CN110419716A - A kind of Brevibacillus laterosporus antibacterial peptide microcapsule preparation method - Google Patents
A kind of Brevibacillus laterosporus antibacterial peptide microcapsule preparation method Download PDFInfo
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Abstract
The present invention provides a kind of Brevibacillus laterosporus antibacterial peptide microcapsule preparation methods.The method of the present invention is using pectin as wall material, and Brevibacillus laterosporus antibacterial peptide is embedded as core material by high pressure homogenization technique, then spray-dried to be prepared into；Wherein, the concentration of pectin and antibacterial peptide ratio is 1:4-4:1, and pH value 3-7, homogeneous revolving speed is 2000-5000r/min, and temperature is 30-60 DEG C, time 10-60min.The method of the present invention is easy to operate, embedding rate it is high up to 98%, it is low in cost, and entire embedding process is without using organic solvent, environmentally protective；Antibacterial peptide microcapsule product obtained can slow release antibacterial peptide play inhibitory effect, while effectively extend the preservation time.Microcapsule antibacterial peptide made from the method for the present invention can be used for the long-effective corrosion of the food including fermented product, in food antiseptic field application potential with higher.
Description
Technical field
The invention belongs to food engineering development fields, and in particular, to a kind of side spore with long-acting slow-release, bacteria resistance function
The microcapsule preparation method of bacillus brevis antibacterial peptide.
Background technique
Food apoilage will lead to economic loss, and the annual whole world is because of damage economic caused by food apoilage according to statistics
It loses and reaches tens billion of members.Meanwhile food apoilage will increase the risk for suffering from food origin disease also to bring food-safety problem.
Therefore, food antiseptic is the important link in food manufacturing.At present in food antiseptic based on the use of chemical preservative, and change
Learn preservative the problems such as there are production processes to easily cause pollution, and product itself degree of safety is low.Therefore, biological preservative is (especially
Microbial source biological preservative) research and development by people favor.
Brevibacillus laterosporus fermentation generates extracellular antiseptic peptide, compared with the Nisin of current merchandized handling, has antibacterial
Wide advantage is composed, bacteriostatic activity is all had to Gram-negative bacteria, gram-positive bacteria, fungi.However, Brevibacillus laterosporus
That there are timeliness is short for bacteriostasis of the antibacterial peptide in food, is susceptible to food composition influence, high dose Brevibacillus laterosporus
Antibacterial peptide directly acts on microbial fermentation product and is easy to inhibit the activity of starter and influence the limitations such as fermented product quality.Cause
This, develops antibacterial peptide microcapsule product, and it is antibacterial to may be implemented slow release long-acting, can reduce and even is eliminated particularly with fermented product
To the inhibiting effect of leavening.
Summary of the invention
The Brevibacillus laterosporus antibacterial peptide antibacterial and high embedding rate the object of the present invention is to provide a kind of slow release long-acting is micro-
Capsule preparation method thereof.
The present invention provides a kind of antibacterial peptide microcapsule preparation method, is to be wrapped using wall material pectin to core material antibacterial peptide
It buries, antibacterial peptide microcapsules is made by spray drying process after embedding.
Specifically, preparation method of the invention the following steps are included:
(1) Brevibacillus laterosporus antibacterial peptide solution is mixed with pectin solution and is mixed according to concentration ratio 1:4-4:1,
It is (2) mixed solution is high-pressure homogeneous,
(3) mixed solution after homogeneous is prepared using spray drying process.
In an embodiment of the present invention, the Brevibacillus laterosporus antibacterial peptide brevilaterin of use is referring to patent ZL
201210019951.4 the method for Brevibacillus laterosporus antibacterial peptide is prepared in；Then by the antibacterial peptide of preparation referring to patent
Method in ZL201210019936.X extracts；To extract obtained antibacterial peptide crude product by rapidly purify chromatographic system into
Row rapidly purifies, and antibacterial peptide after purification is Brevibacillus laterosporus antibacterial peptide brevilaterin.
In preparation method of the invention, the pectin molecule amount is 50000-150000Da.The present invention attempts to use other
Wall material embeds Brevibacillus laterosporus antibacterial peptide, many experiments discovery, using wall material commonly used in the art, such as chitosan, β-ring
Dextrin, gelatin, sodium alginate, soybean protein isolate etc., embedding effect can not show a candle to the embedding effect of pectin.Therefore the application
It is preferred that wall material of the pectin as embedding.
Preferably, in preparation method of the invention, the concentration ratio of the antibacterial peptide solution and pectin solution is 1:2, i.e., anti-
Bacterium peptide solution concentration is 0.4mg/mL, and pectin solution concentration is 0.8mg/mL.The solvent of antibacterial peptide and fruit in the embodiment of the present invention
The solvent of glue is water.
Wherein, the high-pressure homogeneous condition of the step (2) are as follows: pH value 3-7, homogeneous revolving speed are 2000-5000r/min,
Temperature is 30-60 DEG C, time 10-60min.
Preferably, the high-pressure homogeneous condition of the step (2) are as follows: pH value 4-6, temperature are 30-50 DEG C, time 20-
50min。
It is highly preferred that the high-pressure homogeneous condition of the step (2) are as follows: pH value 5.5, temperature are 50 DEG C, and the time is
30min。
In above-mentioned preparation method of the invention, the condition of the spray drying process are as follows: inlet temperature is 180 DEG C, charging
20-40 DEG C of temperature, charging rate 6-12mL/min.
The present invention provides antibacterial peptide microcapsules made from above-mentioned preparation method.
The present invention provides following any applications of antibacterial peptide microcapsules obtained:
(1) application in food, health care product, cosmetics or feed addictive is prepared；
(2) application in food, health care product, cosmetics or feed anticorrosion agent is prepared；
(3) extending food, health care product, cosmetics or the application in the feed shelf-life；
(4) application in food, health care product, cosmetics or feed anticorrosion；
(5) application in fermented food is being prepared.
The present invention provides above-mentioned preparation methods in enhancing antibacterial peptide Microencapsulated Slow effect, extends its preservation time, mentions
Application in high antibacterial peptide embedding rate.
The beneficial effects of the present invention are: the method for the present invention is easy to operate, embedding rate it is high up to 98%, it is low in cost, and
Entire embedding process is environmentally protective without using organic solvent；Antibacterial peptide microcapsule product obtained being capable of slow release antibacterial
Peptide plays inhibitory effect, while effectively extending the preservation time, can reduce the suppression even being eliminated to leavening for fermented product
Production is used.Microcapsule antibacterial peptide made from the method for the present invention can be used for the long-effective corrosion of the food including fermented product, In
Food antiseptic field application potential with higher.
Detailed description of the invention
When antibacterial peptide concentration is 0.4mg/mL in Fig. 1 embodiment 1, concentration of pectin is to antibacterial peptide brevilaterin embedding rate
Influence.
Influence of the pH value to antibacterial peptide embedding rate after antibacterial peptide solution is mixed with pectin solution in Fig. 2 embodiment 3.
Influence of the different temperatures to antibacterial peptide embedding rate in Fig. 3 embodiment 4.
The slow release effect figure of antibacterial peptide microcapsules in Fig. 4 embodiment 5.B represents brevilaterin in figure, and B-P represents micro-
Capsule；(a) it cultivates 12 hours, (b) cultivates 48 hours, (c) cultivate 60 hours.
Influence of the antibacterial peptide microcapsules to bread fermentation character in Fig. 5 embodiment 6.(a) adds the micro- glue of 2000AU/g in figure
The bread of capsule (b) adds the bread of 2000AU/g brevilaterin, (c) adds the bread of 3000AU/g microcapsules, (d) adds
Add the bread of 3500AU/g microcapsules.Bread (2000AU/g to 3500AU/g) fermentation volume of microcapsules is added to without significance difference
It is different, and the bread fermentation volume for being added to 2000AU/g antibacterial peptide brevilaterin reduces 63.57%.
Influence of the antibacterial peptide microcapsules to mould in bread storage in Fig. 6 embodiment 6.
Specific embodiment
The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention..Unless otherwise specified, embodiment
Used in the conventional means that are well known to those skilled in the art of technological means, raw materials used is commercial goods.
In the embodiment of the present application, antibacterial peptide used brevilaterin's the preparation method is as follows: referring to patent ZL
The method of embodiment 1 or embodiment 2 prepares Brevibacillus laterosporus antibacterial peptide in 201210019951.4；Used bacterium germination out
It selects good strains in the field for seed from Brevibacillus laterosporus AS1.2738, Brevibacillus laterosporus AS1.864；Brevibacillus laterosporus AS1.2739.So
The antibacterial peptide of preparation is carried out referring to the method for embodiment 1 or embodiment 2 or embodiment 3 in patent ZL201210019936.X afterwards
It extracts；Obtained antibacterial peptide crude product will be extracted to rapidly purify by rapidly purifying chromatographic system, chromatographic column Flash
Column C18 column (40g), mobile phase A are the aqueous solution of 0.1%TFA, and Mobile phase B is the acetonitrile solution of 0.1%TFA, detection
Wavelength 220nm, sample applied sample amount 5mL, flow velocity 22mL/min, elution process: 0~3min, 30%B；3~8min, 30~80%
B；8~10min, 80%B.Antibacterial peptide after fast purifying is brevilaterin；Then by brevilaterin refined solution
Acetonitrile is removed through rotary evaporation, then freeze-dried method is lyophilized and brevilaterin powder is made.Used in all embeddings
Brevilaterin is the freeze-dried powder after rapidly purifying.
In the embodiment of the present application, embedding rate/%=(A-B)/A × 100%, wherein A indicates the total concentration that antibacterial peptide is added,
B indicates the concentration of remaining (free) antibacterial peptide in supernatant after embedding.Wherein antibacterial peptide concentration is surveyed using high performance liquid chromatography
It is fixed.
Embodiment 1
Antibacterial peptide: Brevibacillus laterosporus antibacterial peptide brevilaterin.
Embedding wall material is respectively as follows: chitosan, pectin, beta-cyclodextrin, sodium alginate, soybean protein isolate
1) chitosan imbedded condition are as follows: then 1mL Tween 80 is added in antibacterial peptide 5mg/mL, chitosan 12mg/mL, pH is
5,3000r/min room temperature homogeneous 30min；2% sodium tripolyphosphate solution is slowly added dropwise, homogeneous solidification 30min is prepared anti-
Bacterium peptide microcapsules.
2) pectin embedding conditions: antibacterial peptide concentration is 0.4mg/mL, concentration of pectin 0.8mg/mL, pH 5, in revolving speed
3000r/min, homogeneous 30min under the conditions of 50 DEG C, is prepared antibacterial peptide microcapsules.
3) beta-cyclodextrin embedding conditions: antibacterial peptide 5mg/mL, cyclodextrin 50mg/mL, under the conditions of 40 DEG C, 3000r/min
1h is embedded, antibacterial peptide microcapsules are prepared.
4) gelatin embedding condition: after emulsifying 30min, nothing is added dropwise in antibacterial peptide 5mg/mL, gelatin 40mg/mL (pH 5)
Water-ethanol 5mL simultaneously continues homogeneous 30min；Then, it moves on in ice-water bath and cools down, while stirring 40min；Finally, it is dense that 10mL is added dropwise
The glutaraldehyde solution that degree is 25%, stirring 20min are solidified, and antibacterial peptide microcapsules are prepared.
5) sodium alginate embedding conditions: antibacterial peptide 5mg/mL, sodium alginate 20mg/mL, it is then slow dropwise while stirring
The calcium chloride solution (20mg/mL) of 1.5 times of volumes is added dropwise, obtains microcapsules.Microcapsules are stirred into solidification 30min, cleaning separates,
Antibacterial peptide microcapsules are prepared.
6) soybean protein isolate embedding conditions: soybean protein isolate 2.5mg/mL, the thermal denaturation 30min at 80 DEG C, it is cooling
Antibacterial peptide (final concentration of 1.25mg/mL) is added after to room temperature, after homogeneous 20min, antibacterial peptide microcapsules are prepared.
Influence of 1 microcapsule wall material of table to antibacterial peptide brevilaterin embedding rate
| Embed wall material | Chitosan | Pectin | Beta-cyclodextrin | Gelatin | Sodium alginate | Soybean protein isolate |
| Embedding rate (%) | 47.37±2.14 | 93.64±1.13 | 45.7±1.23 | 35.23±2.35 | 62.75±1.89 | 53.18±1.57 |
Influence of 2 concentration of embodiment to the microcapsule embedded rate of antibacterial peptide
Antibacterial peptide: Brevibacillus laterosporus antibacterial peptide brevilaterin.
Embedding preparation: antibacterial peptide concentration is 0.4mg/mL, and concentration of pectin 0.1-1mg/mL, solvent is water, and pH is
5.5, the homogeneous 30min under the conditions of revolving speed 3000r/min, 50 DEG C.
Be dried: using spray drying, wherein inlet temperature be 180 DEG C, 30 DEG C of feeding temperature, charging rate 8mL/
min。
As shown in Figure 1, microcapsule embedded rate is 78.69 ± 1.56 when concentration of pectin is 0.4mg/mL；Concentration of pectin is
When 0.6mg/mL, microcapsule embedded rate is 91.95 ± 1.12%；When concentration of pectin is 0.8mg/mL, microcapsule embedded rate is
98.27 ± 0.67%.When concentration of pectin is greater than 0.8mg/mL, the microcapsule embedded rate of antibacterial peptide is close to 100%.
Influence of the embodiment 3pH value to the microcapsule embedded rate of antibacterial peptide
Antibacterial peptide: Brevibacillus laterosporus antibacterial peptide brevilaterin.
Embedding preparation: antibacterial peptide concentration is 0.4mg/mL, concentration of pectin 0.8mg/mL, pH 3-7, in revolving speed 3000r/
Min, homogeneous 30min under the conditions of 50 DEG C.
Be dried: using spray drying, wherein inlet temperature be 180 DEG C, 30 DEG C of feeding temperature, charging rate 8mL/
min。
As shown in Fig. 2, the embedding rate of antibacterial peptide is 93.60 ± 1.47% in antibacterial peptide microcapsules when pH is 5.0；PH is
When 5.5, the embedding rate of antibacterial peptide is 98.27 ± 0.67% in microcapsules；When pH is 6.0, the embedding rate of antibacterial peptide in microcapsules
It is 96.06 ± 2.04%.
Influence of 4 temperature of embodiment to the microcapsule embedded rate of antibacterial peptide
Antibacterial peptide: Brevibacillus laterosporus antibacterial peptide brevilaterin.
Embedding preparation: antibacterial peptide concentration is 0.4mg/mL, concentration of pectin 0.8mg/mL, pH 5.5, in revolving speed 3000r/
Min, homogeneous 30min under the conditions of temperature 50 C.
Be dried: using spray drying, wherein inlet temperature be 180 DEG C, 30 DEG C of feeding temperature, charging rate 8mL/
min。
As shown in figure 3, the embedding rate of antibacterial peptide is 98% when temperature is 50 DEG C；When temperature is 40 DEG C, the embedding of antibacterial peptide
Rate is 90.12 ± 1.25%；When temperature is 30 DEG C, antibacterial peptide embedding rate is 87.99 ± 1.57%.
5 antibacterial peptide Microencapsulated Slow Effect Evaluation of embodiment
The antibacterial peptide brevilaterin microcapsules being prepared when being 5.5 to pH in embodiment 3 carry out slowly releasing effect and comment
Valence.Indicator bacteria chooses Staphylococcus aureus ATCC25923, straight by inhibition zone using punching-agar diffusion method
Diameter detects the bacteriostatic activity of antibacterial peptide microcapsules, as a result, it has been found that by after plate culture 12h, the average antibacterial circle diameter of microcapsules is
The average diameter of microcapsules is 26.1 ± 1.2mm after 15.6 ± 0.8mm, culture 48h, and the inhibition zone of microcapsules is straight after culture 60h
Diameter average value is 28.7 ± 1.3mm；And corresponding antibacterial poly saccharide peptide standard product antibacterial circle diameter does not continue the phenomenon that increasing, it was demonstrated that micro-
Capsule has slowly releasing effect (Fig. 4).
6 antibacterial peptide microcapsules of embodiment are in bread anti-corrosion to the influence of yeast activity
In the present embodiment, bread formula are as follows: butter 18g, white granulated sugar 18g, salt 3g, milk powder 10g, Strong flour 250g, ferment
Female 4g, water 150mL.Saccharomyces cerevisiae is the amount of being routinely added to.4g Saccharomyces cerevisiae is added in every 500g dough.
When being 5.5 to pH in embodiment 3 the antibacterial peptide brevilaterin microcapsules that are prepared for bread anti-corrosion with
Its influence to food fermentation characteristic and anti-corrosion effect is investigated, the antibacterial peptide for adding 2000AU/g as the result is shown is living to Saccharomyces cerevisiae
Property generate and significantly inhibit, it is the solid dough of institutional framework that dough, which does not ferment, sees Fig. 5 (b)；And add 2000AU/g antibacterial
The microcapsule product of peptide, bread fermentation character and un-added (being not added with the microcapsule product of 2000AU/g antibacterial peptide) are without aobvious
Difference is write, sees Fig. 5 (a).It adds the bread of 3000AU/g microcapsules and adds bread and the addition of 3500AU/g microcapsules
The bread fermentation volume of 2000AU/g microcapsules is shown in (a), (c) (d) of Fig. 5 without significant difference.And it is added to 2000AU/g antibacterial
The bread fermentation volume of peptide Brevilaterin reduces 63.57%.Illustrate the antibacterial peptide of the invention containing Brevibacillus laterosporus
Microcapsules can reduce the inhibiting effect even being eliminated to leavening.
The bread anti-corrosion effect of the antibacterial peptide microcapsules of the invention of embodiment 7 is verified
The present embodiment is by new roasted breading slices and to place 5 minutes in air, is then packaged in close in sterile bag
Envelope, is observed continuously and inspects by random samples 7 days, investigates the shadow in antibacterial peptide microcapsules of the invention of different preservation times to fungi number in bread
It rings.
1, bread preservative challenge test method: 3000AU/g obtained when 4 temperature of embodiment is 50 DEG C is added respectively into flour
Brevilaterin microcapsules, 3500AU/g Brevilaterin microcapsules, 1g/Kg calcium propionate, according to conventional bread after mixing
Production method makes bread.Wherein not add the roasted bread of preservative as blank control group.
By the breading slices made (with a thickness of 1cm), weighing, placement, which is put in after five minutes in sterile sealing bag, to be sealed, then
Rusk is put in 28 DEG C of constant incubators and is stored, the fungi number in daily grab sample detection bread, analysis
Anti-corrosion effect of the Brevilaterin microcapsules to bread.
2, it is provided according to national food safety standard GB7099-2015 " cake, bread ", the fungi number limit allowed in bread
Amount≤150CFU/g.Using destructive Acceleration study in actual experiment, carry out being sliced and putting in air after breadmaking
5min is set with contaminant, observes anti-corrosion effect.Common additive calcium propionate in current bread industry is used in experiment to make
For positive control, the amount of addition is also according to the usage amount in practical bread industry.
With addition 1g/Kg calcium propionate, (this is face to the bread fungi number of addition 3000AU/g antibacterial peptide microcapsules as the result is shown
Packet Common Preservatives) bread anti-corrosion effect without significant difference, and the fungi number of substantially less than blank control group, addition
The bread fungi number of 3500AU/g antibacterial peptide microcapsules is substantially less than the fungi number for adding the bread group of 1g/Kg calcium propionate, sees figure
6。
And it is of the invention since the present embodiment is the anti-corrosion effect experiment carried out under the premise of destructive Acceleration study
Antibacterial peptide microcapsules still can maintain the fungi number of bread in national Specification in the case where continuing the Acceleration study to the 3rd day
The upper limit hereinafter, this absolutely prove Brevibacillus laterosporus antibacterial peptide microcapsules made from the application have good slow release effect,
And inhibits fungi ability excellent, can be used for bread industry, remarkable in economical benefits.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
It states, but on the basis of the present invention, it can be made some modifications or improvements, this is apparent to those skilled in the art
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Range.
Claims (10)
1. a kind of antibacterial peptide microcapsule preparation method, which is characterized in that anti-to core material Brevibacillus laterosporus using wall material pectin
Bacterium peptide is embedded, and antibacterial peptide microcapsules are made by spray drying process after embedding.
2. preparation method as described in claim 1, which comprises the following steps:
(1) Brevibacillus laterosporus antibacterial peptide solution is mixed with pectin solution and is mixed according to concentration ratio 1:4-4:1,
It is (2) mixed solution is high-pressure homogeneous,
(3) mixed solution after homogeneous is prepared using spray drying process.
3. preparation method as claimed in claim 1 or 2, which is characterized in that the pectin molecule amount is 50000-150000Da.
4. preparation method as claimed in claim 2, which is characterized in that the concentration ratio of the antibacterial peptide solution and pectin solution is
1:2。
5. preparation method as claimed in claim 2, which is characterized in that the high-pressure homogeneous condition of the step (2) are as follows: pH value is
3-7, homogeneous revolving speed are 2000-5000r/min, and temperature is 30-60 DEG C, time 10-60min.
6. preparation method as claimed in claim 5, which is characterized in that the high-pressure homogeneous condition of the step (2) are as follows: pH value is
4-6, temperature are 30-50 DEG C, time 20-50min.
7. preparation method as claimed in claim 6, which is characterized in that the high-pressure homogeneous condition of the step (2) are as follows: pH value is
5.5, temperature is 50 DEG C, time 30min.
8. preparation method as claimed in claim 1, which is characterized in that the condition of the spray drying process are as follows: into
Mouthful temperature is 180 DEG C, 20-40 DEG C of feeding temperature, charging rate 6-12mL/min.
9. antibacterial peptide microcapsules made from any preparation method of claim 1-8.
10. following any purposes of antibacterial peptide microcapsules as claimed in claim 9:
(1) application in food, health care product, cosmetics or feed addictive is prepared；
(2) application in food, health care product, cosmetics or feed anticorrosion agent is prepared；
(3) extending food, health care product, cosmetics or the application in the feed shelf-life；
(4) application in food, health care product, cosmetics or feed anticorrosion；
(5) application in fermented food is being prepared.
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