CN109452283A - A kind of application of mannosylerythritol lipid in suppressing growth of microorganism - Google Patents
A kind of application of mannosylerythritol lipid in suppressing growth of microorganism Download PDFInfo
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- CN109452283A CN109452283A CN201811292440.3A CN201811292440A CN109452283A CN 109452283 A CN109452283 A CN 109452283A CN 201811292440 A CN201811292440 A CN 201811292440A CN 109452283 A CN109452283 A CN 109452283A
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- microorganism
- staphylococcus aureus
- mannosylerythritol lipid
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- bacillus cereus
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/02—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms
- A01N43/04—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom
- A01N43/14—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings
- A01N43/16—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with one or more oxygen or sulfur atoms as the only ring hetero atoms with one hetero atom six-membered rings with oxygen as the ring hetero atom
Abstract
The invention discloses a kind of application of mannosylerythritol lipid in suppressing growth of microorganism, the microorganism is at least one of staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), Candida albicans (Candida albicans).Mannosylerythritol lipid provided by the invention has preferable inhibitory effect to pathogenic bacterias such as food-borne pathogens staphylococcus aureus, bacillus cereus, and there is extraordinary inhibitory effect to prevailing conditions pathomycete Candida albicans etc., have greatly expanded its application range, it can be combined with its good emulsibility, surface-active, moisture retention, degradability, develop into the antibacterial products such as antiseptics for natural food, food antibacterial film, applied in cosmetics, food, environmental protection, medicine and other fields, there is market potential to be worth.
Description
Technical field
The present invention relates to a kind of application of mannosylerythritol lipid in suppressing growth of microorganism, and belong to natural bacteriostatic agent
Field.
Background technique
Surfactant is a kind of amphipathic molecule, usually by lipophilic group (such as hydrocarbon chain or its substituent) and parent
Water base group's (such as sugar alcohol) composition.It can show good surface and interface activity in gas, solid, liquid surface.One
Determine under concentration, surfactant can be gathered in surface and form orderly molecular self-assembling structure, monolayer, micella, vesica
And the structures such as bilayer.Therefore there is dispersibility, emulsification, wet, washing isoreactivity, be widely used in industrial or agricultural field, have
The title of " industrial monosodium glutamate ".Biosurfactant is by microorganisms such as fungi, bacterium, yeast with sugar, glycerol, grease, carbon aquation
Closing object, agriculture and industry waste etc. has amphipathic, the active compound of excellent surface for what matrix metabolism generated.Biological surface is living
Property agent remove there is the similar interface of chemical surfactant and surface-active, emulsibility etc., also there are other to be better than chemical surface
The property of activating agent.
Biosurfactant can be divided into five classes: glycolipid class, lipopeptid and lipoprotein, phosphatide and fatty acid according to its structure
Class, polymeric surfactant class and microparticle surfaces activating agent class etc..Glycolipid class is most important one kind in biosurfactant,
Its kind is more, there is sophorolipid, seaweed glycolipid, rhamnolipid, cellobiose rouge etc..Mannosylerythritol lipid
(MannosylerythritoI lipids, MELs) is a kind of Glycolipids Biosurfactantss.MELs not only has good
Emulsibility, biological degradability, surface-active, lower critical micelle concentration etc. also have many special physiological activity, such as lure
It can be used as flour improver applied to bread industry for guided cell apoptosis and variation etc., be applied to makeup conduct as moisturizer
Industry has potential application value in environmental protection, food, cosmetics, medicine and other fields.
Mannosylerythritol lipid is mainly derived from microbial fermentation product, production bacterium mainly have mould belong to Ustilago and
Blastocystis Pseudozyma.MELs is mainly using 4-O- β-D- mannopyranose-meso-antierythrite as hydrophilic base portion
Point, using the acetyl group on aliphatic chain or glycosyl as hydrophobic part.Reported MELs has 4 kinds of different types, according on glycosyl
The number and position different classifications of acetyl group are MEL-A (containing there are two acetyl group), MEL-B (C6 conjunctions have acetyl group), MEL-C
(C4 contain acetyl group) and MEL-D (not acetyl-containing), structure is as shown in Figure 1.
Food-borne pathogens get worse sanitarian threat at present, due to the rapid growth of global food trade
And the flowing of population, the change of eating habit, the variation of food processing mode, new food-borne pathogens continuously emerge, more
The serious is global emerging drug resistance pathogenic bacteria to have injured the control to pathogenic bacteria.These have become us must face
Pair one of serious public health problem.
Staphylococcus aureus (Staphylococcus aureus, S.aureus) is people and animals common in global range
The property suffered from pathogenic bacteria altogether can cause a variety of serious diseases in clinical medicine and animal medicine.Since Scotland surgery in 1880 is cured
Since raw Alexander Ogston obtains first plant of bacterial strain from the running sore of patient, staphylococcus aureus has become one kind
Common Gram-positive pathogenic bacterium, of less demanding to growing environment condition, tolerance is strong, is seen everywhere in nature, because
Its all kinds of meat products easy to pollute, so there is the nickname of " thermophilic meat bacterium ".Staphylococcus aureus has stronger invasiveness, can infect
The domestic animals such as pig, ox, sheep and the mankind, and a variety of nosotoxins can be generated, cause ox, sheep mammar gland scorching and poultry staphylococcus
Disease etc. reduces the quality and yield of related meat products and dairy products, the heavy economic losses of aquaculture is caused, to the body of the mankind
Health care belt potentially threatens.
Bacillus cereus is widely distributed, is common in soil, dust and sewage, in vegetable food and much raw prepared food
It is also common in product；It is a kind of aerobic, the Gram-positive bacillus that can also be grown very well under anaerobic conditions.Bacillus cereus
It is conditioned pathogen, causes to poison by food most commonly by diarrhea toxin and vomitoxin is generated.Thallus can be passed through once in a while
Infection causes the diseases such as eye disease, endocarditis, meningitis and the bacteremia of people.
Candida albicans (Candida albicans) is a kind of prevailing conditions pathogenic fungus, can reside in human body difference
Position, such as skin, oral cavity, gastrointestinal tract, cause skin, non-cancer lesion, can also cause the systemic infection of threat to life.Bai Nian
Pearl bacterium pathogenic factor have diversity, including by sticking the identification to host, enhancing virulence by decomposing organism organized enzyme,
Increase the adaptability of the microorganism by Phenotype Transition, form germ tube by being converted between yeast phase and Hyphal form and invade host
Deng.
Summary of the invention
The present invention provides a kind of application of mannosylerythritol lipid in suppressing growth of microorganism, the red moss of the mannose
Sugar alcohol rouge has significant fungistatic effect to common pathogen especially Bacillus cereus, can greatly extend its application.
A kind of application of mannosylerythritol lipid in suppressing growth of microorganism, the microorganism are Staphylococcus aureus
Bacterium (Staphylococcus aureus), bacillus cereus (Bacillus cereus), Candida albicans (Candida
At least one of albicans)；The structural formula of the mannosylerythritol lipid is as follows:
Mannosylerythritol lipid (MELs) is used as glycolipid class natural biological surfactant, and biodegradable is good, and
With excellent surface-active, emulsibility etc., emulsifier, moisturizer, surfactant, anti-agglomerating agent, flower characters can be used as
Modifying agent etc., applied in cosmetics, food, environmental protection, medicine and other fields.
Preferably, the structural formula of the mannosylerythritol lipid is as follows:
Wherein, R1=R2=Ac.
The mannosylerythritol lipid can be to send out from aphid false yeast (Pseudozyma aphidis) DSM70725
It is isolated and purified in zymotic fluid.
Preferably, mannosylerythritol lipid is aphid false yeast DSM70725 using soybean oil as carbon source, it is used in combination
The tunning that ethyl acetate isolates and purifies fermentation liquid.
The application may include steps of: mannosylerythritol lipid is added in culture medium, to culture medium
Middle microbe inoculation；
Wherein: the concentration >=0.5g/L of the mannosylerythritol lipid in the medium, antibacterial timeliness are 12-48h.
Mannosylerythritol lipid is added directly into culture medium and cultivates microorganism, avoids dissolution mannose erythrose
The organic reagent of alcohol ester generates bacteriostasis.
The microorganism can be staphylococcus aureus, mannosylerythritol lipid concentration in the medium >=
2.5g/L, antibacterial timeliness are 12-24h；Preferably, concentration >=10g/L, antibacterial timeliness are 12h, and fungistatic effect is best.
The microorganism can be bacillus cereus, the concentration >=2.5g/ of mannosylerythritol lipid in the medium
L, antibacterial timeliness are 36-48h；Preferably, concentration >=10g/L, antibacterial timeliness are 48h, and fungistatic effect is best.
The microorganism can be Candida albicans, the concentration >=2.5g/L of mannosylerythritol lipid in the medium,
Antibacterial timeliness is 12-24h；Preferably, concentration >=10g/L, antibacterial timeliness are 12h, and fungistatic effect is best.
The present invention also provides above-mentioned mannosylerythritol lipids to prepare the application in microbial inhibitor, micro- life
Object be staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), white
At least one of candida albicans (Candida albicans).Microbial inhibitor is prepared using the mannosylerythritol lipid,
Fungistatic effect is preferable, and the features such as combinable its good emulsibility, surface-active, moisture retention, application field is extensive.
The application that the present invention provides mannosylerythritol lipids in suppressing growth of microorganism.Quantified characterization, MEL-A
There is preferable inhibitory effect to pathogenic bacterias such as staphylococcus aureus, bacillus cereus, and Candida albicans etc. is caused
Characteristic of disease fungi has extraordinary inhibitory effect, has greatly expanded its application range, can be living with its good emulsibility, surface
Property, moisture retention, degradability combine, exploitation is applied to makeup at antibacterial products such as antiseptics for natural food, food antibacterial films
Product, environmental protection, in medicine and other fields, have market potential value at food.
Detailed description of the invention
Fig. 1 is mannosylerythritol lipid structure chart；A type: R1=R2=Ac；Type B: R1=Ac, R2=H；C-type: R1=
H, R2=Ac:n=6~10；D type: R1=R2=H, n=4~14.
Fig. 2 is the influence in solid medium containing various concentration MEL-A to staphylococcus aureus growth.From left to right
The concentration of MEL-A is respectively as follows: blank, 2.5g/L, 5g/L, 10g/L.
Fig. 3 is the influence in fluid nutrient medium containing various concentration MEL-A to staphylococcus aureus growth.
Fig. 4 is the influence grown containing various concentration MEL-A to bacillus cereus in solid medium.Left figure: blank
Control；Right figure: MEL-A 10g/L.
Fig. 5 is the influence that various concentration MEL-A grows bacillus cereus in fluid nutrient medium.
Fig. 6 is influence of the various concentration MEL-A to albicans growth in solid medium.Left figure: blank control；It is right
Figure: MEL-A 10g/L.
Fig. 7 is influence of the various concentration MEL-A to albicans growth in fluid nutrient medium.
Specific embodiment
The preparation of 1 mannosylerythritol lipid of embodiment
Mannosylerythritol lipid is generated by aphid false yeast (Pseudozyma aphidis) DSM70725.Mannose is red
The production of moss sugar alcohol rouge and purification process: aphid false yeast is inoculated into containing 3% yeast extract first, 3% malt extracts
In the activation medium of object, 10% glucose and 5% peptone, and cultivated 36 hours under 28 DEG C and 180rpm.It, will after activation
1mL bacterium solution culture is inoculated by NaNO33g/L, MgSO4·7H2O 0.3g/L, KH2PO40.3g/L, yeast extract 1g/
The seed culture medium of L, glucose 40g/L and distilled water composition.After 28 DEG C and 180rpm are cultivated 2 days, by inoculum from
The heart and with brine twice.By obtained cell inoculation in soybean oil containing 80mL/L, MgSO4·7H2O 0.3g/L,
KH2PO40.3g/L, NaNO33g/L, in the fermentation medium of yeast extract 1g/L.Fermentation process is at 28 DEG C, under 180rpm
It carries out 7 days.After fermentation, the ethyl acetate of same volume is added and extraction fullys shake, obtains mannosylerythritol lipid.
By mixture with 3800rpm centrifugation 15 minutes, separates organic layer and be evaporated under reduced pressure.With methanol: hexamethylene (1: 1, v/
V) after being extracted twice to remove remaining oil & fat acid, MEL-A is purified from crude MEL with silicagel column, and pass through thin layer color
It composes (silica GF254, chloroform: methanol: water=65: 15: 2, v/v) and examines product, the results showed that the mannose antierythrite
Rouge is MEL-A type.
2 bacteriostasis method of embodiment
(1) bacteria suspension is prepared
For staphylococcus aureus and bacillus cereus: aseptically, the bacterium that inclined-plane is saved is inoculated with
Ring picking 1-2 ring, is inoculated into 10mL LB culture medium, trains in the full temperature shaking table that temperature is 37 DEG C and revolving speed is 180r/min
After supporting 12h, 1.8mL bacterium solution is taken out in two 2mL centrifuge tubes, and 8000rpm, centrifugation 3min collects thallus under the conditions of 4 DEG C, then uses
The physiological saline of sterilizing carries out washing resuspension, operation 3 times of above-mentioned centrifuge washing is repeated, finally with sterile normal saline
It is 1 × 10 at concentration7The bacteria suspension of cfu/mL.
For Candida albicans: aseptically, the Candida albicans oese picking 1-2 ring that inclined-plane is saved,
It is inoculated into 10mL LB culture medium, after being cultivated for 24 hours in the full temperature shaking table that temperature is 28 DEG C and revolving speed is 180r/min, takes out
1.8mL bacterium solution is in two 2mL centrifuge tubes, and 8000rpm, centrifugation 3min collects thallus under the conditions of 4 DEG C, then with the physiology salt to sterilize
Water carries out washing resuspension, repeats operation 3 times of above-mentioned centrifuge washing, finally with sterile normal saline at concentration for 1 ×
107The bacteria suspension of cfu/mL.
(2) Antibacterial Activity of mannosylerythritol lipid
A, solid culture
Be formulated with 0.5g/L, 1g/L, 2.5g/L, 5g/L, 10g/L MEL-A agar medium (bacterium be LB culture medium,
Fungi is YPD culture medium), sterilizing.100 μ L bacteria suspensions are added in the agar plate of every group of concentration, and are applied with sterile bead
It smears uniformly.Plate is placed in 37 DEG C of incubators and is cultivated for 24 hours, in parallel three times, the agar plate conduct of MEL-A is not added in every group of experiment
The growing state of bacterium colony is observed in blank control.
B, Liquid Culture
Preparation contains 0.5g/L, 1g/L, 2.5g/L, 5g/L, 10g/L MEL-A and the fluid nutrient medium that MEL-A is not added
(bacterium is LB culture medium, and fungi is YPD culture medium), the fluid nutrient medium of each concentration is dispensed into 6 10mL test tubes, is gone out
Bacterium.200 μ L of bacteria suspension is taken to be inoculated into 3 test tubes of each concentration, in addition 3 test tubes are not inoculated with bacterium solution, make blank control.It will
Test tube is placed in complete warm shaking table and cultivates, and revolving speed 180r/min, bacterium temperature is 37 DEG C, and fungi temperature is 28 DEG C.And 12h,
For 24 hours, sampling microplate reader densitometric at 600nm when 36h, 48h.To prevent the emulsification of MEL-A from causing liquid muddy,
Define OD600=OD600 (inoculation bacterium solution)-OD600 (not being inoculated with bacterium solution).
Bacteriostasis of 3 mannosylerythritol lipid of embodiment to staphylococcus aureus
It is carried out according to the experimental implementation of embodiment 2.As a result it is found that mannose erythrose alcohol ester is in solid medium and liquid
All there is apparent inhibiting effect to staphylococcus aureus in culture medium.As shown in Fig. 2, compared with the control, adding the fine jade of MEL-A
The bacterium colony of staphylococcus aureus is obviously distributed sparse, negligible amounts in rouge plate.And with the raising of concentration, S. aureus L-forms
Clump count is also reduced therewith, illustrates that MEL-A has apparent inhibiting effect to the growth of staphylococcus aureus.According to Fig. 3,
Substantially the growth of staphylococcus aureus has been completely inhibited when MEL-A concentration reaches 10g/L, and can be seen that 12h is
The best antibacterial timeliness of MELs.
Bacteriostasis of 4 mannosylerythritol lipid of embodiment to bacillus cereus
It is carried out according to the experimental implementation of embodiment 2.As a result it is found that mannosylerythritol lipid is in solid medium and liquid
All there is apparent inhibiting effect to bacillus cereus in culture medium.As shown in figure 4, the agar plate containing 10g/L MEL-A
On there is the boxed area of not long bacterium colony, these regions are that MEL-A fails to be dissolved in agar well and appear in plate
Surface.As shown in figure 5, compared with the control, there is slow growth in bacillus cereus in the fluid nutrient medium containing MEL-A,
Even non-growing phenomenon.Solid culture and Liquid Culture all illustrate life of the mannosylerythritol lipid to bacillus cereus
Length is inhibited, and concentration is higher, and fungistatic effect is further obvious.
Bacteriostasis of 5 mannosylerythritol lipid of embodiment to Candida albicans
It is carried out according to the experimental implementation of embodiment 2.As a result it is found that the mannosylerythritol lipid of high concentration is in solid culture
It is inhibited to bacillus cereus in base and fluid nutrient medium.As shown in fig. 6, the agar containing 10g/L MEL-A is flat
Occurs the boxed area of not long bacterium colony on plate, these regions are that MEL-A fails to be dissolved in agar and appear in flat well
Plate surface.As shown in fig. 7, compared with the control, when MEL-A concentration reaches 2.5g/L, there is slow growth in Candida albicans
The phenomenon that, illustrate that certain density MELs is inhibited to the growth of Candida albicans.
In summary, MEL-A has good inhibition to food-borne pathogens staphylococcus aureus, bacillus cereus
Its effect grown, and with the raising of concentration, fungistatic effect is further obvious.The growth of Common fungi Candida albicans also by
To the inhibition of MEL-A.For MELs as Glycolipids Biosurfactantss, biodegradable is good, and has good surface living
Property, emulsibility and significant bacteriostasis etc., can be used as emulsifier, moisturizer, surfactant, anti-agglomerating agent, flour product
Quality improving agent etc., applied in cosmetics, food, environmental protection, medicine and other fields.And since MEL-A has to common pathogen
Significant fungistatic effect can be developed into the antibacterial products such as antiseptics for natural food, food antibacterial film, before wide application
Scape.
Claims (7)
1. a kind of application of mannosylerythritol lipid in suppressing growth of microorganism, which is characterized in that the microorganism is gold
Staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), Candida albicans
At least one of (Candida albicans)；The structural formula of the mannosylerythritol lipid is as follows:
2. application as described in claim 1, which is characterized in that the structural formula of the mannosylerythritol lipid is as follows:
Wherein, R1=R2=Ac.
3. application as described in claim 1, which comprises the steps of: mannosylerythritol lipid to be added to
In culture medium, to inoculation of medium microorganism；
Wherein: the concentration >=0.5g/L of the mannosylerythritol lipid in the medium, antibacterial timeliness are 12-48h.
4. application as claimed in claim 3, which is characterized in that the microorganism is staphylococcus aureus, the red moss of mannose
Concentration >=the 2.5g/L of sugar alcohol rouge in the medium, antibacterial timeliness are 12-24h.
5. application as claimed in claim 3, which is characterized in that the microorganism is bacillus cereus, mannose erythrose
Concentration >=the 2.5g/L of alcohol ester in the medium, antibacterial timeliness are 36-48h.
6. application as claimed in claim 3, which is characterized in that the microorganism is Candida albicans, mannose antierythrite
Concentration >=the 2.5g/L of rouge in the medium, antibacterial timeliness are 12-24h.
7. mannosylerythritol lipid as described in claim 1 is preparing the application in microbial inhibitor, the microorganism
It is read for staphylococcus aureus (Staphylococcus aureus), bacillus cereus (Bacillus cereus), white
At least one of pearl bacterium (Candida albicans).
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Cited By (3)
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| CN112352901A (en) * | 2020-09-16 | 2021-02-12 | 浙江大学 | Application of synergy of mannosylerythritol lipids and ultrasound in inhibition of growth of drug-resistant bacteria |
| CN113243407A (en) * | 2021-04-19 | 2021-08-13 | 浙江大学 | Application of mannosylerythritol lipid in preparation of frozen dough improver |
| CN113924884A (en) * | 2021-11-09 | 2022-01-14 | 广西壮族自治区农业科学院 | Fruit retention treatment method for improving storage and transportation resistance of sunshine rose |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112352901A (en) * | 2020-09-16 | 2021-02-12 | 浙江大学 | Application of synergy of mannosylerythritol lipids and ultrasound in inhibition of growth of drug-resistant bacteria |
| CN112352901B (en) * | 2020-09-16 | 2022-06-10 | 浙江大学 | Application of synergy of mannosylerythritol lipids and ultrasound in inhibition of growth of drug-resistant bacteria |
| CN113243407A (en) * | 2021-04-19 | 2021-08-13 | 浙江大学 | Application of mannosylerythritol lipid in preparation of frozen dough improver |
| CN113924884A (en) * | 2021-11-09 | 2022-01-14 | 广西壮族自治区农业科学院 | Fruit retention treatment method for improving storage and transportation resistance of sunshine rose |
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