CN108669213A - A kind of nisin thermal sensitive liposomes and its preparation method and application - Google Patents
A kind of nisin thermal sensitive liposomes and its preparation method and application Download PDFInfo
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- CN108669213A CN108669213A CN201810387040.4A CN201810387040A CN108669213A CN 108669213 A CN108669213 A CN 108669213A CN 201810387040 A CN201810387040 A CN 201810387040A CN 108669213 A CN108669213 A CN 108669213A
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- 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
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Abstract
The invention discloses a kind of nisin thermal sensitive liposomes and its preparation method and application, and preparation method is：(1) it takes phosphatide and cholesterol to be dissolved in organic solvent, is solution one；(2) nisin is added to wiring solution-forming two in phosphate buffer；Solution one is instilled in solution two；(3) revolving forms lipid membrane in round-bottomed flask inner wall in rotating instrument, and phosphate buffer is added, and hydration forms multi-layer vesicles liposome, continues water-bath and keeps the temperature to obtain stable liposome turbid liquor；(4) it repeats to filter by filter membrane, obtains nisin thermal sensitive liposomes.The release that nisin is controlled by adjusting temperature significantly reduces degradation of the protease to nisin in meat product process of manufacture while ensureing bacteriostasis, improve the stability and utilization rate of nisin, unnecessary loss is reduced, cost is reduced, production method is simple.
Description
Technical field
The invention belongs to field of food, it is related to a kind of thermal sensitive liposome and its preparation method and application, more particularly to one
Kind nisin (nisin) thermal sensitive liposome and its preparation method and application.
Background technology
Nineteen sixty-five Bangham is found that a kind of closed phospholipid bilayer system, referred to as liposome for the first time.Liposome is by two
The spherical bilayer vesicle that parent's property immobilized artificial membrane is formed, inside is with aqueous core and surface is with hydrophobic layer.Due to good
Good biological degradability, biocompatibility and sustained release, liposome are suggested quickly as the concept of drug delivery system.
Temperature sensitive liposome is delivered drugs into as carrier in human body, discharges drug by the diseased region in high temperature, there is targeting to control
The effect for the treatment of is the good dosage form of chemotherapeutics, antitumor drug, antiseptic.Since phosphatide has Thermo-sensitive, liposome can be with
It is discharged near phase transition temperature encapsulates molecule.Under room temperature, liposome membrane close-packed arrays, encapsulating molecule is difficult to be released；Up to phase
After temperature, membrane structure changes, and permeability increases, and encapsulating molecule can largely discharge.Using this characteristic, phosphatide can be used for
The preparation of temperature sensitive type liposome.
Currently, in the food industry, liposome can be used as nutritional ingredient novel form, protection carrier or slow-released carrier application
In process of manufacture.Such as nisin (nisin) liposome is prepared using proliposome in cheesemaking, make
Nisin stability increases, aspect of cheese time lengthening.But compared with traditional liposomal, proliposome is more suitable
Encapsulating fat-soluble medicine is closed, and preparation process is more complicated, water dispersible is poor.
34 amino acid residues (3.5kDa) of one kind that nisin (nisin) is secreted by certain lactic acid bacterias form
Cationic antibacterial peptide, to gram-positive bacteria such as Listeria, Bacillus acidi lactici, clostridium botulinum, staphylococcus aureus, waxy
Bacillus etc. shows the antibacterial activity of wide spectrum.Currently, nisin is as a kind of natural antiseptic agent without toxic, side effects, in U.S.
More than 50 a countries and regions such as state, European Union, China, are widely used to field of food.However, straight in food processing process
It connects and nisin is added, it can be easily degraded by proteases, and stability and antimicrobial acivity reduce, and utilization rate reduces.Chinese patent discloses
Number CN104920584A discloses a kind of chitosan-nisin core-shell particles and the preparation method and application thereof, i.e., in acid condition
Embedding is carried out to nisin and forms inclusion compound, its stability is improved, enhances its antibacterial activity.This microspherulite diameter is larger, and grain size
It is inhomogenous, it is easy to form condensation product, difficulty is brought for practical application.
China Patent Publication No. CN107668473A discloses a kind of nisin polylysines antimicrobial nano particle and its preparation
Nisin is carried out embedding using the electrostatic interaction of natural polymers and forms polylysine antimicrobial nano particle, improved by method
Stability of the nisin in food, keeps its bioactivity.This electrostatic interaction may be by charged ion in biotic environment
Influence, can need further to be studied applied to industrial production.In addition, polylysine is expensive, life is further increased
Produce cost.
Invention content
The purpose of the present invention is overcome the deficiencies of the prior art and provide one kind nisin can be effectively prevent by egg in food
White enzyme degrades and improves the nisin thermal sensitive liposomes of fungistatic effect.
Second object of the present invention is to provide a kind of at low cost, preparation side of nisin thermal sensitive liposomes simple for process
Method.
Third object of the present invention is to provide a kind of applications of nisin thermal sensitive liposomes.
Technical scheme of the present invention is summarized as follows：
A kind of preparation method of nisin thermal sensitive liposomes, includes the following steps：
(1) in mass ratio (3-15):1 ratio takes phosphatide and cholesterol to be dissolved in organic solvent, makes the phospholipid concentration
It is solution one for 2.5-50g/L；
(2) nisin is added to the solution for being made into a concentration of 0.25g/L in pH=5.7 phosphate buffers, is solution two；
Solution one is instilled in solution two, it is 1 to make the mass ratio of nisin and phosphatide in solution one:(5-20)；
(3) liquid for obtaining step (2) is being rotated in rotating instrument in round-bottomed flask inner wall formation lipid membrane, is added
The pH=7.4 phosphate buffers for entering 0.4-1 times of phosphatide quality are hydrated 20-40min at 40-60 DEG C, form multi-layer vesicles lipid
Body continues to obtain stable liposome turbid liquor in 40-60 DEG C of water-bath heat preservation 30-60min；
(4) suspension obtained by step (3) is repeated into filtering 6-10 times by 0.2-0.45 μm of filter membrane, obtains nisin temperature-sensitives
Liposome.
Phosphatide is preferably phospholipid of natural soybean, egg PC, Hydrogenated Soybean Phosphatidylcholine, single palmityl phosphatide
Phatidylcholine, dipalmitoylphosphatidylcholine, 1- stearoyl -2- hydroxyl-sn- glycerol-3-phosphocholines and 1,2- distearyls -
It is one or more in sn- glycerol-3-phosphocholines.
Organic solvent is preferably chloroform, methanol or absolute ethyl alcohol.
Nisin thermal sensitive liposomes prepared by the above method.
Application of the above-mentioned nisin thermal sensitive liposomes in food antiseptic.
Advantages of the present invention：
The release that nisin is controlled by adjusting temperature significantly reduces meat product while ensureing bacteriostasis
Degradation of the different protease to nisin, improves the stability and utilization rate of nisin in process of manufacture, and it is unnecessary to reduce
Loss, reduce cost.Production method is simple, is not necessarily to additional base group modification, and the operation of temperature control release can make
Nisin gives full play to fungistatic effect.
Description of the drawings
Fig. 1 is the phase transition temperature of nisin thermal sensitive liposomes prepared by embodiment 1.
Fig. 2 is temperature sensitive release of the nisin thermal sensitive liposomes of the preparation of embodiment 1 under the conditions of trypsase.
Fig. 3 is temperature sensitive release of the nisin thermal sensitive liposomes of the preparation of embodiment 1 under the conditions of fresh meat stuffing.
Specific implementation mode
Further detailed description is done to the present invention below in conjunction with specific embodiment.The following example is merely illustrative
The ground description and interpretation present invention, and it is not necessarily to be construed as limiting the scope of the invention.It is all to be based on the above of the present invention
In the range of the technology realized is encompassed by the present invention is directed to protect.
Unless otherwise indicated, the raw materials and reagents used in embodiment are commercial goods, or can pass through known formula
It is prepared by method.
Embodiment 1
A kind of preparation method of nisin thermal sensitive liposomes, includes the following steps：
(1) in mass ratio 5:1 ratio takes phospholipid of natural soybean and cholesterol to be dissolved in chloroform, makes the crude soya bean phosphorus
Lipid concentration is 10g/L, is solution one；
(2) nisin is added to the solution for being made into a concentration of 0.25g/L in pH=5.7 phosphate buffers, is solution two；
Solution one is instilled in solution two, it is 1 to make the mass ratio of nisin and phospholipid of natural soybean in solution one:10；
(3) liquid that step (2) obtains is rotated in rotating instrument in round-bottomed flask inner wall and forms lipid membrane, be added
The pH=7.4 phosphate buffers of 0.8 times of phospholipid of natural soybean quality are hydrated 30min at 50 DEG C, form multi-layer vesicles liposome,
Continue to obtain stable liposome turbid liquor in 50 DEG C of water-bath heat preservation 45min；
(4) suspension obtained by step (3) is repeated into filtering 8 times by 0.3 μm of filter membrane, obtains nisin thermal sensitive liposomes.
Embodiment 2
A kind of preparation method of nisin thermal sensitive liposomes, includes the following steps：
(1) in mass ratio 3:1 ratio takes egg PC and cholesterol to be dissolved in methanol, makes the yolk phospholipid
A concentration of 2.5g/L of phatidylcholine is solution one；
(2) nisin is added to the solution for being made into a concentration of 0.25g/L in pH=5.7 phosphate buffers, is solution two；
Solution one is instilled in solution two, it is 1 to make the mass ratio of nisin and one mesolecithal phosphatidyl choline of solution:5；
(3) liquid that step (2) obtains is rotated in rotating instrument in round-bottomed flask inner wall and forms lipid membrane, be added
The pH=7.4 phosphate buffers of 0.4 times of egg PC quality are hydrated 20min at 60 DEG C, form multi-layer vesicles lipid
Body continues to obtain stable liposome turbid liquor in 60 DEG C of water-bath heat preservation 30min；
(4) suspension obtained by step (3) is repeated into filtering 6 times by 0.2 μm of filter membrane, obtains single layer nisin temperature-sensitive lipids
Body.
It is demonstrated experimentally that with dipalmitoylphosphatidylcholine, 1- stearoyl -2- hydroxyl-sn- glycerol-3-phosphocholines or 1,
2- distearyl-sn- glycerol-3-phosphocholines substitute the egg PC of the present embodiment, prepared by other same the present embodiment
Obtain corresponding nisin thermal sensitive liposomes.
Embodiment 3
A kind of preparation method of nisin thermal sensitive liposomes, includes the following steps：
(1) in mass ratio 15:1 ratio takes phosphatide and cholesterol to be dissolved in absolute ethyl alcohol, makes the phospholipid concentration be
50g/L is solution one；
(2) nisin is added to the solution for being made into a concentration of 0.25g/L in pH=5.7 phosphate buffers, is solution two；
Solution one is instilled in solution two, it is 1 to make the mass ratio of nisin and phosphatide in solution one:20；
(3) liquid that step (2) obtains is rotated in rotating instrument in round-bottomed flask inner wall and forms lipid membrane, be added
The pH=7.4 phosphate buffers of 1 times of phosphatide quality are hydrated 40min at 40 DEG C, form multi-layer vesicles liposome, continue at 40 DEG C
Water-bath heat preservation 60min obtains stable liposome turbid liquor；
(4) suspension obtained by step (3) is repeated into filtering 10 times by 0.45 μm of filter membrane, obtains nisin thermal sensitive liposomes,
Phosphatide is mass ratio 1：1 Hydrogenated Soybean Phosphatidylcholine and monopalmitoylphosphatidylcholine.
Embodiment 4
The phase transition temperature of nisin thermal sensitive liposomes
The nisin thermal sensitive liposomes that embodiment 1 is prepared carry out phase transition temperature measurement.Use differential scanning calorimetry
(DSC) phase transition temperature of the liposome is measured.5 DEG C/min of heating speed, 20 DEG C~60 DEG C of temperature range are set.
Fig. 1 is the phase transition temperature measurement result of nisin thermal sensitive liposomes of the present invention.
As shown in Figure 1, the nisin thermal sensitive liposome phase transition temperatures that embodiment 1 is prepared are 35.12 DEG C.
Embodiment 5
Temperature sensitive release of nisin thermal sensitive liposomes under the conditions of trypsase
The nisin thermal sensitive liposomes that embodiment 1 is prepared are subjected to temperature sensitive release under the conditions of trypsase.
Trypsase is added in the nisin thermal sensitive liposomes that embodiment 1 obtains, controls enzyme concentration 5mg/mL, 4 DEG C of conditions
Lower stirring 30min (simulation meat processing operation).It is sampled under 4 DEG C, 80 DEG C (heating water baths), 100 DEG C (heating water baths) respectively
Supernatant is taken after 500 μ L, 2000r/min centrifugations 2min, obtains sample.It is equal to prepare nisin mass used with embodiment 1
Nisin powder as a control group；It is compareed using the nisin thermal sensitive liposomes that embodiment 1 obtains as temperature.Come with potency experiment
Evaluation gained nisin thermal sensitive liposome antibacterial abilities compare nisin activity height according to inhibition zone size.
One, the inclined-planes LB with Sarcina lutea are taken, 2mL physiological saline is added, is in vitro taken out repeatedly with pipette
It inhales, the lawn on inclined-plane is made to elute, be uniformly mixed, bacteria suspension is spare.200 μ L of bacteria suspension are taken to be added to 6mL sterile physiologicals
Vortex oscillation mixing in brine obtains diluted bacteria suspension, and 390 μ L Tween-20s are added to 70 DEG C of 26mL potency culture mediums, mix
It is even, when it is cooled to 50 DEG C, the diluted bacteria suspensions of 6mL to be poured into detection culture medium conical flask, side bevelling one direction shakes up,
It is poured into horizontal positioned sterilized petri dishes, after its solidification, is uniformly got on above-mentioned tablet with sterile diameter 7mm card punch
8 holes.20mm is cannot be less than when punching with media surface vertical angle, pitch of holes, and the spacing of Kong Yukong will be as possible
It is equal.
Take nisin standard items, dissolved with the HCl/water solution of 0.02mol/L, be made a concentration of 200,100,50,25IU/mL
Titer.The sample of the titer of the various concentration of 80 μ L and 80 μ L is put in hole respectively respectively with pipettor.Lid
Upper plate lid is sealed mouth with sealed membrane, and careful level moves to 37 DEG C and just sets culture for 24 hours.Vernier caliper measurement antibacterial circle diameter.
Using the logarithm of nisin titer potency as ordinate, antibacterial circle diameter is abscissa, and standard curve is made.Sample to be tested
Nisin potency is substituted into calibration curve equation and is calculated by the antibacterial circle diameter of sample.
Fig. 2 is bacteriostatic activity result of the nisin thermal sensitive liposomes of the present invention under the conditions of trypsase.
As shown in Fig. 2, protease has hydrolysis to nisin, for free nisin, most of nisin is dropped
Solution, so the bacteriostatic activity detected is low；More than denaturation temperature, protease loses activity, the denaturation temperature of general protease
Be 80 DEG C or so, so 80 DEG C, the bacteriostatic activities that detect of 100 DEG C of free nisin be higher than 4 DEG C.Nisin liposomes are come
It says, 4 DEG C of phase transition temperature or less liposome protects nisin not to be degraded by enzymes, so the bacteriostatic activity detected is relatively low；It is warm-natured becoming
Degree or more, enzyme loses activity, and a large amount of nisin are released in solution, so the bacteriostatic activity detected is high.In addition, with and without pancreas
Protease exists to the bacteriostatic activity of nisin liposomes without larger impact.Illustrate nisin thermal sensitive liposomes below phase transition temperature
It protects nisin not to be easily degraded by proteases, active nisin can be discharged under hot conditions.80 DEG C and 100 DEG C of measurement results it is close,
Major part protease has inactivated when proving 80 DEG C.
Embodiment 6
Temperature sensitive release of nisin thermal sensitive liposomes under the conditions of fresh meat stuffing
The nisin thermal sensitive liposomes that embodiment 1 is prepared are subjected to temperature sensitive releasing research under the conditions of fresh meat stuffing.Ginseng
The research step for examining embodiment 5 is added fresh unlike the addition trypsase of embodiment 5 in nisin thermal sensitive liposomes
Meat stuffing controls meat stuffing concentration 10mg/mL, simulates practical meat products processing process.
Fig. 3 is bacteriostatic activity result of the nisin thermal sensitive liposomes of the present invention under the conditions of fresh meat stuffing.
As shown in figure 3, measurement result under the conditions of fresh meat stuffing is similar to Example 3, can lead to the same conclusion.
Embodiment 7
Sausage makes
Sausage process controls 10 DEG C of temperature hereinafter, being now in nisin thermal sensitive liposomes phase transition temperature hereinafter, can be with
Nisin is protected not to be easily degraded by proteases.Meat is twisted into meat stuffing first, nisin thermal sensitive liposomes are added, and (nisin is most in meat stuffing
Big usage amount is 0.5g/Kg) and other prepare the common auxiliary material used in sausage, continue to stir evenly, it is marinated for 24 hours.Then will salt down
The meat stuffing made, which is cut, to be mixed uniform pack and enters casing, 80 DEG C of boiling 30min.At this time more than nisin thermal sensitive liposome denaturation temperatures,
Protease loses activity, and a large amount of nisin are released in solution, plays bacteriostasis.Quantitative package is vacuumized after cooling.In order to
It extends the shelf life, the product after packaging can heat 10min re-pasteurizations at 85-90 DEG C.
It these results suggest that the nisin thermal sensitive liposomes of the present invention while ensureing bacteriostasis, can be effectively reduced
Degradation of the different protease to nisin in food production process.It can be used as natural antiseptic agent in meat processing operation, lead to
The release for overregulating temperature control nisin, improves stability and utilization rate, reduces unnecessary loss, reduce cost.
More than, embodiments of the present invention are illustrated.But the present invention is not limited to the above embodiments.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution, improvement and etc. done should be included in the guarantor of the present invention
Within the scope of shield.
Claims (5)
1. a kind of preparation method of nisin thermal sensitive liposomes, it is characterized in that including the following steps：
(1) in mass ratio (3-15):1 ratio takes phosphatide and cholesterol to be dissolved in organic solvent, makes the phospholipid concentration be
2.5-50g/L being solution one；
(2) nisin is added to the solution for being made into a concentration of 0.25g/L in pH=5.7 phosphate buffers, is solution two；It will be molten
Liquid one instills in solution two, and it is 1 to make the mass ratio of nisin and phosphatide in solution one:(5-20)；
(3) liquid for obtaining step (2) is being rotated in rotating instrument in round-bottomed flask inner wall formation lipid membrane, and phosphorus is added
The pH=7.4 phosphate buffers of 0.4-1 times of lipid amount are hydrated 20-40min at 40-60 DEG C, form multi-layer vesicles liposome, after
Continue and obtains stable liposome turbid liquor in 40-60 DEG C of water-bath heat preservation 30-60min；
(4) suspension obtained by step (3) is repeated into filtering 6-10 times by 0.2-0.45 μm of filter membrane, obtains nisin temperature-sensitive lipids
Body.
2. according to the method described in claim 1, it is characterized in that the phosphatide be phospholipid of natural soybean, egg PC,
Hydrogenated Soybean Phosphatidylcholine, monopalmitoylphosphatidylcholine, dipalmitoylphosphatidylcholine, 1- stearoyl -2- hydroxyls-sn-
It is one or more in glycerol-3-phosphocholine and 1,2- distearyl-sn- glycerol-3-phosphocholines.
3. according to the method described in claim 1, it is characterized in that the organic solvent is chloroform, methanol or absolute ethyl alcohol.
4. nisin thermal sensitive liposomes prepared by the method for one of claim 1-3.
5. application of the nisin thermal sensitive liposomes of claim 4 in food antiseptic.
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| CN113455531A (en) * | 2021-07-19 | 2021-10-01 | 西安海斯夫生物科技有限公司 | Preservative liposome for baked food and preparation method thereof |
| CN115191617A (en) * | 2022-07-13 | 2022-10-18 | 江南大学 | Preparation method of sodium alginate-stabilized hypoglycemic collagen peptide liposome |
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| CN108669213B (en) | 2021-06-08 |
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