CN109805412A - A kind of compound probiotic intestinal-specific control delivery and preparation method thereof - Google Patents
A kind of compound probiotic intestinal-specific control delivery and preparation method thereof Download PDFInfo
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- CN109805412A CN109805412A CN201910104921.5A CN201910104921A CN109805412A CN 109805412 A CN109805412 A CN 109805412A CN 201910104921 A CN201910104921 A CN 201910104921A CN 109805412 A CN109805412 A CN 109805412A
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Abstract
The invention discloses a kind of compound probiotic intestinal-specific control delivery and preparation method thereof, probiotics actication of culture, the preparation of indigo fruit polysaccharide constructs the multiple element compound control delivery, probiotics embedding.Obtained system solution is stirred continuously, room temperature is cooled to, is then 5 DEG C in ice bath environment decline low temperature, curing agent CaCl is added2Precipitation to be precipitated is persistently stirred after solution, is collected by filtration after centrifugation, with the brine microballoon of sterilizing, obtains wet capsule.The present invention not only possesses the hydrophobic self assembly characteristic of protein, and has the good water solubility of polysaccharide, it can be used to embed and convey compound probiotic and form intestinal-specific control delivery, intergranular aggregation, sedimentation can be effectively suppressed in the space steric effect of macromolecular, and there is good biocompatibility, stronger resistance to compression, heat resistance and environmental suitability, realize probiotics in the Targeting delivery of colon site.
Description
Technical field
The present invention relates to a kind of functional food more particularly to a kind of compound probiotic intestinal-specific control delivery and its
Preparation method.
Background technique
Probiotics is a kind of active microorganism beneficial to human body, and simultaneously growth and breeding can be colonized in the enteron aisle of human body,
Human body intestinal canal flora is adjusted, the microecological balance in human body intestinal canal is maintained, to promote health, prevents disease.But it is beneficial
Raw bacterium is more sensitive to external environment, and the unfavorable conditions such as the gastric acid of low pH and bile can seriously affect the work of probiotics in human body
Property, the viable count for causing probiotics to enter in enteron aisle reduces, influences it and be colonized and play beneficial function.
Currently, constructing carrier systems by single macromolecular carrier mostly both at home and abroad, such as separated using some soybean
Albumen, gelatinization cornstarch, gelatin etc. are used as carrier, prepare microcapsules or capsule of nano, nanometer emulsion, nano-lipid
Body, microballoon and polymer nano-particle etc. embed probiotics and are loaded, and realize the transport of probiotics in vivo.However,
Probiotics is more sensitive to environment, during preparing the carrier systems such as nanoemulsions, needs strict control preparation condition such as
Temperature, oxygen, illumination and transiting state metal etc., and its active force and action mode between different polymer dielectrics is not to the utmost
Identical, environmental condition is larger to the stability influence of carrier system.Moreover, solution system is easy dispersion and forms coagulation phenomenon,
It causes the quick release (burst release) of probiotics and is unfavorable for its proliferation and utilizes.
Summary of the invention
Technical problem to be solved by the present invention lies in: how to realize the slow release of control delivery, provides a kind of multiple
Probiotics intestinal-specific control delivery and preparation method thereof is closed, this water soluble protein of oralbumin and polysaccharide are passed through into layer
The mode of layer self assembly constructs nano combined carrier systems.
The present invention is to solve above-mentioned technical problem, a kind of compound probiotic enteron aisle of the invention by the following technical programs
Target controlling and releasing system preparation method, comprising the following steps:
(1) probiotics actication of culture:
Under sterile conditions, the MRS liquid that the freeze-dried powder bacterium powder of a variety of probiotics is inoculated into sterilized cooling is trained
Activation, Anaerobic culturel in base are supported, separation abandons supernatant, collects thallus, then being washed with sterile saline, and supernatant is abandoned in centrifugation again
Liquid collects plastc ring；
(2) preparation of indigo fruit polysaccharide:
Fresh indigo fruit is taken, indigo fruit powder is dissolved in distilled water after crushing, ultra sonic bath mentions, and anhydrous second is then added
Alcohol is stood, and removes supernatant, and filter residue is dissolved with distilled water, removing protein, and sediment is freeze-dried up to indigo fruit Polyose extraction
Object；
(3) the multiple element compound control delivery is constructed:
Dry oralbumin is dissolved in distilled water, is then added pectin solution, oralbumin and pectin are matched
It is while magnetic agitation that oralbumin and pectin binary is compound than pH is transferred to 7 after the two mixing for 1:0.5~1
Object: indigo fruit polysaccharide solution is added dropwise according to volume ratio 2:1 in indigo fruit polysaccharide solution, overnight to guarantee abundant water at 4 DEG C
It closes；
(4) probiotics embeds:
The plastc ring that step (1) is collected into is added dropwise to the compound that step (3) is prepared with syringe
In, it is continuously stirred under magnetic stirring apparatus to promote to be sufficiently mixed, then adjusting pH value is 4.6, to obtain the control of load probiotics
Release system.
In the step (1), a variety of probiotics are bifidobacterium longum, the bifidobacterium breve, youth of mass ratio 1:1:1:1:1
Bifidobacterium, saccharomycete, lactic acid bacteria.
In the step (2), detailed process is as follows for the preparation of indigo fruit polysaccharide:
Fresh indigo fruit is taken, indigo fruit powder is dissolved in distilled water after crushing, solid-liquid ratio 1:15 is placed in 40 DEG C
60min is extracted in ultrasonic water bath, and it is 175w that ultrasonic power, which is arranged, the dehydrated alcohol that 3~5 times of volumes are added after extraction is stood
10h, then 4000rpm removes supernatant after being centrifuged 20min, and filter residue is dissolved with distilled water, with Sevag [V (chloroform): V
(n-butanol)=4:1] reagent removing protein, sediment is freeze-dried up to indigo fruit polyoses extract.
In the step (3), dry oralbumin is dissolved in distilled water, it is while magnetic agitation that egg white is white
Indigo fruit polysaccharide solution is added dropwise according to volume ratio 2:1 in albumen and indigo fruit polysaccharide solution, abundant to guarantee overnight at 4 DEG C
Hydration.
The volume ratio of thallus liquid and compound is 1:1 in the step (4).
Control delivery made from a kind of compound probiotic intestinal-specific control delivery preparation method as mentioned.
A method of wet capsule being prepared using control delivery, detailed process is as follows: obtained system solution is constantly stirred
It mixes, is cooled to room temperature, be then 5 DEG C in ice bath environment decline low temperature, curing agent CaCl is added2It is persistently stirred after solution to heavy
Precipitation goes out, and is collected by filtration after centrifugation, with the brine microballoon of sterilizing, obtains wet capsule.
Indigo fruit category Angiospermae can effectively be gone containing functional components such as a large amount of anthocyanin, organic acid, polysaccharide
Except human free radical, antioxidant and anti-aging, reducing blood sugar and blood fat.Indigo fruit polyoses content is abundant, and series of studies shows indigo fruit
Polysaccharide have improve the immunity of the human body, be antifatigue, is hypoglycemic, anticancer, the physiological functions such as antibacterial, radiation protection.At present to indigo fruit
The research of anthocyanin is more, and indigo fruit polysaccharide researches are less.Using indigo fruit polysaccharide as one of polysaccharide carrier of control delivery,
The effect of probiotics can not only be embedded, but also as a kind of function factor, reducing blood sugar and blood fat can be played.
The present invention has the advantage that the present invention not only possesses the hydrophobic self assembly characteristic of protein compared with prior art,
And have the good water solubility of polysaccharide, it can be used to embed and convey compound probiotic formation intestinal-specific control delivery, greatly
The space steric effect of molecule can be effectively suppressed intergranular aggregation, sedimentation, and have good biocompatibility, stronger
Resistance to compression, heat resistance and environmental suitability can effectively prevent the diffusion and drop of pepsin in simulation gastro-intestinal Fluid environment
Solution, and in intestinal juice simulated gastrointestinal condition, gradually probiotics can be released with the time, realize probiotics in colon
The Targeting delivery at position.Therefore compound probiotic can be improved to the tolerance of environment in the foundation of control delivery, guarantees probiotics
Stablize metabolism and vigor, can discharge and rise in value in enteron aisle, beneficial human health.
Detailed description of the invention
Fig. 1 is that the release conditions of 3h embodiment 1 and 2 gained microcapsules of embodiment in simulated intestinal fluid are handled through simulate the gastric juice
Schematic diagram.
Specific embodiment
It elaborates below to the embodiment of the present invention, the present embodiment carries out under the premise of the technical scheme of the present invention
Implement, the detailed implementation method and specific operation process are given, but protection scope of the present invention is not limited to following implementation
Example.
Embodiment 1
The control delivery of the present embodiment the preparation method is as follows:
(1) probiotics actication of culture: aseptically operating, by bifidobacterium longum, bifidobacterium breve, youth bifid bar
The freeze-dried powder bacterium powder of this five kinds of probiotics of bacterium, saccharomycete, lactic acid bacteria is seeded in the MRS fluid nutrient medium of sterilized cooling,
Vessel port is sealed, is stood, under the conditions of 37 DEG C, constant temperature incubation to liquid turns turbid.By the strain after activation, MRS liquid is accessed
37 DEG C of Anaerobic culturels of body culture medium, will cultivate resulting five in stationary phase kind probiotics be centrifuged (4000rpm,
After 15min), supernatant is abandoned, thallus is collected, is washed with 0.9g/100mL sterile saline, be centrifuged again (4000rpm,
15min), supernatant is abandoned, plastc ring is collected.
(2) preparation of indigo fruit polysaccharide: taking fresh indigo fruit, and indigo fruit powder is dissolved in distilled water (material after crushing
Liquor ratio is 1:15), it is placed in 40 DEG C of ultrasonic water bath and extracts 60min, and it is 175w that ultrasonic power, which is arranged,.3~5 are added after extraction
The dehydrated alcohol of times volume stands 10h, and then 4000rpm removes supernatant after being centrifuged 20min, and filter residue is dissolved with distilled water, uses
Sediment is freeze-dried and mentions up to indigo fruit polysaccharide by Sevag [V (chloroform): V (n-butanol)=4:1] reagent removing protein
Take object.Optimization technique is 24.5% with Phenol sulfuric acid procedure measurement indigo fruit polysaccharide yield.
(3) building of binary complex control delivery: dry oralbumin is dissolved in distilled water, in magnetic agitation
While according to the proportion (oralbumin/indigo fruit polysaccharide solution) 1:0.5 indigo fruit polysaccharide solution is added dropwise, at 4 DEG C
Overnight to guarantee sufficiently hydration.
(4) plastc ring being collected into the embedding of probiotics: is added dropwise to oralbumin/indigo with syringe
In the binary complex of fruit polysaccharide (thallus liquid: binary complex=1:1), continuously stirred under magnetic stirring apparatus to promote sufficiently
Mixing, then adjusting pH value is 4.6, to obtain the control delivery of load probiotics.
Embodiment 2
The control delivery of the present embodiment the preparation method is as follows:
(1) probiotics actication of culture: aseptically operating, by bifidobacterium longum, bifidobacterium breve, youth bifid bar
The freeze-dried powder bacterium powder of this five kinds of probiotics of bacterium, saccharomycete, lactic acid bacteria is seeded in the MRS fluid nutrient medium of sterilized cooling,
Vessel port is sealed, is stood, under the conditions of 37 DEG C, constant temperature incubation to liquid turns turbid.By the strain after activation, MRS liquid is accessed
37 DEG C of Anaerobic culturels of body culture medium, will cultivate resulting five in stationary phase kind probiotics be centrifuged (4000rpm,
After 15min), supernatant is abandoned, thallus is collected, is washed with 0.9g/100mL sterile saline, be centrifuged again (4000rpm,
15min), supernatant is abandoned, plastc ring is collected.
(2) preparation of indigo fruit polysaccharide: taking fresh indigo fruit, and indigo fruit powder is dissolved in distilled water (material after crushing
Liquor ratio is 1:15), it is placed in 40 DEG C of ultrasonic water bath and extracts 60min, and it is 175w that ultrasonic power, which is arranged,.3~5 are added after extraction
The dehydrated alcohol of times volume stands 10h, and then 4000rpm removes supernatant after being centrifuged 20min, and filter residue is dissolved with distilled water, uses
Sediment is freeze-dried and mentions up to indigo fruit polysaccharide by Sevag [V (chloroform): V (n-butanol)=4:1] reagent removing protein
Take object.Optimization technique is 24.5% with Phenol sulfuric acid procedure measurement indigo fruit polysaccharide yield.
(3) building of ternary complex control delivery: dry oralbumin is dissolved in distilled water, fruit is then added
The proportion of sol solution, oralbumin and pectin are as follows: 0.5-1.PH is transferred to 7 after the two mixing, is pressed while magnetic agitation
Indigo fruit polysaccharide solution is added dropwise according to proportion (oralbumin/pectin binary complex: indigo fruit polysaccharide solution) 1:0.5,
Overnight to guarantee sufficiently hydration at 4 DEG C.
(4) plastc ring being collected into the embedding of probiotics: is added dropwise to the white egg of pectin/egg white with syringe
In the ternary complex of white/indigo fruit polysaccharide (thallus liquid: ternary complex=1:1), continuously stirred under magnetic stirring apparatus with
Promote to be sufficiently mixed, then adjusting pH value is 4.6, to obtain the control delivery of load compound probiotic.
Test process:
One, embedding rate measures:
The measurement of resulting control delivery progress embedding rate will be distinguished in embodiment 1 and embodiment 2:
Viable count × 100% before embedding rate (%)=(embed before viable count-be not embedded viable count)/embeds
Viable count before embedding: after actication of culture, bacterium colony counting is carried out.
The viable count not being embedded: taking system solution, is collected by filtration precipitating, and with brine, collects filtrate, survey
Determine viable count (CFU/ml)
The embedding rate and room temperature viable count of embodiment 1 and embodiment 2 see for oneself table 1.
Microcapsules index in 1 embodiment 1 of table and embodiment 2
As can be seen from Table 1: the embedding rate of microcapsules is reachable in embodiment 1: 84%+1.0%, and room temperature viable bacteria amount reaches 8
×109, average grain diameter 1.58mm.The embedding rate of microcapsules is reachable in embodiment two: 97%+1.2%, and room temperature viable bacteria amount reaches
6×1010, average grain diameter 1.67mm.
Two, the microencapsulation of compound probiotic control delivery:
The system solution of above-mentioned preparation is cooled to room temperature in the case where constantly stirring, then declines low temperature in ice bath environment
It is 5 DEG C, persistently stirs precipitation to be precipitated after curing agent CaCl2 solution is added, be collected by filtration after centrifugation, with the physiological saline of sterilizing
Microballoon is washed, wet capsule is obtained.Wet capsule is placed in -80 DEG C of refrigerator after 2~4h of pre-freeze, vacuum freeze drier is placed in
In, the powder of microcapsules is prepared to get compound probiotic ternary microcapsules are arrived in -80 DEG C of 24~48h of freeze-drying.At random
It is several to extract the compound probiotic slow-release microcapsule prepared in experiment, measures the partial size of each microcapsules respectively with vernier caliper,
It is averaged.
The average grain diameter of embodiment 1 and embodiment 2 is shown in Table 1.
Three, in complex probiotic microcapsule thallus release:
1g microcapsule product is weighed, is added in the PBS buffer solution that 9mLpH value is 7.4, water-bath is shaken to complete at 37 DEG C
Full disintegration, and number of viable is measured using gradient dilution the method for plate culture count.
Four, release of the complex probiotic microcapsule in simulation human body alimentary canal environment
By five kinds of probiotics gymnobacterias, 1 microcapsules of embodiment, each 2g of 2 microcapsules of embodiment, simulate the gastric juice 48mL is added.It is mixed
After closing uniformly, 37 DEG C are put into, is handled in the shaking table of 180r/min, 0.5mL mixed liquor is drawn in 0h, 2h, while 0.5mL is added
Gastric juice.After mixed liquor gradient dilution, selects suitable gradient that 0.1mL is sucked out and apply plate count, unit CFU/mL.Stomach will be used
The mixed liquor of probiotics gymnobacteria, 1 microcapsules of embodiment, 2 microcapsules of embodiment after liquid processing 2h, 4000rpm are centrifuged 5min, receive
Collect sediment, the model bile of 28mL is added, after mixing, is put into 37 DEG C, is handled in the shaking table of 180r/min, in 30min
When draw 0.5mL mixed liquor, while 0.5mL bile is added.After mixed liquor gradient dilution, suitable gradient is selected to be sucked out
0.1mL applies plate count, unit CFU/mL.
Five, intestinal juice detecting for slowly-releasing property:
By through the bile processing gymnobacteria of 30min, 1 microcapsules of embodiment, 2 microcapsules of embodiment mixed liquor, 4000rpm from
Heart 5min collects sediment, after 48mL simulated intestinal fluid is added, is uniformly mixed.37 DEG C are put into, is handled in the shaking table of 180r/min
It after 45min, is spaced at regular intervals, viable count, unit CFU/mL is measured by sampling.And by measurement microcapsules through artificial intestines
The total viable count of liquid before and after the processing calculates simulated intestinal fluid treated thallus release rate.
The viable count of viable count in release rate (%)=simulated intestinal fluid/after simulated gastric fluid handles 3h in microcapsules ×
100%.
Thallus release rate in embodiment 1 and embodiment 2 is shown in Fig. 1.
Fig. 1 is that the release feelings of 3h embodiment 1 and 2 gained microcapsules of embodiment in simulated intestinal fluid are handled through simulate the gastric juice
Condition.It can be seen that probiotics rate of release is very fast in 1 microcapsules of embodiment, it is 88% that release rate was reached at 6 hours.It is real
It applies example 2 and has reached slow release in simulated intestinal fluid, it is 96% that maximum release rate was reached at 8 hours, higher than the release of embodiment 1
Rate with embodiment 1 so compare, and compound probiotic slow-release microcapsule prepared by embodiment 2 has preferable sustained release performance, and accumulation is released
The time is put up to 8 hours.
It is as shown in table 2 that two kinds of complex probiotic microcapsules discharge the case where thallus in simulated gastrointestinal tract.
As shown in Table 2, it is handled by the gastric juice of 2h, gymnobacteria cell concentration is from 1010CFU/mL has decreased to 107CFU/mL,
Have dropped 3 orders of magnitude.After microcapsules handle 2h in gastric juice in embodiment 1, cell concentration is from 1010CFU/mL drops to
108CFU/mL has dropped 2 orders of magnitude.Illustrate in 1 microcapsules of embodiment, thallus is not released completely, in embodiment 1
Microcapsules have certain slow release effect.After microcapsules handle 2h in gastric juice in embodiment 2, cell concentration is from 1010CFU/mL
Drop to 109CFU/mL has dropped 1 order of magnitude.Illustrate that microcapsule embedded rate is higher in embodiment 2, and its weak acid resistant
It is relatively strong, handled by 2h gastric juice, each sample is directly entered intestinal juice, after intestinal juice handles 3h, cell concentration in each sample with into
The initial cell concentration for entering intestinal juice is consistent, and is illustrated that intestinal juice does not damage gymnobacteria, is handled by bile, gymnobacteria and embodiment
Composite micro-capsule cell concentration is from 10 in 110CFU/mL drops to 106CFU/mL has dropped 4 orders of magnitude.Illustrate gymnobacteria to gallbladder
The tolerance of salt is poor, and composite micro-capsule is from 10 in embodiment 210CFU/mL drops to 109CFU/mL has dropped 1 order of magnitude,
Illustrate that composite micro-capsule plays the role of centainly being sustained in embodiment 2, has thallus release in part after cholate, so still maintaining
Higher cell concentration.2 microcapsules of embodiment pass through entire simulated gastrointestinal tract cell concentration from 1.4 × 1010CFU/mL drops to
1.9×108CFU/mL, microcapsules play slow release effect, and the quantity of compensation gastrointestinal tract loss probiotics is maintained at cell concentration
The higher order of magnitude, so that prebiotic effect is played, and 1 effect of embodiment is slightly weak.
2 complex probiotic microcapsule of table discharges the case where thallus in simulated gastrointestinal tract
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (7)
1. a kind of compound probiotic intestinal-specific control delivery preparation method, which comprises the following steps:
(1) probiotics actication of culture:
Under sterile conditions, the freeze-dried powder bacterium powder of a variety of probiotics is inoculated into the MRS fluid nutrient medium of sterilized cooling
Middle activation, Anaerobic culturel, separation abandon supernatant, collect thallus, then being washed with sterile saline, and supernatant is abandoned in centrifugation again, is received
Collect plastc ring；
(2) preparation of indigo fruit polysaccharide:
Fresh indigo fruit is taken, indigo fruit powder is dissolved in distilled water after crushing, ultra sonic bath mentions, and it is quiet that dehydrated alcohol is then added
It sets, removes supernatant, filter residue is dissolved with distilled water, removing protein, and sediment is freeze-dried up to indigo fruit polyoses extract；
(3) the multiple element compound control delivery is constructed:
Dry oralbumin is dissolved in distilled water, is then added pectin solution, the proportion of oralbumin and pectin is
PH is transferred to 7 after the two mixing by 1:0.5~1, by oralbumin and pectin binary complex while magnetic agitation: blue
Indigo fruit polysaccharide solution is added dropwise according to volume ratio 2:1 in indigo fruit polysaccharide solution, overnight to guarantee sufficiently hydration at 4 DEG C；
(4) probiotics embeds:
The plastc ring that step (1) is collected into is added dropwise in the compound that step (3) is prepared with syringe,
It is continuously stirred under magnetic stirring apparatus to promote to be sufficiently mixed, then adjusting pH value is 4.6, to obtain the controlled release body of load probiotics
System.
2. a kind of compound probiotic intestinal-specific control delivery preparation method according to claim 1, which is characterized in that institute
State in step (1), a variety of probiotics be the bifidobacterium longum of mass ratio 1:1:1:1:1, bifidobacterium breve, bifidobacterium adolescentis,
Saccharomycete, lactic acid bacteria.
3. a kind of compound probiotic intestinal-specific control delivery preparation method according to claim 1, which is characterized in that institute
It states in step (2), detailed process is as follows for the preparation of indigo fruit polysaccharide:
Fresh indigo fruit is taken, indigo fruit powder is dissolved in distilled water after crushing, solid-liquid ratio 1:15 is placed in 40 DEG C of ultrasound
60min is extracted in water-bath, and it is 175w that ultrasonic power, which is arranged, the dehydrated alcohol that 3~5 times of volumes are added after extraction stands 10h, so
4000rpm removes supernatant after being centrifuged 20min afterwards, and filter residue is dissolved with distilled water, with Sevag [V (chloroform): V (n-butanol)
=4:1] reagent removing protein, sediment is freeze-dried up to indigo fruit polyoses extract.
4. a kind of compound probiotic intestinal-specific control delivery preparation method according to claim 1, which is characterized in that institute
It states in step (3), dry oralbumin is dissolved in distilled water, by oralbumin and indigo while magnetic agitation
Indigo fruit polysaccharide solution is added dropwise according to volume ratio 2:1 in fruit polysaccharide solution, overnight to guarantee sufficiently hydration at 4 DEG C.
5. a kind of compound probiotic intestinal-specific control delivery preparation method according to claim 1, which is characterized in that institute
Stating the volume ratio of thallus liquid and compound in step (4) is 1:1.
6. made from a kind of compound probiotic intestinal-specific control delivery preparation method as claimed in any one of claims 1 to 5
Control delivery.
7. a kind of method for preparing wet capsule using control delivery as claimed in claim 6, which is characterized in that detailed process is such as
Under: obtained system solution is stirred continuously, room temperature is cooled to, is then 5 DEG C in ice bath environment decline low temperature, solidification is added
Agent CaCl2Precipitation to be precipitated is persistently stirred after solution, is collected by filtration after centrifugation, with the brine microballoon of sterilizing, is obtained
Wet capsule.
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Application publication date: 20190528 |