CN110272979A - Putrefactive microorganisms suppressing method for bean paste - Google Patents
Putrefactive microorganisms suppressing method for bean paste Download PDFInfo
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Abstract
The invention discloses a kind of putrefactive microorganisms suppressing methods for bean paste, using the bean cotyledon for occurring producing inflatable tank as sample, bacterial strain is separated, purified, is numbered, then carries out producing gas verifying, the bacterial strain of gas is produced in screening, extract the DNA of aerogen, PCR amplification, electrophoresis, sequencing, sequencing result carries out the analysis of Blast sequence, determines aerogen kind；Select several preservatives, using cylinder plate method bacteriostatic experiment, filter out several preservatives of good antimicrobial effect, then each preservative MIC is measured, orthogonal experiment factor level table is formulated according to preservative MIC and using limitation, orthogonal experiment obtains optimal horizontal combination, compound preservative is obtained, further progress bacteriostatic experiment verifies the fungistatic effect of compound preservative.Using Antimicrobial method of the invention, the bag that can rise for swell caused by a variety of different putrefactive microorganisms prepares the compound preservative that various microorganisms are all had with excellent fungistatic effect.
Description
Technical field
The present invention relates to Antimicrobial methods, more particularly it relates to which a kind of corruption for bean paste is micro-
Biological inhibition method.
Background technique
Bean paste is deep to be liked by general public as the most distinctive flavouring in southwest.The fermentation of bean cotyledon
Journey can be divided into earlier fermentation, fermentation phase and fermentation later period.Earlier fermentation is starter-making stage, in this stage mould in occupation of absolute
Advantage, the various enzymes secreted by aspergillus decompose, the macromolecular in conversion bean cotyledon is small molecule for subsequent micro-organisms
Fermentation.As step the most key in bean cotyledon production process, the quality of fermentation directly determines product quality.From koji-making to hair
Ferment, different phase have different function bacteriums to participate in, and type and quantity suffer from biggish difference.The fermentation phase is due to joined
A large amount of salt is not suitable for the growth of yeast and mold, mainly bacillus megaterium (Bacillus megaterium), plant
The bacterial strains such as object lactobacillus (Lactobacillus plantarum), bacillus amyloliquefaciens (B.amyloliquefaciens) exist
Effect (Jianxin Zhao, 2010)；The fermentation later period is mainly the microorganisms such as bacillus, lactobacillus, saccharomycete (Li Zhi
China, 2014).Although having carried out quality control in production process, bean cotyledon is in storage, putrefactive microorganisms meeting therein
Generating gas using remaining nutriment secondary fermentation leads to swell, influences product quality.
Summary of the invention
The present invention, by separation, purifying, molecular biology identification, is determined and is produced to there is the bean cotyledon of swell problem as raw material
The type of gas microorganism then stores the type primary dcreening operation preservative of interior environment and aerogen according to bean cotyledon, passes through Dan Yin
Plain bacteriostatic experiment selects the preferable preservative of fungistatic effect, then optimizes the formula of Compositional antiseptic agent by orthogonal,
The Compositional antiseptic agent, which is directed to the aerogen in the bean cotyledon for swell problem occur, has good inhibitory effect.
The invention adopts the following technical scheme:
A kind of putrefactive microorganisms suppressing method for bean paste, it is characterised in that the following steps are included:
(1) strain isolation
A variety of common strain isolation culture mediums are prepared, are sterilized；
Using the bean cotyledon for occurring producing inflatable tank as sample, 10 times of serial dilutions are carried out to sample with sterile saline
(such as 10-2、10-3、10-4、10-5、10-6、10-7), each multiple dilution 0.1mL is drawn respectively to be coated on each culture medium, Meng
Add and draw red culture based on 27 DEG C of cultures, other cultures are based on 37 DEG C of cultures；
(2) bacterial strain purifies
Classification number is carried out to bacterium colony according to the morphological feature of bacterium colony in culture medium, is inoculated into PCA training with method of scoring respectively
It supports on base, carries out purebred culture；
(3) gas confirmatory experiment is produced
Purifying bacterial strain is carried out producing gas confirmatory experiment with glucose fermentation culture medium, primarily determines the number of aerogen
Amount, the aerogen bacterium solution primarily determined is inoculated in the bean cotyledon just to have dispatched from the factory and carries out culture observation, to aerogen into
Row is verified again, determines aerogen；
(4) PCR amplification and sequence alignment
It is fallen in the EP pipe of 1.5mL using sterile bamboo stick picking aerogen single bacterium, referring to Biotech DNA kit
Specification extracts thallus DNA；
Using the thallus DNA extracted as template, PCR amplification, amplification condition: 95 DEG C of denaturation 5min；Circulation: 95 DEG C of denaturation
1min, 50 DEG C of annealing 1min, 72 DEG C of extension 2min, 35 recycle；72 DEG C of extension 10min；
Gained PCR product is detected at the standard conditions using 2.0% (m/V) agarose gel electrophoresis, electrophoresis time
30min；And successful PCR stoste will be expanded and be used to be sequenced；Sequencing result is inputted into National Center for Biotechnology Information
GenBank database carries out the analysis of Blast sequence, determines aerogen kind；
(5) compound preservative is screened
1. cylinder plate method bacteriostatic experiment
It will be made into the solution that concentration is 0.5% (m/V) for examination preservative, bacteriostatic experiment is carried out using cylinder plate method, it is micro- to produce gas
The bacteria suspension concentration of biology is 107-108Cfu/mL measures the diameter of inhibition zone in 37 DEG C of cultures, from for examination preservative afterwards for 24 hours
Several preservatives that antibacterial circle diameter is maximum and antimicrobial stability is good are selected to carry out the measurement of MIC value；
2. the measurement of MIC value
The preservative filtered out is configured to series of concentrations gradient respectively, measures its MIC value for being directed to aerogen, behaviour
Make method with reference to step (1), more each preservative is directed to the MIC value of different aerogen, selects MIC value in aerogen
Maximum bacterial strain carries out subsequent preservative orthogonal building experiment；
3. preservative orthogonal building is tested
The factor level of orthogonal experiment is formulated using limitation according to the preservative of each preservative MIC value and GB2760-2014
Table carries out orthogonal experiment, carries out range analysis and variance analysis to Orthogonal experiment results, determines each factor to the shadow of fungistatic effect
Size is rung, and the level for selecting each factor optimal is combined, obtains compound preservative；
4. confirmatory experiment
Bacteriostatic experiment is carried out using compound preservative, obtains antibacterial circle diameter, antibacterial circle diameter drop when by orthogonal experiment
Sequence arrangement, calculates the average value of the biggish antibacterial circle diameter of the first half, carries out with the antibacterial circle diameter that this bacteriostatic experiment obtains
Compare, if the antibacterial circle diameter of this experiment is greater than or equal to the average value and subtracts 2mm, illustrates that it has had antibacterial
Effect is then added into the bean cotyledon just to have dispatched from the factory, and control group is arranged, and is cultivated in 37 DEG C, in the observation period of 10d
It is interior, the production gas situation of sample is recorded, if control group, which has, produces gas phenomenon, and adds the bean cotyledon of compound preservative without gas phenomenon is produced, then
The compound preservative inhibits suitable for putrefactive microorganisms.
It is described for examination preservative include Nipagin complex esters, Nisin, dehydroactic acid sodium, sodium Diacetate, fumaric acid and sorb
Sour potassium, but it is not limited to these types of preservative.
The morphological feature according to bacterium colony in culture medium to bacterium colony carry out classification number refer to by colony shape, edge,
Gloss, quality, color and the identical bacterium colony of transparency are divided into one kind, and every one kind is numbered.
A variety of common strain isolation culture mediums include PCA culture medium, MRS culture medium, TSA culture medium, YPD culture
Base, YM culture medium, glucose fermentation culture medium and rose bengal medium.
PCA culture medium: tryptone 5.0g, yeast extract 2.5g, glucose 1.0g, agar 15.0g, distilled water
7.0 scholar 0.2 of 1000ml, pH.
MRS culture medium: peptone 10.0g, beef extract 10.0g, yeast extract 5.0g, diammonium hydrogen citrate 2.0g, glucose
20.0g, Tween 80 1.0mL, sodium acetate 5.0g, dipotassium hydrogen phosphate 2.0g, magnesium sulfate 0.58g, manganese sulfate 0.25g, agar
18.0g, distilled water 1 000mL, pH 6.2~6.6.
TSA culture medium: tryptone 15.0g, phytone 5.0g, sodium chloride 30.0g, agar 15.0g, distilled water are settled to
7.3 scholar 0.2 of 1000mL, pH.
YPD culture medium: 10g yeast extract, 20g peptone, 20g glucose, 20g agar powder, distilled water 1000ml.
YM culture medium: yeast powder 0.3% (m/m), malt flour 0.3%, peptone 0.5%, agar 2%, remaining is water.
Glucose fermentation culture medium: peptone 2.7g, sodium chloride 5.0g, 0.2% (m/V) bromine Moschus phenol orchid 0.03g, agar
3g, KH2PO40.3g, glucose 10.0g, water 1000mL, pH 7.0.
Rose bengal medium: peptone 5g, glucose 10g, potassium dihydrogen phosphate 1g, magnesium sulfate 0.5g, agar 20g, 1/
3000 rose-bengal solution 100mL, distilled water 1000mL, chloramphenicol 0.1g.
Glucose in each culture medium adds after sterilization.
Using Antimicrobial method of the invention, can rise bag system for swell caused by a variety of different putrefactive microorganisms
It is standby to go out to all have various microorganisms the compound preservative of excellent fungistatic effect.
Detailed description of the invention
Fig. 1 is the gel electrophoresis figure of pcr amplification product.
Fig. 2 is the antibacterial screening experiment result of single factor test.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Embodiment 1
Strain isolation and purifying
PCA culture medium, MRS culture medium, TSA culture medium, YPD culture medium, YM culture medium, glucose hair are prepared as requested
Ferment culture medium and rose bengal medium, 121 DEG C of sterilizings.
The bean cotyledon for occurring producing inflatable tank is provided as sample by Dan Dan bean paste Group Plc, Sichuan Province.
10g sample is taken, 10 times of serial dilutions (10 are carried out to sample with sterile saline-2、10-3、10-4、10-5、10-6、10-7), each multiple dilution 0.1mL is drawn respectively and is coated on each culture medium, and rose bengal medium is cultivated in 27 DEG C, other trainings
It supports based on 37 DEG C of cultures.
The morphological feature for observing bacterium colony in culture medium, by colony shape, edge, gloss, quality, color and transparency phase
Same bacterium colony is divided into one kind, is numbered to every one kind, is inoculated on PCA culture medium with method of scoring respectively, carries out purebred culture.
By isolating and purifying, combining form is observed, and filters out 26 plants of microorganisms in Cong Chanqi bean cotyledon sample, and number is PX001- respectively
PX026。
Produce gas confirmatory experiment
Purifying bacterial strain is carried out using Durham's fermentation tube with glucose fermentation culture medium to produce gas confirmatory experiment, discovery is shared
Two plants of bacterium show gas generation property, respectively PX012, PX019, both aerogen bacterium solutions are inoculated in the beans just to have dispatched from the factory
Culture observation is carried out in valve, aerogen is verified again, determines that PX012, PX019 are aerogen.
PCR amplification and sequence alignment
Raw material and equipment: DNA kit Biotech Biotechnology Co., Ltd；Sigma company, the R-Nase enzyme solution U.S.；
Bacterium the primer is primer Eu27f (5 '-AGAGTTTGATCCTGGCTCAG-3 '), lower primer 1490R (5 '-on universal primer
GGTTACCTTGTTACGACTT-3 '), Taq archaeal dna polymerase TIANGEN Biotech (Beijing) Co., Ltd.；The Zhejiang Nisin silver as
Biotechnology Engineering Co., Ltd.Nipagin complex esters, Nisin, dehydroactic acid sodium, sodium Diacetate, fumaric acid and potassium sorbate
Chengdu Ke Long chemical company.
5422 type desk centrifuge Eppendorf companies；AG22331 type Polymerized human serum albumin Eppendorf company；
Bio-Rad company, the T2A type gel imaging system U.S.；DYY-8C type electrophoresis apparatus Liuyi Instruments Plant, Beijing；The vertical height of G154DWS type
Press steam sterilization pan Zealway (Xiamen) Instrument Inc..
Using the single colonie of sterile bamboo stick picking aerogen PX012, PX019 respectively in the EP pipe of 1.5mL, reference
The specification of Biotech DNA kit extracts thallus DNA.
Using the thallus DNA extracted as template, PCR amplification, reaction system: 16 μ L of dd water, 10 to thaw on ice ×
2 μ L of Taq buffer, 2 μ L of d NTP, the 25mmolL to thaw on ice-1MgCl21.5 μ L, upper and lower primer and each 1 μ L of template,
0.5 μ L of Taq archaeal dna polymerase；Amplification condition: 95 DEG C of denaturation 5min；Circulation: 95 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C
Extend 2min, 35 circulations；72 DEG C of extension 10min.
Gained PCR product is detected at the standard conditions using 2.0% (m/V) agarose gel electrophoresis, electrophoresis time
30min, as a result as shown in Figure 1；And successful PCR stoste will be expanded and sent to the progress of Chengdu Qing Ke Zi Xi Bioisystech Co., Ltd
Sequencing；Sequencing result input National Center for Biotechnology Information GenBank database is subjected to the analysis of Blast sequence, really
Fixed output quota gas microorganism kind, the results are shown in Table 1.
1 Analysis of Microbial Diversity of table
| Strain number | Sequence length | Homologous strain (sequence accession number) | Similitude |
| PX012 | 1377bp | Bacillus amyloliquefaciens(KX242411) | 100% |
| PX019 | 1396bp | Bacillus licheniformis(KU551126) | 100% |
By electrophorogram and alignment, determine that two plants of aerogenic bacterias are respectively as follows: PX012 bacillus amyloliquefaciens；PX019
Bacillus licheniformis.
Screen compound preservative
1. cylinder plate method bacteriostatic experiment
It will be for trying preservative (Nipagin complex esters, Nisin, dehydroactic acid sodium, sodium Diacetate, fumaric acid and potassium sorbate)
It is made into the solution that concentration is 0.5% (m/V), bacteriostatic experiment, the bacterium of aerogen (PX012, PX019) are carried out using cylinder plate method
Suspension concentration is 107-108Cfu/mL measures the diameter of inhibition zone in 37 DEG C of cultures afterwards for 24 hours, selects from for examination preservative antibacterial
Several preservatives that loop diameter is maximum and antimicrobial stability is good carry out the measurement of MIC value；Antimicrobial stability refers to well same anti-corrosion
When agent repeatedly carries out bacteriostatic experiment to same bacterial strain, the difference of multiple antibacterial circle diameter maxima and minimas of acquisition is less than or waits
In 8mm.As a result as shown in Figure 2.
Figure it is seen that dehydroactic acid sodium and potassium sorbate are bad to the fungistatic effect of two plants of bacterium.As in food most
Common preservative, potassium sorbate have a very wide range of application, are better than gram sun to the fungistatic effect of Gram-negative bacteria
Property bacterium, and its fungistatic effect is poor under conditions of partial neutral.Bean cotyledon belongs to weakly acidic condition, wherein being mostly bacillus, Gu Shan
Potassium sorbate is bad to its inhibitory effect, and antibacterial circle diameter is fluctuated from almost without phenomenon to 9mm, and fungistatic effect is unstable.
Dehydroactic acid sodium has good inhibiting effect to bacterium, while more preferably to the inhibiting effect of mould, saccharomycete, and by pH value
Influence is smaller, and for different strain to its tolerance difference, therefore to PX019 bacterium, antibacterial circle diameter is completely unobvious, inhibits to make
With very weak, and to the inhibiting effect substantially 12mm or so of PX012 bacterium, but fungistatic effect be weaker than Nipagin complex esters,
Nisin, sodium Diacetate and fumaric acid, but it is better than potassium sorbate.Nisin is as a kind of narrow spectrum preservative of bion, to G+'s
Fungistatic effect is better than G- bacterium.Parabens preservative has preferable fungistatic effect as a kind of spectrum bacteriostatic agent, to G+,
Result of study shows that it all has preferable fungistatic effect to bacillus subtilis, mould etc..Sodium Diacetate is as a kind of good
Mould inhibitor has good inhibiting effect to fungi, mould, bacterium.Fumaric acid can preferably inhibit mould in food,
Bacterium, at the same it is small to sensibility such as temperature, pH value.Experimental data also indicates that, Nipagin complex esters, Nisin, sodium Diacetate and richness
Horse four kinds of preservatives of acid are preferable to the inhibitory effect of aerogenic bacteria strain, and the size of antibacterial circle diameter is relatively concentrated, concentrate on respectively 22mm,
25mm, 19mm, 17mm or so, fungistatic effect are stablized, therefore these four preservatives is selected to carry out combine experiment.
2. the measurement of MIC value
Four kinds of Nipagin complex esters, Nisin, sodium Diacetate and fumaric acid preservatives are configured to series of concentrations ladder respectively
Degree measures its MIC value for being directed to aerogen, operating method reference strain isolation and purification step, more each preservative needle
To the MIC value of different aerogen, select the subsequent preservative of the maximum bacterial strain progress of MIC value in aerogen orthogonal multiple
With experiment.The MIC value of each preservative is as shown in table 2.
The MIC value of each preservative of table 2
By the MIC value of 2 four kinds of preservatives of table it is found that each preservative stablizes the fungistatic effect of two plants of aerogen,
The fungistatic effect of middle Nisin is best, is then followed successively by sodium Diacetate, Nipagin complex esters and fumaric acid.Nisin and sodium Diacetate
MIC value stablize, be 0.05 ‰ and 0.40 ‰；And Nipagin complex esters and fumaric acid are slightly weaker than the inhibitory effect of PX012
PX019.The MIC value of comprehensive four kinds of preservatives selects PX012 bacterium to carry out the orthogonal of preservative as the object bacterium of subsequent experimental
Combine experiment.
3. preservative orthogonal building is tested
The factor level of orthogonal experiment is formulated using limitation according to the preservative of each preservative MIC value and GB2760-2014
Table, as shown in table 3.Orthogonal experiment is carried out, range analysis and variance analysis are carried out to Orthogonal experiment results, as a result such as table 4 and table 5
It is shown, it determines influence size of each factor to fungistatic effect, and the level for selecting each factor optimal is combined, obtains compound anti-
Rotten agent.
3 orthogonal experiment factor level table of table
4 Orthogonal experiment results range analysis of table
By range analysis: the influence size to fungistatic effect are as follows: Nisin > Nipagin complex esters > sodium Diacetate
> fumaric acid.Optimum combination are as follows: A4B4C3D3, i.e. Nisin 0.2 ‰, Nipagin complex esters 0.2 ‰, sodium Diacetate 0.6 ‰, richness
Horse acid 3.0 ‰.
5 Orthogonal experiment results variance analysis of table
A. level of signifiance a=0.05* indicates to influence experimental result significant
By variance analysis: influence of the Nisin to experimental result is more significant, next is sodium Diacetate, nipalgin is multiple
Close ester and fumaric acid.The optimum combination of Orthogonal experiment results are as follows: A4B4C3D3, i.e. Nisin 0.2 ‰, Nipagin complex esters
0.2 ‰, sodium Diacetate 0.6 ‰, fumaric acid 3.0 ‰.
It is mostly bacterium, bacillus category, the aerogen whole identified from the point of view of the microorganism separated in bean cotyledon
For bacillus category.Bacillus licheniformis (B.licheniformis) is the common strain in bean cotyledon fermentation, is that nature decomposes
The common microbiological of organic matter in environment, often isolated in traditional food, major function is to assist during the fermentation
The protein and starch in fungi decomposing food are helped, creates conditions and is formed flavor substance for the growth of other microorganisms, solve
Dominant bacteria one of of the bacillus amyloliquefaciens as the fermentation phase is synthesized organic with very strong starch-splitting and using albumen
The ability of acid and biacetyl, by the starch fast decoupled in system, promotes the quick hair of bean cotyledon in the fermentation process of bean cotyledon
Ferment, while aroma substance is generated, such as organic acid, diacetyl substance.But research shows that its excessive proliferation can decompose largely
Nutriment, while niff is generated, adverse effect is also easy to produce to fermentation.Bacillus licheniformis and bacillus amyloliquefaciens
It is to be brought into system by being attached to the surface of raw material mostly, it is numerous using the nutriment progress in system in fermentation stage
It grows, fragrant production ester is produced in the fermentation process of bean cotyledon, promotes the formation of flavor substance.In later period storage, lichens gemma bar
Bacterium, bacillus amyloliquefaciens carry out secondary fermentation, generation gas using remaining sugar, starch in bean cotyledon system etc..
4. confirmatory experiment
Compound preservative is carried out bacteriostatic experiment, experimental subjects PX012, experiment knot according to 0.1 ‰ concentration by the present invention
Fruit shows antibacterial circle diameter average out to 22mm, and fungistatic effect is obvious, and (antibacterial circle diameter descending arrangement when by orthogonal experiment, calculates
The average value of the biggish antibacterial circle diameter of the first half is 21.6mm, the antibacterial circle diameter of this bacteriostatic experiment and the average value
It is not much different, illustrates its fungistatic effect having had), then compound preservative is added to just to go out according to the ratio of 0.1g/Kg
In the bean cotyledon of factory, another group is added preservative according to factory's original production process, is cultivated for 37 DEG C in constant-temperature table, in 10d
Observation period in, record the production gas situation of sample, as the result is shown: according to factory's original formulation addition preservative sample in the 4th day
Slight production gas phenomenon is begun with, production gas situation aggravates within the 6th day；And the sample for adding compound preservative does not occur within the observation period
Produce gas phenomenon.
Although reference be made herein to invention has been described for explanatory embodiment of the invention, however, it is to be understood that ability
Field technique personnel can be designed that a lot of other modification and implementations, these modifications and implementations will fall in the application public affairs
Within the scope and spirit opened.It more specifically, can be to the group of theme combination layout in range disclosed in the present application
A variety of variations and modifications are carried out at component and/or layout.In addition to variations and improvements to the component parts and or layout,
To those skilled in the art, other purposes also will be apparent.
Claims (4)
1. a kind of putrefactive microorganisms suppressing method for bean paste, it is characterised in that the following steps are included:
(1) strain isolation
A variety of common strain isolation culture mediums are prepared, are sterilized；
Using the bean cotyledon for occurring producing inflatable tank as sample, 10 times of serial dilutions are carried out to sample with sterile saline, respectively
It draws each multiple dilution 0.1mL to be coated on each culture medium, rose bengal medium is cultivated in 27 DEG C, other cultures are based on 37
DEG C culture；
(2) bacterial strain purifies
Classification number is carried out to bacterium colony according to the morphological feature of bacterium colony in culture medium, is inoculated into PCA culture medium with method of scoring respectively
On, carry out purebred culture；
(3) gas confirmatory experiment is produced
Purifying bacterial strain is carried out producing gas confirmatory experiment with glucose fermentation culture medium, primarily determines the quantity of aerogen,
The aerogen bacterium solution primarily determined is inoculated in the bean cotyledon just to have dispatched from the factory and carries out culture observation, aerogen is carried out again
Secondary verifying, determines aerogen；
(4) PCR amplification and sequence alignment
It is fallen in the EP pipe of 1.5mL using sterile bamboo stick picking aerogen single bacterium, is extracted using BiotechDNA kit
Thallus DNA；
Using the thallus DNA extracted as template, PCR amplification, amplification condition: 95 DEG C of denaturation 5min；
Circulation: 95 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 2min, 35 recycle；72 DEG C of extension 10min；
Gained PCR product is detected at the standard conditions using 2.0% (m/V) agarose gel electrophoresis, electrophoresis time
30min；And successful PCR stoste will be expanded and be used to be sequenced；Sequencing result is inputted into National Center for Biotechnology Information
GenBank database carries out the analysis of Blast sequence, determines aerogen kind；
(5) compound preservative is screened
1. cylinder plate method bacteriostatic experiment
It will be made into the solution that concentration is 0.5% (m/V) for examination preservative, bacteriostatic experiment, aerogen are carried out using cylinder plate method
Bacteria suspension concentration be 107-108Cfu/mL measures the diameter of inhibition zone in 37 DEG C of cultures afterwards for 24 hours, selects from for examination preservative
Several preservatives that antibacterial circle diameter is maximum and antimicrobial stability is good carry out the measurement of MIC value；
2. the measurement of MIC value
The preservative filtered out is configured to series of concentrations gradient respectively, measures its MIC value for being directed to aerogen, operation side
Method refers to step (1), and more each preservative is directed to the MIC value of different aerogen, selects MIC value in aerogen maximum
Bacterial strain carry out the experiment of subsequent preservative orthogonal building；
3. preservative orthogonal building is tested
The factor level table for formulating orthogonal experiment using limitation according to the preservative of each preservative MIC value and GB2760-2014, into
Row orthogonal experiment carries out range analysis and variance analysis to Orthogonal experiment results, determines that influence of each factor to fungistatic effect is big
It is small, and the level for selecting each factor optimal is combined, and obtains compound preservative；
4. confirmatory experiment
Bacteriostatic experiment is carried out using compound preservative, obtains antibacterial circle diameter, antibacterial circle diameter descending row when by orthogonal experiment
Column calculate the average value of the biggish antibacterial circle diameter of the first half, and the antibacterial circle diameter obtained with this bacteriostatic experiment is compared,
If the antibacterial circle diameter of this experiment, which is greater than or equal to the average value, subtracts 2mm, illustrate its fungistatic effect having had,
It is then added into the bean cotyledon just to have dispatched from the factory, and control group is set, cultivated in 37 DEG C, within the observation period of 10d, record
The production gas situation of sample if control group, which has, produces gas phenomenon, and adds the bean cotyledon of compound preservative without gas phenomenon is produced, then this is compound anti-
Rotten agent inhibits suitable for putrefactive microorganisms.
2. the putrefactive microorganisms suppressing method according to claim 1 for bean paste, it is characterised in that described for examination
Preservative includes Nipagin complex esters, Nisin, dehydroactic acid sodium, sodium Diacetate, fumaric acid and potassium sorbate.
3. the putrefactive microorganisms suppressing method according to claim 1 for bean paste, it is characterised in that the basis
In culture medium the morphological feature of bacterium colony to bacterium colony carry out classification number refer to by colony shape, edge, gloss, quality, color and
The identical bacterium colony of transparency is divided into one kind, and every one kind is numbered.
4. the putrefactive microorganisms suppressing method according to claim 1 for bean paste, it is characterised in that described a variety of
Common strain isolation culture medium includes PCA culture medium, MRS culture medium, TSA culture medium, YPD culture medium, YM culture medium, glucose
Fermentation medium and rose bengal medium.
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