CN105494626A - Bacteriostatic protective agent as well as preparation method and application - Google Patents
Bacteriostatic protective agent as well as preparation method and application Download PDFInfo
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- CN105494626A CN105494626A CN201510868520.9A CN201510868520A CN105494626A CN 105494626 A CN105494626 A CN 105494626A CN 201510868520 A CN201510868520 A CN 201510868520A CN 105494626 A CN105494626 A CN 105494626A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C3/00—Preservation of milk or milk preparations
- A23C3/08—Preservation of milk or milk preparations by addition of preservatives
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C2240/00—Use or particular additives or ingredients
- A23C2240/15—Use of plant extracts, including purified and isolated derivatives thereof, as ingredient in dairy products
Abstract
The invention discloses a bacteriostatic protective agent as well as a preparation method and an application thereof. The preparation method comprises steps as follows: Phellinus baumii fruiting bodies are added to water after being crushed, and boiling water extraction is performed; an obtained mixture is filtered, and residues and an extract liquid are obtained; after the extract liquid is concentrated, a supernatant is taken, and a concentrated liquid is obtained; ethyl alcohol is added to the obtained concentrated liquid, standing is performed, precipitates are collected, water is added for dissolution, a supernatant is taken, and the bacteriostatic protective agent is obtained. The preparation method is convenient to operate and can better meet the requirement of industrial production. The bacteriostatic protective agent prepared with the preparation method is derived from edible fungi, is safe and reliable, has broad spectrum bacteriostatic performance, has no inhibition effect on growth of probiotics, increases the survival rate of beneficial microorganisms while inhibiting harmful microorganisms and has broad market prospect and application value.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of antibacterial protective agent and preparation method and application.
Background technology
At present, the population of 2% is worldwide had to suffer from food origin disease every year, 25% microbial contamination of World of Food supply.Although China is for alimentary codex more and more opening, be then more and more stricter to food safety requirements.Along with the raising of people's level of consumption and consciousness, food that is natural, safety becomes the major consumers trend of consumer.And natural bacteriostatic composition mainly divides animal sources antipathogenic composition, Natural antibacterial constituents from plant origin and microbial source antipathogenic composition.Due to microbial source bacteriostatic agent there is easy acquisition, source is wide, cost is low, be convenient to expand the many advantages such as cultivations, one that becomes that food preservative in recent years develops important direction.
The antibacterial mode of dairy products comprises the endogenous composition effect of dairy products, as the organic acid etc. that there is the antibacterial peptide of broad-spectrum antibacterial characteristic, lactoferrin and hydrolysate thereof breast iron element, fermentable produces, scholar's research is separately had to find, the fermentation such as lactobacillus delbruockii subspecies bulgaricus, streptococcus thermophilus probiotics can be substituted in the most putrefactivebacterias grown in enteron aisle, reduce easy the to be corrupt metabolin produced by spoilage organisms, extend the shelf-life of dairy products.
Except the antibacterial system that product is endogenous, add to product from other source extraction natural bacteriostatic compositions and also become dairy products corrosion-resistant important means.This type of external source antipathogenic composition mainly comprises antibacterial class enzyme preparation, natural essential oil, Polyphenols etc.Be that nisin (Nisin), Nisin can control harmful bacteria effectively as studied thorough, the most most widely used at present, particularly heat-resisting bacillus and the Growth and reproduction of clostridium, to reach the object extending different dairy products shelf life.But, compared with the antibacterial system of dairy products endogenous, the external source antipathogenic composition of interpolation exist sphere of action little, do not have a drawback of broad-spectrum antibacterial.And for ensureing the security of food, by the antibacterial substance limitednumber specifying can make an addition in dairy products, especially dairy products are because it has the characteristics such as certain moisture and nutrition, microbial contamination and putrid and deteriorated is easily subject in processing and storage process, Nisin is because having the features such as consumption is few, antibacterial ability is strong, it is the antibacterial additive that uniquely can be used for dairy products, other component additive agents may produce certain toxicity to human body, limit the exploitation of the antibacterial substance of dairy products to a certain extent, for the dairy products developing long shelf life bring certain difficulty.So it is wide that present stage lacks a kind of sphere of action, tool broad spectrum antibacterial, efficiently, easily obtain, can large-scale production be applied to the external source antipathogenic composition of dairy products.
Summary of the invention
The technical problem to be solved in the present invention also exists for the current external source antipathogenic composition for adding in dairy products that not have broad-spectrum antibacterial, a sphere of action little; the defects such as the kind of safety non-toxic is few can be guaranteed; the invention provides a kind of antibacterial protective agent and its preparation method and application; this antibacterial protective agent derives from microorganism; wide and the tool broad-spectrum antibacterial of sphere of action, natural, safety; preparation method is simple and reliable, is applicable to large-scale production and applies in dairy market.
For solving the problems of the technologies described above, one of technical scheme provided by the invention is, a kind of antibacterial protectant preparation method, and described preparation method comprises the following steps:
1) be added to the water after Phellinus (Phellinusbaumii) fructification being pulverized, carry out extracting in boiling water;
2) by step 1) filtration of gained mixture, obtain residue and extract respectively;
3) by step 2) after gained extract is concentrated, gets supernatant, obtain concentrate;
4) in step 3) add ethanol in gained concentrate, leave standstill, collecting precipitation, is dissolved in water, and gets supernatant, obtains antibacterial protective agent.
Step 1 of the present invention) be: be added to the water after Phellinus (Phellinusbaumii) fructification is pulverized, carry out extracting in boiling water.Wherein, described Phellinus fructification is that Phellinus (Phellinusbaumii) cultivating bacterial spawn obtains.Described cultivation condition is conventional, as long as can obtain Phellinus fructification by Phellinus bacterial classification.Phellinus is as a kind of traditional medicinal fungi of preciousness, and it is recorded in the earliest " property of medicine opinion ", and Traditional Chinese Medicine thinks that Phellinus taste is sweet pungent, is a kind of edible and medicinal fungi that in the field of food of international approval, one after another is the highest.At present, the biologically active for Phellinus fructification water extract has many reports, as anti-oxidant, anti-ageing, improve many biological effects such as immunity, anti-sudden change, anti-fibrosis, anti-pneumonia.Described Phellinus fructification preferably before pulverizing through super-dry, described drying is conventional drying methods.Described pulverizing is conventional, preferably for using pulverizer to pulverize fructification, more preferably for Phellinus fruit body powder is broken to about 0.5 ~ 3cm
3particle, best for Phellinus fructification is crushed into about 1cm
3particle.Described water is conventional, and being preferably deionized water, is more preferably the deionized water of 10 ~ 25 times of volumes, and be the deionized water of 20 ~ 15 times of volumes best, described volume multiple is relative to Phellinus fructification volume.The extraction time of described extracting in boiling water is conventional, and being preferably 1 ~ 3 hour (h) be more preferably 1.5 ~ 2.5 hours, is 2 hours best.
Step 2 of the present invention) be: by step 1) filtration of gained mixture, obtain residue and extract respectively.Wherein, described filtration is conventional, preferably uses 4 layers of gauze to filter.
Preparation method of the present invention is in step 3) before preferably also comprise step 2 ') by step 2) and described in residue again carry out extracting in boiling water, by gained extract and step 2) gained extract merges.
Step 3 of the present invention) be: by step 2) after gained extract is concentrated, gets supernatant, obtain concentrate.Wherein, described simmer down to routine is preferably under a reduced pressure, use Rotary Evaporators reduced pressure concentration is carried out to extract, be more preferably in the process of reduced pressure concentration control temperature not higher than 50 DEG C.Described supernatant is that conventional method obtains, preferably for extract concentrated after obtain after centrifugal segregation precipitation, more preferably for extract concentrated after obtain through 4000r/min centrifugal 10min removal precipitation.
Step 4 of the present invention) be: in step 3) add ethanol in gained concentrate, leave standstill, collecting precipitation, is dissolved in water, and gets supernatant, obtains antibacterial protective agent.Wherein, described ethanol is conventional, and preferably for concentration of alcohol after adding ethanol is 60% ~ 95%, described percentage is percent by volume.The described time left standstill is conventional, and being preferably 12 ~ 30 hours, is 24 hours best, and dwell temperature is conventional, is preferably room temperature.The method of described collecting precipitation is conventional, and preferably after leaving standstill, pour out supernatant fraction, remainder is through the centrifugal 10min collecting precipitation of 4000r/min.Described supernatant adquisitiones and step 3) described in supernatant adquisitiones identical.
Preferably, preparation method's step 4 of the present invention) described in supernatant namely obtain antibacterial protective agent through freeze drying.
Preparation method of the present invention is preferably in step 4) after also comprise step 5), by step 4) the antibacterial protective agent of gained dialyses, obtain the solution after dialysing, be antibacterial protective agent.Wherein, described dialysis is conventional, and dialysis temperature is preferably 4 DEG C, and dialysis time is conventional, and being preferably 0 ~ 24 hour, is more preferably 4 ~ 24 hours, is 12 ~ 24 hours best.
Preferably preparation method of the present invention is in step 5) namely solution after described dialysis obtain antibacterial protective agent through freeze drying.Preferably also comprise step 6 after abovementioned steps): by step 5) described in antibacterial protective agent add water and dissolve, obtain the antibacterial protective agent solution of variable concentrations, described antibacterial protective agent solution concentration is preferably 0.5mg/mL ~ 100mg/mL.
For solving the problems of the technologies described above, two of technical scheme provided by the invention is, a kind of antibacterial protective agent, and described antibacterial protective agent is prepared by aforementioned preparation process and obtains.
In the present invention, when aforementioned preparation process preferably includes step 6) time, i.e. the corresponding antibacterial protective agent solution preparing variable concentrations.Described antibacterial protective agent solution concentration is preferably 0.5mg/mL ~ 100mg/mL.The operative temperature of described antibacterial protective agent or described antibacterial protective agent solution is conventional, is preferably 4 ~ 37 DEG C.
For solving the problems of the technologies described above, three of technical scheme provided by the invention is, antibacterial protective agent is in the application of dairy products.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can be combined, obtain the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: this invention exploits a kind of antibacterial protectant preparation method, this preparation method is easy and simple to handle, can meet the demand of suitability for industrialized production preferably.The antibacterial protective agent utilizing this preparation method to prepare derives from edible fungi, safe and reliable, there is broad-spectrum antibacterial, Escherichia coli can be suppressed in various degree, staphylococcus aureus, salmonella, bacillus subtilis, the growth of the harmful bacterias such as Listeria monocytogenes, another to brewer's yeast, white mould, the fungal microbes such as mould also have certain inhibitory action, but to lactobacillus bulgaricus, streptococcus thermophilus, the unrestraint effect of the beneficial bacterium growths such as Bifidobacterium, so, antibacterial protective agent of the present invention protects beneficial microorganism survival rate at suppression harmful microbe simultaneously, this is particularly important in dairy products, so, there is wide market using value in antibacterial protective agent provided by the invention.
Accompanying drawing explanation
Fig. 1 is that the antibacterial protective agent solution of variable concentrations is to the effect of brewer's yeast growth inhibition.
Fig. 2 is that the antibacterial protective agent solution of variable concentrations is to the effect of pasteurization milk flora sum.
Detailed description of the invention
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Room temperature involved in the present invention is 0 ~ 40 DEG C.
The antibacterial protective agent of embodiment 1 is to the suppression of Gram-negative bacteria
After Phellinus cultivating bacterial spawn obtains fructification, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 10 times of volumes, extracting in boiling water 3h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Added by ethanol in concentrate, be 95%, after left at room temperature 24h, pour out supernatant fraction to ethanol final concentration, the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out freeze drying, gets freeze-dry extract and be antibacterial protective agent, be made into 100mg/mL solution, be antibacterial protective agent solution.
Prepared by solid broth bouillon (i.e. broth agar culture medium): get nutrient broth 1.8g, agar 1.2g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Prepared by liquid broth: get nutrient broth 1.8g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Get Gram-negative bacteria Escherichia coli (Escherichiacoli), after bacillus ceylonensis A (ShigellaCastellani), salmonella (salmonella) carry out bacterial classification recovery respectively, thalline is inoculated in shaken cultivation 18h at liquid broth 37 DEG C, regulates after cultivating bacterial classification concentration in bacterium liquid to be 10
5~ 10
6individual/mL, get 1mL regulate after bacterium liquid mix with the broth agar culture medium be in a liquid state after down in culture dish; After culture medium (flat board) solidifies, the antibacterial protective agent solution of 20 μ L100mg/mL and 20 μ L sterilized waters are dropped in respectively and is mixed with on harmful microbe culture medium, with 20 μ L sterilized waters as a control group (blank); Measure dull and stereotyped inhibition zone size after quiescent culture 36h at 37 DEG C, the large I of inhibition zone reflects antibacterial protective agent bacteriostasis, and inhibition zone is larger, bacteriostasis stronger (as shown in table 1).When the antibacterial protective agent of the present invention exists, be mixed with harmful microbe flat board and can observe inhibition zone, the antibacterial protective agent of the present invention of can reaching a conclusion thus effectively can suppress Gram-negative bacteria.
Table 1100mg/mL activity suppresses situation to Gram-negative bacteria
The antibacterial protective agent of embodiment 2 is to the suppression of gram-positive bacteria
After Phellinus cultivating bacterial spawn obtains fructification, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 25 times of volumes, extracting in boiling water 1h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Added by ethanol in concentrate, be 65%, after left at room temperature 24h, pour out supernatant fraction to ethanol final concentration, the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out dialysis 24h, and after dialysis, solution carries out freeze drying, gets freeze-dry extract and is antibacterial protective agent, be made into 0.5mg/mL solution and be antibacterial protective agent solution.
Medium preparing is with reference to embodiment 1.
Get after gram-positive bacteria Listeria monocytogenes (Listeriamonocytogenes), staphylococcus aureus (Staphylococcusaureus), micrococcus luteus (Micrococcusluteus) and bacillus subtilis (Bacillussubtilis) carry out bacterial classification recovery respectively, thalline is inoculated in shaken cultivation 18h at liquid broth 37 DEG C, regulates after cultivating bacterial classification concentration in bacterium liquid to be 10
5~ 10
6individual/mL, get 1mL regulate after bacterium liquid mix with the broth agar culture medium be in a liquid state after down in culture dish; After culture medium solidifying, the antibacterial protective agent solution of 20 μ L0.5mg/mL is dropped in and is mixed with on harmful microbe culture medium, with 20 μ L sterilized waters as a control group; Dull and stereotyped inhibition zone size is measured after quiescent culture 36h, inhibition zone size reaction protective agent bacteriostasis (as shown in table 2) at 37 DEG C.When the antibacterial protective agent of the present invention exists, be mixed with harmful microorganism staphylococcus aureus, the flat board of bacillus subtilis can observe inhibition zone.
Table 20.5mg/mL activity suppresses situation to gram-positive bacteria
The antibacterial protective agent of embodiment 3 is to the suppression of gram-positive bacteria
After Phellinus cultivating bacterial spawn obtains fructification, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 15 times of volumes, extracting in boiling water 2h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Being added by ethanol in concentrate, is 80% to ethanol final concentration, after left at room temperature 24h, and the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out dialysis 12h, and after dialysis, solution carries out freeze drying, gets freeze-dry extract and is antibacterial protective agent, be made into 20mg/mL solution and be antibacterial protective agent solution.
Medium preparing is with reference to embodiment 1 preparation method.
Get after gram-positive bacteria Listeria monocytogenes, staphylococcus aureus, micrococcus luteus and bacillus subtilis carry out bacterial classification recovery respectively, thalline is inoculated in shaken cultivation 18h at liquid broth 37 DEG C, regulates after cultivating bacterial classification concentration in bacterium liquid to be 10
5~ 10
6individual/mL, get 1mL regulate after bacterium liquid mix with the broth agar culture medium be in a liquid state after down in culture dish; After culture medium solidifying, 20 μ L20mg/mL (different in concentration and table 3 herein, which is correct concentration to ask inventor to confirm) antibacterial protective agent solution is dropped in and is mixed with on harmful microbe culture medium, with 20 μ L sterilized waters as a control group; Dull and stereotyped inhibition zone size is measured after quiescent culture 36h, inhibition zone size reaction protective agent bacteriostasis (as shown in table 3) at 37 DEG C.When the antibacterial protective agent of the present invention exists, be mixed with harmful microbe flat board and can observe inhibition zone.The antibacterial protective agent of the known the present invention of integrated embodiment 2 and 3 effectively can suppress gram-positive bacteria.
Table 320mg/mL activity suppresses situation to gram-positive bacteria
The antibacterial protective agent of embodiment 4 is to the suppression of mould
After Phellinus cultivating bacterial spawn obtains fructification, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 10 times of volumes, extracting in boiling water 1.5h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Being added by ethanol in concentrate, is 75% to ethanol final concentration, after left at room temperature 24h, and the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out dialysis 4h, and after dialysis, solution carries out freeze drying, and get freeze-dry extract and be antibacterial protective agent, wiring solution-forming is antibacterial protective agent solution.
Solid PDA medium is prepared: get PDA agar powder 4g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Get mould (Penicilliumcandidum), white mould bacterial strain (Geotrichumcandidum) respectively, carry out activating and carry out solid expansion and cultivate, antibacterial protective agent solution after sterilizing (121 DEG C of autoclaving 15min) is mixed with PDA culture medium, antibacterial protective agent final concentration is made to be respectively 0.1mg/mL, 1mg/mL, 10mg/mL, pour culture dish into, after flat board (culture medium) solidifies, with the PDA solid medium of blank as a control group (blank), picking mould respectively, white mould bacterium is inoculated in plate center, cultivate after 7 days for 30 DEG C, measure mould respectively, the diameter of white mould in flat board, be negative correlation (result is as shown in table 4) to the inhibitory action of fungus growth and fungus growth loop diameter size.When antibacterial protective agent of the present invention exists, fungus growth loop diameter reduces relative to blank; so the antibacterial protective agent of the present invention can the growth of effective mould fungus inhibition; and fungus growth loop diameter increases along with the antibacterial protective agent concentration of the present invention and reduces, the larger fungistatic effect of antibacterial protective agent concentration is better as can be seen here.
The antibacterial protective agent of table 4 is to the inhibitory action of mould
The antibacterial protective agent of embodiment 5 is to the suppression of mould
After Phellinus cultivating bacterial spawn obtains fructification, fructification is carried out drying, dried fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 10 times of volumes, extracting in boiling water 1.5h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Being added by ethanol in concentrate, is 75% to ethanol final concentration, after left at room temperature 24h, and the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out dialysis 4h, and after dialysis, solution carries out freeze drying, and get freeze-dry extract and be antibacterial protective agent, wiring solution-forming is antibacterial protective agent solution.
Carry out the mould Inhibition test identical with embodiment 4; when finding that antibacterial protective agent of the present invention exists, fungus growth loop diameter reduces relative to blank, obtains the conclusion identical with embodiment 4: the antibacterial protective agent of the present invention can the growth of effective mould fungus inhibition.
The antibacterial protective agent of embodiment 6 is to the suppression of yeast
After Phellinus cultivating bacterial spawn obtains fructification, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 20 times of volumes, extracting in boiling water 2h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Added by ethanol in concentrate, be 95%, after left at room temperature 12h, pour out supernatant fraction to ethanol final concentration, the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out dialysis 24h, and after dialysis, solution carries out freeze drying, and get freeze-dry extract and be antibacterial protective agent, wiring solution-forming is antibacterial protective agent solution.
Get brewer's yeast bacterial classification (Saccharomycescerevisiae) to bring back to life; saccharomyces cerevisiae after picking activation expands to be cultivated; and get 180 μ L yeast fermentation broths and mix with the antibacterial protective agent dissolution homogeneity of 20 μ L; be placed in bioscreen10*10 orifice plate (purchased from Bioscreen company of Finland); antibacterial protective agent final concentration in mixture is made to be respectively 4mg/mL, 20mg/mL, 40mg/mL; using water as blank; 30 DEG C of concussions continuously; measure the light absorption value of wide wavelength and 600nm; interval 20min reading, METHOD FOR CONTINUOUS DETERMINATION 2000min.Yeast concentration is directly proportional to light absorption value; the light absorption value recorded is as shown in Fig. 1 and table 5; obtain antibacterial protective agent fungistatic effect of the present invention as follows: deposit in case at antibacterial protective agent of the present invention; light absorption value is little; saccharomycete concentration is low; so antibacterial protective agent of the present invention can significantly suppress brewer's yeast to grow, and the larger antibacterial protective agent fungistatic effect of concentration is better.
Growth curve of yeast light absorption value under the effect of table 5 variable concentrations bacteriostatic agent
The antibacterial protective agent of embodiment 7 is to the effect of pasteurization milk flora sum
After Phellinus cultivating bacterial spawn obtains fructification, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 10 times of volumes, extracting in boiling water 2.5h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Added by ethanol in concentrate, be 90%, after left at room temperature 30h, pour out supernatant fraction to ethanol final concentration, the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out freeze drying, gets freeze-dry extract and be antibacterial protective agent, wiring solution-forming is antibacterial protective agent solution.
Antibacterial protective agent solution after sterilizing is mixed with fresh milk; the final concentration of antibacterial protective agent in cow's milk is made to be respectively 0mg/mL, 0.5mg/mL, 1mg/mL, 5mg/mL; pasteurize is carried out after homogeneous; preserve at being placed in 4 DEG C; total number of bacteria in cow's milk is measured, METHOD FOR CONTINUOUS DETERMINATION 2 weeks (result is as Suo Shi Fig. 2 and table 6) every 24h.Deposit in case there being antibacterial protective agent of the present invention; fresh-keeping cow's milk flora sum is lower than there is not antibacterial protectant control group of the present invention; visible, antibacterial protective agent of the present invention effectively can control preservation milk Ruzhong flora sum, plays the effect of Shelf-life.And along with the raising of antibacterial protectant concentration of the present invention, the preservation milk Ruzhong flora sum preserving same time is lower, so the higher fungistatic effect of antibacterial protective agent concentration is better.
Flora sum change under the effect of table 6 variable concentrations bacteriostatic agent
The antibacterial protective agent of embodiment 8 is to the effect of beneficial bacterium
After the fructification obtain Phellinus cultivating bacterial spawn, fruit body powder is broken to about 1cm
3particle, in the fruit body powder of drying minces, add the deionized water of 15 times of volumes, extracting in boiling water 2h, filter residue carries out second extraction; Merge extracted twice liquid and carry out reduced pressure concentration, the centrifugal 10min of 4000r/min removes precipitation; Added by ethanol in concentrate, be 60%, after left at room temperature 24h, pour out supernatant fraction to ethanol final concentration, the centrifugal 10min collecting precipitation of 4000r/min, sediment fraction adds deionized water dissolving, and the centrifugal 10min of 4000r/min removes insoluble matter, gets supernatant; Supernatant is carried out dialysis 24h, and after dialysis, solution carries out freeze drying; Get Phellinus water-soluble extractive and be antibacterial protective agent, use deionized water to dissolve, be antibacterial protective agent solution.
Solid MRS culture medium is prepared: get MRS powder (being purchased from German Merck company) 6.82g, agar 1.2g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Solid M17 culture medium is prepared: get M17 powder (being purchased from German Merck company) 4.25g, agar 1.2g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Liquid MRS culture medium is prepared: get MRS powder (being purchased from German Merck company) 6.82g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Liquid M17 culture medium is prepared: get M17 powder (being purchased from German Merck company) 4.25g, add 100mL deionized water dissolving, after 121 DEG C of sterilizing 20min, stand-by.
Get lactobacillus bulgaricus (Lactobacillusbulgaricus), streptococcus thermophilus (Streptococcusthermophilus) and Bifidobacterium (Bifidobacterium) three kinds of useful bacterial strains respectively, recovery, after lactobacillus bulgaricus, Bifidobacterium use liquid MRS culture medium, streptococcus thermophilus use liquid M17 culture medium expansion cultivation, regulating and cultivating cell concentration in rear bacterium liquid is 10
5~ 10
6cfu/mL, get 1mL regulate after bacterium liquid mix with the solid MRS culture medium (solid M17 culture medium, according to bacterial classification select) be in a liquid state after down in culture dish; After culture medium (flat board) solidifies; 20 μ L concentration are respectively the antibacterial protective agent solution of 1mg/mL, 10mg/mL, 50mg/mL to be dropped on culture medium; after suitable condition of culture 48h; measure antibacterial circle diameter; result is as shown in table 7: do not produce visible inhibition zone for lactobacillus bulgaricus, streptococcus thermophilus after adding the antibacterial protective agent of the present invention; only on Bifidobacterium flat board, observe less inhibition zone compared with Gao Shicai in concentration, thus antibacterial protective agent of the present invention to beneficial bacterium without remarkable inhibitory action.
The antibacterial protective agent of table 7 is to the effect of beneficial bacterium
The present invention adopts boil water extraction method, antipathogenic composition in Phellinus fructification is extracted, and first Application is in the research for the antibacterial aspect of dairy products, the present invention have chosen harmful bacteria common and representative in dairy products, fungi as antibacterial object, find Phellinus fructification water extract by research, the fresh-keeping harmful microorganism of fresh milk grows can effectively to suppress Escherichia coli, staphylococcus aureus, salmonella, bacillus subtilis, Listeria monocytogenes, brewer's yeast, white mould, mould etc. to be unfavorable within the scope of 0.5 ~ 100mg/mL activity; Within the scope of 0 ~ 50mg/mL activity; to lactobacillus bulgaricus, streptococcus thermophilus and Bifidobacterium beneficial bacterium without remarkable inhibitory action; therefore can be understood as; Phellinus water extract of the present invention not damaging harmful microbe growth in effective suppression dairy products under beneficial microorganism prerequisite, can be applied to the exploitation of dairy products as antibacterial protective agent.
Comparative example 1Nisin and the antibacterial protective agent of the present invention are to the effect of different flora
Culture medium preparation is with reference to embodiment 1, embodiment 4 and embodiment 8;
Get lactobacillus bulgaricus, streptococcus thermophilus, staphylococcus aureus, bacillus subtilis and Escherichia coli respectively; After carrying out bacterial classification recovery respectively, thalline is inoculated in fluid nutrient medium (liquid broth, liquid MRS culture medium, liquid M17 culture medium; Select according to cultivated bacterial classification, choice criteria is with embodiment 1 and 8) shaken cultivation 18h at 37 DEG C, to regulate after cultivating bacterial classification concentration in bacterium liquid to be 10
5~ 10
6individual/mL, gets 1mL and regulates rear bacterium liquid and the solid medium (broth agar culture medium, solid MRS culture medium, the solid M17 culture medium that are in a liquid state; According to cultivated bacterial classification select, choice criteria is with embodiment 1 and 8) mix after down in culture dish; After culture medium (flat board) solidifies, be that the antibacterial protective agent solution of 0.5mg/mL and 1mg/mL (obtaining according to the method preparation in embodiment 2) and Nisin solution drop in respectively and is mixed with on harmful microbe culture medium by 20 μ L concentration; Measure following (table 8): the Nisin of dull and stereotyped inhibition zone size result at 37 DEG C after quiescent culture 36h to Escherichia coli (Gram-negative bacteria) without inhibition zone, unrestraint effect, and protective agent of the present invention there is inhibitory action to Escherichia coli.
Table 8Nisin solution and the antibacterial protective agent of the present invention are to the effect of Gram-positive and negative bacterium
Operation according in same embodiment 4: get mould, white mould bacterial strain respectively, carries out activating and carries out solid expansion and cultivate, antibacterial protective agent solution (obtaining according to the method preparation in embodiment 4) after sterilizing and Nisin solution are mixed with PDA culture medium respectively, antibacterial protective agent and Nisin final concentration is made to be respectively 0.1mg/mL, 1mg/mL, pour culture dish into, after culture medium solidifying, with the PDA solid medium of blank as a control group (blank), picking mould respectively, white mould bacterium is inoculated in plate center, cultivate after 7 days for 30 DEG C, measure mould respectively, the diameter of white mould in flat board, be negative correlation (result is as shown in table 9) to the inhibitory action of fungus growth to fungus growth loop diameter size: when Nisin exists, the growth loop diameter of mould is similar with blank group, namely Nisin is to mould unrestraint effect, and the present invention's antibacterial protective agent is when existing, fungus growth loop diameter comparatively control group reduces, especially when concentration is higher, fungus growth loop diameter is about the half of control group.
Table 9Nisin solution and antibacterial protective agent are to the effect of mould
Visible, Nisin antimicrobial spectrum is narrower, although have good inhibitory action for gram-positive bacteria, bacteriostasis is due to bacteriostatic agent of the present invention, and Nisin is for Gram-negative bacteria and the equal unrestraint effect of mould.And bacteriostatic agent of the present invention effectively can suppress the growth of Gram-negative bacteria and mould.
The bacteriostasis of comparative example 2 Phellinus liquid fermentation hypha extract
Phellinus bacterial classification carries out liquid fermentation and obtains mycelium; the extracting method in embodiment 1 is used to obtain hypha extract; hypha extract and the antibacterial protective agent of the present invention is utilized to carry out the bacteriostatic test identical with comparative example 1; change the Nisin in comparative example 1 into hypha extract to test; obtain this comparative example, related experiment operation is with reference to comparative example 1.Result is as Table 10-11: under this comparative example concentration, do not observe inhibition zone when hypha extract exists on Gram-negative bacteria flat board, then can observe when the antibacterial protective agent of the present invention exists; Inhibition zone can be observed when hypha extract exists on gram-positive bacteria flat board, but when concentration is identical, the inhibition zone that hypha extract group obtains is less than the antibacterial protective agent group of the present invention; When hypha extract exists, the growth loop diameter of mould is greater than the growth loop diameter of mould when the antibacterial protective agent of the present invention under same concentration exists, so comparatively hypha extract is good for the antibacterial protective agent fungistatic effect of the present invention.
Table 10 hypha extract and the antibacterial protective agent of the present invention are to the effect of Gram-positive and negative bacterium
Table 11 hypha extract and the antibacterial protective agent of the present invention are to the effect of mould
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. an antibacterial protectant preparation method, it is characterized in that, it comprises the following steps:
1) be added to the water after Phellinus (Phellinusbaumii) fructification being pulverized, carry out extracting in boiling water;
2) by step 1) filtration of gained mixture, obtain residue and extract respectively;
3) by step 2) after gained extract is concentrated, gets supernatant, obtain concentrate;
4) in step 3) add ethanol in gained concentrate, leave standstill, collecting precipitation, is dissolved in water, and gets supernatant, obtains antibacterial protective agent.
2. preparation method as claimed in claim 1, is characterized in that, step 1) described in Phellinus fructification will through super-dry before pulverizing, or described water is the deionized water of 10 ~ 25 times of volumes, and described volume multiple is relative to Phellinus fructification volume.
3. preparation method as claimed in claim 1, it is characterized in that, step 1) described in extraction time of extracting in boiling water be 1 ~ 3 hour, it is preferably 1.5 ~ 2.5 hours, it is more preferably 2 hours, or described preparation method is in step 3) before also comprise step 2 '), described step 2 ') for by step 2) and described in residue again carry out extracting in boiling water, by gained extract and step 2) gained extract merges.
4. preparation method as claimed in claim 1, it is characterized in that, step 3) described in supernatant be described extract concentrated after remove precipitation through the centrifugal 10min of 4000r/min and obtain, or step 4) described in add ethanol after concentration of alcohol be 60% ~ 95%, described percentage is percent by volume.
5. preparation method as claimed in claim 1, is characterized in that, step 4) described in the time left standstill be 12 ~ 30 hours, be preferably 24 hours, or step 4) namely gained supernatant obtain antibacterial protective agent through freeze drying.
6. preparation method as claimed in claim 1, it is characterized in that, described preparation method is in step 4) after also comprise step 5) by step 4) gained supernatant dialyses, obtains the solution after dialysis, be antibacterial protective agent.
7. preparation method as claimed in claim 6; it is characterized in that, step 5) described in time of dialysis be 0 ~ 24 hour, be preferably 4 ~ 24 hours; be more preferably 12 ~ 24 hours, or step 5) described in dialysis after solution namely obtain antibacterial protective agent through freeze drying.
8. preparation method as claimed in claim 7; it is characterized in that; described preparation method is in step 5) after also comprise step 6); described step 6) for by step 5) described in antibacterial protective agent add water and dissolve; obtain the antibacterial protective agent solution of variable concentrations, or described antibacterial protective agent solution concentration is 0.5mg/mL ~ 100mg/mL.
9. an antibacterial protective agent, is characterized in that, described antibacterial protective agent is prepared by the preparation method as described in claim 1-8 any one claim and obtains.
10. the application of antibacterial protective agent in dairy products as claimed in claim 9.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030059985A (en) * | 2002-01-05 | 2003-07-12 | 주식회사 빙그레 | Preventive Yogurt against Adult Diseases and Method for Producing the same |
| CN1502251A (en) * | 2002-11-26 | 2004-06-09 | 陈克铨 | Composite baterial milk products and preparation proess thereof |
| CN101356940A (en) * | 2007-08-02 | 2009-02-04 | 内蒙古伊利实业集团股份有限公司 | Liquid milk added with active polysaccharide |
-
2015
- 2015-12-01 CN CN201510868520.9A patent/CN105494626A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20030059985A (en) * | 2002-01-05 | 2003-07-12 | 주식회사 빙그레 | Preventive Yogurt against Adult Diseases and Method for Producing the same |
| CN1502251A (en) * | 2002-11-26 | 2004-06-09 | 陈克铨 | Composite baterial milk products and preparation proess thereof |
| CN101356940A (en) * | 2007-08-02 | 2009-02-04 | 内蒙古伊利实业集团股份有限公司 | Liquid milk added with active polysaccharide |
Non-Patent Citations (3)
| Title |
|---|
| 王忠民等: "《葡萄多糖的抑菌作用研究》", 《食品科学》 * |
| 窦茜茜: "《桑黄多糖的提取、纯化、理化性质与活性研究及片剂制备》", 《中国优秀硕士学位论文全文数据库（电子期刊）》 * |
| 郑洪健等: "《桑黄子实体提取物抑菌和抗氧化活性初步研究》", 《中国食品科学技术学会第八届年会暨第六届东西方食品业高层论坛论文摘要集》 * |
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