CN105432766B - A kind of quorum sensing inhibitor of aquatic products Pseudomonas fluorescens and preparation method thereof - Google Patents
A kind of quorum sensing inhibitor of aquatic products Pseudomonas fluorescens and preparation method thereof Download PDFInfo
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- CN105432766B CN105432766B CN201510717836.8A CN201510717836A CN105432766B CN 105432766 B CN105432766 B CN 105432766B CN 201510717836 A CN201510717836 A CN 201510717836A CN 105432766 B CN105432766 B CN 105432766B
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- tree peony
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23B—PRESERVING, e.g. BY CANNING, MEAT, FISH, EGGS, FRUIT, VEGETABLES, EDIBLE SEEDS; CHEMICAL RIPENING OF FRUIT OR VEGETABLES; THE PRESERVED, RIPENED, OR CANNED PRODUCTS
- A23B4/00—General methods for preserving meat, sausages, fish or fish products
- A23B4/14—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12
- A23B4/18—Preserving with chemicals not covered by groups A23B4/02 or A23B4/12 in the form of liquids or solids
- A23B4/20—Organic compounds; Microorganisms; Enzymes
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
Abstract
The present invention relates to a kind of aquatic products fluorescent Pseudomonads body-sensings to answer inhibitor and preparation method thereof.The quorum sensing inhibitor can be applied to the exploitation of aquatic products composite preservative；The preparation method is using the root bark of tree peony as raw material, the root bark of tree peony is pre-processed, ultrasonic wave added concussion extraction is carried out after organic solvent is added, filters and collect filtrate, by the filtrate rotary evaporation, crude extract is obtained, dehydrated alcohol is then added and carries out alcohol precipitation, filters removal filter residue, filtrate rotary evaporation is obtained into root bark of tree peony extracting solution, the root bark of tree peony extracting solution is freeze-dried, obtains Cortex Moutan extract, the Cortex Moutan extract is the quorum sensing inhibitor.Raw material sources of the invention are abundant, and preparation method is simple；Cortex Moutan extract can interfere with the intervention school-based of aquatic products Pseudomonas fluorescens in the present invention, blocks it to cause the expression of the rotten factor, can be used for the purpose of preservation of fishery and composite preservative.
Description
Technical field
The invention belongs to food storage technical field of preservation of fresh, and in particular to a kind of group's sense of aquatic products Pseudomonas fluorescens
Answer inhibitor and preparation method thereof.
Background technique
Fresh aquatic products are rich in nutriment and moisture, and musculature is fragile, and content of soluble protein is high, and pH value is close
It is neutral.But fresh aquatic products are if without effective Preservation Treatment that it is easy to appear corruption.Cause the master that aquatic products are putrid and deteriorated
Wanting reason includes the effect of enzyme, microorganism and fat oxidation etc..Wherein, microorganism passes through decomposing protein, lipid and carbohydrate etc.
Nutriment carries out growth and breeding, causes aquatic products corrupt, asks so as to cause huge economic loss and serious public health
Topic.Aquatic products corruption is not to be caused by all microbial activities, but specific spoilage organisms play key effect.Pseudomonad
Category is the dominant spoilage organisms of aquatic products, wherein most of is Pseudomonas fluorescens.The activity of Pseudomonas fluorescens can generate bad
Smell causes unacceptable on aquatic products sense organ.In addition, Pseudomonas fluorescens is also one of conditioned pathogen of fish, often
Cause red skin disease.
In recent years, quorum sensing (Quorum sensing, QS) system is found to be research food spoilage in spoiled food
Mechanism provides new direction.QS participate in regulating and controlling microbial a variety of life habits and various physiology courses, as bioluminescence,
The synthesis of antibiotic, the formation of biomembrane, the expression of virulence factor and biological swarming etc..It is existing research shows that QS participate in
Food spoilage process regulates and controls protein relevant to food spoilage, fat, pectin, chitin etc. and decomposes enzymatic activity.Therefore, lead to
The intervention school-based for crossing interference food spoilage inhibits the expression of deteriorative factor, this provides new direction for food fresh keeping.
The generation of bacterium QS phenomenon is blocked to be known as quorum sensing inhibition or interference by different modes.Quorum sensing inhibitor
(Quorum sensing inhibitor, QSI) is broadly divided into two classes: natural QSI and artificial synthesized QSI.And day
Right QSI concentration is low and small toxicity, is the hot spot of current people's research.Currently, the natural QSI of research discovery both at home and abroad has ocean algae
Extract of class, fruits and vegetables class and Chinese herbal medicine etc., such as thallus laminariae, broccoli, pineapple, Phyllanthus embical fruit, windmill and laurel etc..
In addition, gingerol, andrographolide, 4',5,7-trihydroxyflavone and the cinnamic acid etc. in curcumin, ginger in turmeric also have QSI active.
However, these QSI inhibiting effect focus primarily upon the pathogenic of pseudomonas aeruginosa, Escherichia coli and serratia marcescens etc. and
Saprogenicity, to the inhibition of Pseudomonas fluorescens saprogenicity, there is not been reported.
The root bark of tree peony throughout our country to have cultivation as a kind of Chinese medicine more, have effects that clearing heat and cooling blood, promoting blood circulation to remove blood stasis, and
Toxic side effect is small.Currently, the patent about the root bark of tree peony focus mostly in its active constituent extraction and its medical field application.
But application of the Cortex Moutan extract in terms of aquatic products fluorescent Pseudomonads body-sensing answers inhibitor yet there are no open report so far.
Summary of the invention
In order to solve the above-mentioned technical problem, the invention discloses a kind of inhibition of the quorum sensing of aquatic products Pseudomonas fluorescens
Agent and preparation method thereof, is raw material using the Chinese medicine root bark of tree peony, Cortex Moutan extract is obtained by a series of processing, by verifying, institute
It is inhibited to the quorum sensing to Pseudomonas fluorescens to state Cortex Moutan extract, by interfering aquatic products Pseudomonas fluorescens
Intervention school-based, block it to cause the expression of the rotten factor, can be used for the exploitation of preservation of fishery and composite preservative.
The present invention is achieved by the following technical solutions:
A kind of preparation method of the quorum sensing inhibitor of aquatic products Pseudomonas fluorescens, the preparation method are with the root bark of tree peony
Organic solvent is added in raw material, shakes extractive technique using ultrasonic wave added, obtains Cortex Moutan extract, the Cortex Moutan extract includes
Gallic acid and Paeonol；The Cortex Moutan extract is quorum sensing inhibitor, and the quorum sensing inhibitor can reduce glimmering
The corrupt ability of light pseudomonad, is used for the exploitation of preservation of fishery and composite preservative；Through examining, gallic acid and pellet
Skin phenol has inhibitory activity to the quorum sensing of Pseudomonas fluorescens.
Further, the preparation method includes root bark of tree peony pretreatment, ultrasonic wave added concussion is extracted, alcohol precipitation and freeze-drying walk
Suddenly.
Further, the preparation method specifically: using the root bark of tree peony as raw material, the root bark of tree peony is pre-processed, is obtained red
Pi Fen carries out ultrasonic wave added concussion extraction after organic solvent is added, filters and collect filtrate, the filtrate is carried out rotation steaming
Hair obtains crude extract, and dehydrated alcohol is then added and carries out alcohol precipitation, filters removal filter residue, and the filtrate rotary evaporation acquisition root bark of tree peony is mentioned
Liquid is taken, the root bark of tree peony extracting solution is freeze-dried, obtains Cortex Moutan extract.
Further, the specific steps of the preparation method:
(1) root bark of tree peony pre-processes: the root bark of tree peony being dried in 30-40 DEG C of constant incubator, is then crushed, root bark of tree peony powder is obtained；
(2) ultrasonic wave added concussion is extracted: will rapid (1) treated the root bark of tree peony powder and the organic solution according to solid-liquid ratio
1:6-1:10 is mixed, first ultrasonic extraction 10-15min, and then 3-6h is extracted in concussion, is filtered removal residue, is collected filtrate；
The organic solution is any one in methanol solution, ethanol solution and acetone soln, and the concentration of the methanol solution is
60%-90% (W/V), the concentration of the ethanol solution are 50%-90% (W/V), and the concentration of the acetone soln is 60%-
80% (W/V)；
(3) alcohol precipitation: the filtrate that step (2) are collected carries out rotary evaporation, obtains crude extract；In the crude extract
Dehydrated alcohol is added and carries out alcohol precipitation, the alcohol precipitation time is 8-14h, wherein the volume ratio of the crude extract and the dehydrated alcohol is
1:5-10；It is filtered after alcohol precipitation, removes filter residue, filtrate is subjected to rotary evaporation, obtains root bark of tree peony extracting solution；
(4) it is freeze-dried: the root bark of tree peony extracting solution that step (3) obtain being freeze-dried, the root bark of tree peony is obtained and mentions
Take object.
Further, in step (2), the ultrasonic power of the ultrasonic extraction is 200-300W, the item that the concussion is extracted
Part are as follows: 30-40 DEG C of temperature, concussion speed 150-180r/min.
A kind of preparation method of composite preservative, the composite preservative include quorum sensing inhibitor, the compound guarantor
Fresh dose of preparation method first determines whether the freshness of aquatic products, is then determined according to the freshness of the aquatic products described compound
The formula of antistaling agent, specific step is as follows for the preparation method of the composite preservative:
1) Comprehensive Assessment of aquatic products: synthesis is carried out according to the color of aquatic products, smell, tissue morphology, tissue elasticity and is commented
Point, obtain comprehensive grading value, the highest of the comprehensive grading value be divided into 5 points and it is minimum be divided into 1 point, and commented according to the synthesis
The aquatic products are divided into fresh, comparatively fresh and stale three ranks by score value；Due to stale rank aquatic products
Through being not suitable for eating, so only adding the composite preservative to fresh and two ranks of comparatively fresh aquatic products to protect
It is fresh；
2) preparation of composite preservative raw material and solvent: the composite preservative raw material is constituted are as follows: the quorum sensing suppression
Preparation, chitosan and tea polyphenols；Constitute the composite preservative solvent are as follows: 0.1mol/L acetum and 0.2mol/L sodium chloride
The mixed solution of solution；
3) determination of composite fresh-keeping agent prescription: according to step (1) to the Comprehensive Assessment of aquatic products as a result, working as the aquatic products
When being divided into fresh rank, the formula of the composite preservative are as follows: by weight, 10 parts of the quorum sensing inhibitor,
Described chitosan 1-2 parts, tea polyphenols 1-2 parts described, 30-60 parts of the composite preservative solvent；When the aquatic products are divided
When for comparatively fresh rank, the formula of the composite preservative are as follows: by weight, 10 parts of the quorum sensing inhibitor, described
4-5 parts of chitosan, tea polyphenols 3-5 parts described, 20-30 parts of the composite preservative solvent.
Further, in the Comprehensive Assessment of step (1) aquatic products, the aquatic products are divided according to the comprehensive grading value
For fresh, comparatively fresh and the foundation of stale three ranks are as follows: be divided into the corresponding synthesis of aquatic products of fresh rank
Score value is 4-5 point, including 4 points；Being divided into the corresponding comprehensive grading value of aquatic products of comparatively fresh rank is 3-4 points, packet
It includes 3 points but does not include 4 points；The corresponding comprehensive grading value of aquatic products for being divided into stale rank is not include lower than 3 points
3 points.
Advantageous effects of the invention:
(1) raw material sources of the invention are abundant, and preparation method is simple；
(2) Cortex Moutan extract prepared has quorum sensing inhibitory activity, can effectively interfere aquatic products fluorescence false single
The intervention school-based of born of the same parents bacterium also can be used alone or achieve the purpose that preservation of fishery with other antistaling agents compound use；
(3) Cortex Moutan extract of the invention shows the quorum sensing inhibitory activity of Pseudomonas fluorescens: the root bark of tree peony extracts
Object all has suppression to the formation of the biofilm of Pseudomonas fluorescens, the generation of protease and the quorum sensings such as cluster and swimming
Production is used, and above-mentioned quorum sensing is to cause the rotten factor, and therefore, the cause that the Cortex Moutan extract can reduce Pseudomonas fluorescens is rotten
Ability.
Detailed description of the invention
Fig. 1 is a kind of step flow chart of the quorum sensing inhibitor preparation method of aquatic products Pseudomonas fluorescens；
Fig. 2 is the total number of bacterial colonies of control group and the root bark of tree peony processing group flesh of fish under different storage times；
Fig. 3 is the pH value of control group and the root bark of tree peony processing group flesh of fish under different storage times；
Fig. 4 is TBA (thiobarbituricacidα-) value of control group and the root bark of tree peony processing group flesh of fish under different storage times；
Fig. 5 is TVB-N (Volatile Base Nitrogen) value of control group and the root bark of tree peony processing group flesh of fish under different storage times.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is explained in further detail.It should be appreciated that specific embodiment described herein is used only for explaining the present invention, and
It is not used in the restriction present invention.
On the contrary, the present invention covers any substitution done on the essence and scope of the present invention being defined by the claims, repairs
Change, equivalent method and scheme.Further, in order to make the public have a better understanding the present invention, below to of the invention thin
It is detailed to describe some specific detail sections in section description.Part without these details for a person skilled in the art
The present invention can also be understood completely in description.
Embodiment 1
The root bark of tree peony is dried in 30-40 DEG C of constant incubator, is ground into powder, obtains root bark of tree peony powder, by the root bark of tree peony powder with
The methanol solution of 75% (W/V) is mixed according to solid-liquid ratio 1:8, ultrasonic extraction 10min first, and then 6h is extracted in concussion, described super
The ultrasonic power that sound extracts is 200-300W, the condition that the concussion is extracted are as follows: 30-40 DEG C of temperature, concussion speed 150-180r/
min；Then removal residue is filtered, filtrate is collected；Obtain by the filtrate rotary evaporation, obtain crude extract；It is slightly mentioned described
Dehydrated alcohol is added in liquid and carries out alcohol precipitation, the alcohol precipitation time is 8h, wherein the volume ratio of the crude extract and the dehydrated alcohol is
1:5；It is filtered after alcohol precipitation, removes filter residue, filtrate is subjected to rotary evaporation, obtains root bark of tree peony extracting solution；By the root bark of tree peony
Extracting solution is freeze-dried, and the Cortex Moutan extract is obtained；The present embodiment is first during preparing the Cortex Moutan extract
It shakes and extracts first with ultrasonic wave added, improve the recovery rate of the Cortex Moutan extract；And root bark of tree peony drying temperature and concussion are extracted
Temperature is arranged at 30-40 DEG C, ensure that the activity of the Cortex Moutan extract of preparation, prevents the pellet for making to extract since temperature is excessively high
The inhibitory effect of the quorum sensing of bark extract confrontation aquatic products Pseudomonas fluorescens is affected；Last freeze-drying step
The activity of the Cortex Moutan extract of preparation is further protected.
The Cortex Moutan extract dry powder of acquisition is resuspended through deionized water, crosses 0.22 μm of sterilised membrane filter degerming；It will remove
The Cortex Moutan extract re-suspension liquid that mass concentration after bacterium is 0.39-6.25mg/mL is examined with reporting bacterial strain chromabacterium biolaceum CV026
It surveys, i.e., after being activated overnight chromabacterium biolaceum CV026 twice, is inoculated in by 2% access amount containing the 20 fresh LB of μ g/mL kanamycins
In meat soup, 150r/min shake culture 16-18h is mixed with the 100mL LB culture medium for containing 10 μm of ol/L C4-HSL, is put down
Plate is punched with the punch that diameter is 6mm after to be solidified, the re-suspension liquid of various concentration is added into hole, sees after 30 DEG C of culture 12h
It examines as a result, control group is sterile water.Testing result shows that Cortex Moutan extract is able to suppress the generation of its purpurin, shows the root bark of tree peony
Extract has quorum sensing inhibitory activity to reporting bacterial strain chromabacterium biolaceum CV026, and inhibiting rate reaches as high as 89.30%.
Wherein, C4-HSL is a kind of signaling molecule, can freely enter and leave the cell wall and cell membrane of mushroom, when its concentration reaches
It can be bacterium by the formation of starting related virulence factor expression and biomembrane in conjunction with endobacillary gene to after certain value
The key factor that external biological film is formed.
Influence of the Cortex Moutan extract to Pseudomonas fluorescens biofilm is measured, i.e., twice by Pseudomonas fluorescens activation
Afterwards, it is inoculated into fresh LB meat soup by 1% access amount, 200 μ L bacterium solutions and 50 μ L various concentrations is added in each hole of 96 orifice plates
Cortex Moutan extract makes its final concentration of 1.56-6.25mg/mL.Negative control is sterile LB meat soup.After 28 DEG C of culture 48h, remove
Culture solution in hole is cleaned 3-5 times with 250 μ L sterile purified waters, with the violet staining 5min of 250 μ L 0.4%, with sterile steaming
After distilled water is cleaned 3 times, it is placed in the dry 10min of 60 DEG C of thermostatic drying chambers, 96 orifice plates is taken out, 250 μ L 95% is added in each hole
Ethyl alcohol measures OD value with microplate reader after standing 10min under 595nm wavelength.The results show that Cortex Moutan extract can be effectively
Inhibit the biofilm of Pseudomonas fluorescens, inhibiting rate reaches as high as 71.74%.
The influence that measurement Cortex Moutan extract generates Pseudomonas mycoproteinase, that is, press 10% skimmed milk power and 1.5%
Agar powder makes defatted milk agar plate, 105 DEG C of sterilizing 15min.When culture medium is down to 40 DEG C or so, inverted plate, plate coagulates
After Gu, punched with the punch that diameter is 6mm, it is then past to be separately added into aseptic culture medium in the air, be activated overnight twice
Pseudomonas bacterium solution, bacterium solution+inhibitor and bacterium solution+C4-HSL, 28 DEG C of cultures for 24 hours, measure proteolysis loop diameter.As a result it shows
Show, Cortex Moutan extract can effectively inhibit the generation of Pseudomonas fluorescens extracellular protease, and inhibiting rate reaches as high as 59%.
Influence of the Cortex Moutan extract to fluorescent Pseudomonads collection and swimming is measured, i.e., various concentration root bark of tree peony crude extract is through going
Ionized water is resuspended, then after 0.22 μm of membrane filtration degerming, respectively with agar medium (1% egg that is cooled to 40 DEG C of clusters
White peptone, 0.5% sodium chloride, 0.5% agar and 0.5% glucose) and swimming agar medium (1% tryptone, 0.5%
Sodium chloride and 0.3% agar) it mixes, inverted plate, make the final concentration of 1.56-6.25mg/mL of Cortex Moutan extract in plate.It is past cold
But plate center is added 5 μ L and is activated overnight Pseudomonas bacterium solution twice, sterile wind drying, and 28 DEG C of constant temperature incubation 48h are seen
Examine Pseudomonas fluorescens migration situation.Crude extract is not added in negative control.As a result, it has been found that with the increasing of Cortex Moutan extract mass concentration
Add, the diameter of fluorescent Pseudomonads collection and swimming migration area is gradually reduced, and reaches the level of signifiance compared with the control group.Cluster fortune
Dynamic inhibiting rate is up to 60.71%, and the inhibiting rate of swimming reaches 40.15%.
Embodiment 2
The root bark of tree peony is dried in 30-40 DEG C of constant incubator, is ground into powder, obtains root bark of tree peony powder, by the root bark of tree peony powder with
The methanol solution of 75% (W/V) is mixed according to solid-liquid ratio 1:10, ultrasonic extraction 15min first, and then 3h is extracted in concussion, described super
The ultrasonic power that sound extracts is 200-300W, the condition that the concussion is extracted are as follows: 30-40 DEG C of temperature, concussion speed 150-180r/
min；Then removal residue is filtered, filtrate is collected；Obtain by the filtrate rotary evaporation, obtain crude extract；It is slightly mentioned described
Dehydrated alcohol is added in liquid and carries out alcohol precipitation, the alcohol precipitation time is 8h, wherein the volume ratio of the crude extract and the dehydrated alcohol is
1:10；It is filtered after alcohol precipitation, removes filter residue, filtrate is subjected to rotary evaporation, obtains root bark of tree peony extracting solution；By the root bark of tree peony
Extracting solution is freeze-dried, and the Cortex Moutan extract is obtained.
The Cortex Moutan extract dry powder of acquisition is resuspended through deionized water, crosses 0.22 μm of sterilised membrane filter degerming.It will remove
The Cortex Moutan extract re-suspension liquid that mass concentration after bacterium is 6.25mg/mL is with Aeromonas hydrophila (corrupt turbot is isolated)
It is detected, and measures it to the formation of Aeromonas hydrophila biofilm, protease generation, the influence of cluster and swimming.As a result
Show the Cortex Moutan extract to the inhibiting rate of Aeromonas hydrophila up to 54.32%, to Aeromonas hydrophila extracellular protein enzyme activity
The inhibiting rate of property is up to 42.3%, to the inhibiting rates of Aeromonas hydrophila crowd hazards and swimming respectively up to 46.4% and 22.81%.
Embodiment 3
The preparation method is the same as that of Example 1 for Cortex Moutan extract used in the present embodiment, and it is fresh-keeping to be used as aquatic products-turbot
Research, specific research method are as follows:
Fresh and alive turbot trash ice is freezed to death, the flesh of fish is taken out, is blotted with filter paper after sterile water wash clean, sterile fish block respectively at
1min (the root bark of tree peony is impregnated in produced Pseudomonas fluorescens bacterium solution (control group) and Pseudomonas fluorescens bacterium solution+Cortex Moutan extract
Extract-treated groups), it takes out use filter paper suck dry moisture immediately, packing, 4 DEG C of storages measure total number of bacterial colonies, the pH of the flesh of fish
Value, TVB-N value and TBA value.
As shown in Figure 2, the total number of bacterial colonies of Cortex Moutan extract processing group and control group is not significantly different, and illustrates the root bark of tree peony
There is no the growths for inhibiting Pseudomonas fluorescens for extract.
After fish sudden death, internal glycogen generates lactic acid through glycolysis, and declines the pH value of muscle, it is also possible to due to air
Middle CO2 and microbial metabolism generation CO2, which enters, forms H2CO3 in the flesh of fish, reduce so as to cause the pH value of the flesh of fish.With under freshness
Drop, protein are decomposed, and generate as the volatility alkali such as ammonia and trimethylamine composition compound, these substances are mostly alkalinity, finally
The pH value of the flesh of fish is caused to rise.From the figure 3, it may be seen that raised trend after first reducing is presented in pH value, and the root bark of tree peony mentions in two experimental groups
It takes the pH value of object processing group to be consistently lower than control group, to illustrate that the Cortex Moutan extract can delay microbial metabolism, makes it
Freshness is able to maintain the long period, and delays the decomposition of protein in the flesh of fish.
TBA (thiobarbituricacidα-) value: TBARS value (TBA value) is the effective of detection Oxidation of Fat and Oils product
Method, it is widely used in measurement lipid food, especially meat and the rancid degree of aquatic products fat oxidation, is to judge flesh of fish rouge
The important indicator of fat degree of oxidation.TBA value is bigger, and fat oxidation is more, as shown in Figure 4, the Cortex Moutan extract processing group
TBA value is significantly lower than control group, therefore the Cortex Moutan extract can inhibit oxidation fatty in the flesh of fish.
TVB-N (Volatile Base Nitrogen) value: total volatile basic nitrogen (TVB-N) be animal food in storage by
The collective effect of endogenous enzymes and bacterium in muscle, breaks down proteins and the alkaline nitrogen substance such as ammonia and amine for generating.
TVB-N value is bigger to illustrate that protein degradation is more, and flesh of fish corruption is also more serious.As shown in Figure 5, the Cortex Moutan extract can have
Effect inhibits the degradation of protein.
But work as Cortex Moutan extract as antistaling agent in use, tradition different with the Fresh Keeping Mechanics of traditional antistaling agent
Antistaling agent be mainly pass through inhibit bacterium growth achieve the purpose that it is fresh-keeping.And inhibitor is by interfering spoilage organisms deteriorative factor
The way of production, reduce its corrupt ability, reach fresh-keeping purpose, will not influence the growth of spoilage organisms, this fresh-keeping mechanism energy
The generation for enough preventing drug resistance is a kind of novel bio-preservative.
Embodiment 4
A kind of preparation method of composite preservative, the composite preservative apply a kind of aquatic products Pseudomonas fluorescens
Quorum sensing inhibitor prepare, the preparation method of the composite preservative pass through first evaluation judges the fresh of aquatic products
Degree, then determines the formula of the composite preservative according to the freshness of the aquatic products, in the aquatic products in the present embodiment
It is Spanish mackerel, is purchased from seafood market.
Specific step is as follows for the preparation method of the composite preservative:
1) Comprehensive Assessment of Spanish mackerel: carrying out the Spanish mackerel stripping and slicing that will be bought to Spanish mackerel, spare；Evaluate the evaluation by special training
Group (5 people), is integrated according to the evaluation form of 1 Spanish mackerel of table according to the color of Spanish mackerel block, smell, tissue morphology, tissue elasticity
Scoring, obtains comprehensive grading value, as shown in table 1, the highest of the comprehensive grading value be divided into 5 points and it is minimum be divided into 1 point, and press
The Spanish mackerel is divided into fresh, comparatively fresh and stale three ranks according to the comprehensive grading value；Due to stale rank
Spanish mackerel it is unsuitable edible, so only to fresh and two ranks of comparatively fresh Spanish mackerels add the composite preservative with into
Row Preservation Treatment；
The aquatic products are divided into fresh, comparatively fresh and stale three ranks according to the comprehensive grading value
Foundation are as follows: be divided into the corresponding comprehensive grading value of aquatic products of fresh rank as 4-5 points, including 4 points；It is divided into comparatively fresh
The corresponding comprehensive grading value of the aquatic products of rank is 3-4 point, including 3 points but do not include 4 points；It is divided into stale rank
The corresponding comprehensive grading value of aquatic products is lower than 3 points, does not include 3 points.
By above-mentioned evaluation, Spanish mackerel comprehensive grading value is 3.5 points in the present embodiment, belongs to comparatively fresh rank.
Table 1: Spanish mackerel block evaluation form
2) preparation of composite preservative raw material and solvent: the composite preservative raw material is constituted are as follows: the quorum sensing suppression
Preparation, chitosan and tea polyphenols；Constitute the composite preservative solvent are as follows: 0.1mol/L acetum and 0.2mol/L sodium chloride
The mixed solution of solution；
Chitosan and tea polyphenols belong to traditional antistaling agent, are able to suppress the growth of spoilage organisms；Wherein, chitosan has good
Good film forming characteristics and strong antibacterial characteristics, antifungal mechanism: the chitosan of small molecule can be with penetrating cell wall and cell membrane
Into cell interior, the duplication of DNA and the synthesis of protein are influenced, and macromolecular chitosan is then in cell appearance at macromolecule
Film hinders the metabolism of microorganism, finally makes bacterial death, and the groups such as amino in chitosan and metal ion-chelant are made
With the intake of inhibition microorganism microelement reduces microelement in conjunction with the nutriment necessary to growing, influences microorganism
Metabolism；Tea polyphenols have extensive bacteriostasis property, to gram-positive cocci, produce bacillus, Gram-negative, aerobic ball
Bacterium and bacillus, facultative Bacteroides nodosus, coccus and apparent inhibiting effect is all had with regard to bacillus.Antifungal mechanism: the phenol in tea polyphenols
Hydroxyl occurs in conjunction with hydrogen with the amino of the protein molecule in bacterium or carboxyl, and with protein hydrophobic binding can also occur for phenyl ring,
This multiple spot, which combines, causes microbial metabolism to be obstructed, and tea polyphenols destroy the structure of bacterial cell membrane.
The selection of the composite preservative is not arbitrary, and the selected composite preservative solvent can guarantee shell
The abundant dissolution of glycan.Dissolution conditions are: with magnetic agitation about half an hour, most 2 hours, not having to heating, chitosan is complete
Dissolution, stands overnight and does not have precipitating, stability is good.
3) determination of composite fresh-keeping agent prescription: according to step (1) to the Comprehensive Assessment of aquatic products as a result, Spanish mackerel is divided
For comparatively fresh rank；Aquatic products after death, the variation of quality can be divided into 4 stages: it is stiff → solution deadlock → self-dissolving → corruption
It loses.The rotten of aquatic products is mainly since the mass propagation of microorganism causes ruining for quality, mainly in self-dissolving stage and corruption
Stage.So aquatic products have been in the primary stage of self-dissolving stage or putrefaction stage, water when aquatic products are in comparatively fresh rank
Micro organism quantity in product has begun a large amount of procreations, in order to reach good fresh-keeping effect, it is necessary to improve the compound guarantor
The total content of chitosan and tea polyphenols in fresh dose with bactericidal effect, makes the total content of the chitosan and tea polyphenols not
Lower than the content of Cortex Moutan extract (the quorum sensing inhibitor)；Matching for composite preservative raw material described in the present embodiment is set
Side are as follows: it is by weight, 10 parts of the quorum sensing inhibitor, chitosan 4-5 parts described, tea polyphenols 3-6 parts described, it is described multiple
Close 30 parts of antistaling agent solvent.
But if quality is in stiff and solves the stiff stage, in aquatic products when aquatic products are in comparatively fresh rank
Microorganism is a large amount of not yet to be bred, and in the aquatic products composite preservative used at this time, is correspondinglyd increase the quorum sensing and is inhibited
Agent can reach good fresh-keeping effect, although the inhibitor can not reduce the quantity of flora, Microflora is kept
Good fresh-keeping level can be reached in existing level.In this case, the composite preservative raw material can be set
Formula are as follows: by weight, 10 parts of the quorum sensing inhibitor, 4 parts of the chitosan, 3 parts of the tea polyphenols are described compound
30 parts of antistaling agent solvent.
Claims (6)
1. a kind of preparation method of the quorum sensing inhibitor of aquatic products Pseudomonas fluorescens, which is characterized in that the preparation side
Organic solvent is added using the root bark of tree peony as raw material in method, shakes extractive technique using ultrasonic wave added, obtains Cortex Moutan extract, the root bark of tree peony
Extract includes gallic acid and Paeonol；The Cortex Moutan extract is quorum sensing inhibitor, and the quorum sensing inhibits
Agent can reduce the corrupt ability of Pseudomonas fluorescens, be used for the exploitation of preservation of fishery and composite preservative.
2. a kind of preparation method of the quorum sensing inhibitor of aquatic products Pseudomonas fluorescens according to claim 1, special
Sign is that the preparation method includes root bark of tree peony pretreatment, ultrasonic wave added concussion extraction, alcohol precipitation, freeze-drying step.
3. a kind of preparation method of the quorum sensing inhibitor of aquatic products Pseudomonas fluorescens according to claim 2, special
Sign is, the preparation method specifically: using the root bark of tree peony as raw material, the root bark of tree peony is pre-processed, obtains root bark of tree peony powder, addition has
Ultrasonic wave added concussion is carried out after solvent to extract, filters and collect filtrate, the filtrate is subjected to rotary evaporation, acquisition slightly mentions
Then liquid is added dehydrated alcohol and carries out alcohol precipitation, filter removal filter residue, filtrate rotary evaporation is obtained root bark of tree peony extracting solution, will be described
Root bark of tree peony extracting solution is freeze-dried, and Cortex Moutan extract is obtained.
4. a kind of preparation method of the quorum sensing inhibitor of aquatic products Pseudomonas fluorescens according to claim 3, special
Sign is, the specific steps of the preparation method:
(1) root bark of tree peony pre-processes: the root bark of tree peony being dried in 30-40 DEG C of constant incubator, is then crushed, root bark of tree peony powder is obtained；
(2) ultrasonic wave added concussion is extracted: will rapid (1) treated the root bark of tree peony powder and the organic solution according to solid-liquid ratio 1:6-
1:10 is mixed, first ultrasonic extraction 10-15min, and then 3-6h is extracted in concussion, is filtered removal residue, is collected filtrate；It is described
Organic solution is any one in methanol solution, ethanol solution and acetone soln, and the concentration of the methanol solution is 60%-
90%, the concentration of the ethanol solution is 50%-90%, and the concentration of the ethanol solution is 60%-80%；
(3) alcohol precipitation: the filtrate that step (2) are collected carries out rotary evaporation, obtains crude extract；It is added in the crude extract
Dehydrated alcohol carries out alcohol precipitation, and the alcohol precipitation time is 8-14h, wherein the volume ratio of the crude extract and the dehydrated alcohol is 1:5-
10；It is filtered after alcohol precipitation, removes filter residue, filtrate is subjected to rotary evaporation, obtains root bark of tree peony extracting solution；
(4) it is freeze-dried: the root bark of tree peony extracting solution that step (3) obtain being freeze-dried, the Cortex Moutan extract is obtained.
5. a kind of preparation method of the quorum sensing inhibitor of aquatic products Pseudomonas fluorescens according to claim 4, special
Sign is, in step (2), the ultrasonic power of the ultrasonic extraction is 200-300W, the condition that the concussion is extracted are as follows: temperature
30-40 DEG C, concussion speed 150-180r/min.
6. a kind of preparation method of composite preservative, the composite preservative includes quorum sensing inhibitor, which is characterized in that institute
The preparation method for stating composite preservative first determines whether the freshness of aquatic products, is then determined according to the freshness of the aquatic products
The formula of the composite preservative, specific step is as follows for the preparation method of the composite preservative:
1) Comprehensive Assessment of aquatic products: carrying out comprehensive score according to the color of aquatic products, smell, tissue morphology, tissue elasticity, will
The aquatic products are divided into fresh, comparatively fresh and stale three ranks；
2) preparation of composite preservative raw material and solvent: the composite preservative raw material is constituted are as follows: the quorum sensing inhibitor,
Chitosan and tea polyphenols；Constitute the composite preservative solvent are as follows: 0.1mol/L acetum and 0.2mol/L sodium chloride solution
Mixed solution；
3) determination of composite fresh-keeping agent prescription: according to step (1) to the Comprehensive Assessment of aquatic products as a result, when the aquatic products are drawn
When being divided into fresh rank, the formula of the composite preservative are as follows: by weight, 10 parts of the quorum sensing inhibitor, described
1-2 parts of chitosan, tea polyphenols 1-2 parts described, 30-60 parts of the composite preservative solvent；When the aquatic products be divided into compared with
When fresh rank, the formula of the composite preservative are as follows: by weight, 10 parts of the quorum sensing inhibitor, the shell are poly-
3-5 parts of 4-5 parts sugared, the described tea polyphenols, 20-30 parts of the composite preservative solvent,
The quorum sensing inhibitor is Cortex Moutan extract, and the Cortex Moutan extract includes gallic acid and Paeonol.
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| CN107183146A (en) * | 2017-06-15 | 2017-09-22 | 中国农业大学 | Quorum quenching enzymes and bacteriocin joint anti-rot and fresh-keeping method |
| CN110663764A (en) * | 2019-11-20 | 2020-01-10 | 晋中学院 | Fruit and vegetable fresh-keeping coating containing moutan bark and preparation method thereof |
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