main technologic parameters of freeze-drying are in described step (6)：- 50~-70 DEG C of pre-freeze 12-24h
Afterwards, -60~80 DEG C of lyophilized 24-72h.
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