CN110327302A - A kind of chitosan-pectin compound system preparation method and applications enhancing cowberry anthocyanin stability - Google Patents
A kind of chitosan-pectin compound system preparation method and applications enhancing cowberry anthocyanin stability Download PDFInfo
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Abstract
The invention discloses a kind of chitosan-pectin compound system preparation methods for enhancing cowberry anthocyanin stability, taking concentration is the aqueous pectin solution of 0.01~0.1mg/ml, cowberry anthocyanin is added into aqueous pectin solution, add the chitosan solution that concentration is 0.02~0.12mg/ml, it stirs evenly, obtains mixed solution A；4~25 DEG C, by the mixed solution A after homogenate, ultrasonic disperse and homogeneous vacuum freeze drying, obtain chitosan-pectin compound system.The chitosan-pectin compound system has different degrees of protective effect for the oxidative stress of Caenorhabditis elegans, heat shock, ultraviolet damage and acrylamide toxicity；Cause rat kidney cell apoptosis that there is certain inhibiting effect acrylamide, can be used for preparing food or health care product.The storage-stable of bioactive ingredients cowberry anthocyanin can be improved in the present invention, the destruction of anthocyanin molecular structure caused by reducing because of conditions such as environmental factors.
Description
Technical field
The present invention relates to Food Science and Engineering technical fields, steady more specifically to a kind of enhancing cowberry anthocyanin
The preparation method and applications of qualitative chitosan-pectin compound system.
Background technique
Cowberry (Vaccinium vitis-idaea Linn), ericad, fruit is berry, blue or red,
Subcircular.Anthocyanin class substance rich in cranberry, anthocyanin are that anthocyanidin is combined into sugar with glycosidic bond
Flavonoids is water-soluble natural pigment, bright in luster, safe and non-toxic, has natural and natural colorant
Double effects, can be applied to enhance human immunity, delay senescence, prevent cardiovascular disease, improve eyesight, anti-mutation, drop
Blood lipid, the plurality of health care functions such as protect liver.Therefore, the industrialization prospect of anthocyanin is boundless.
However, the stability of anthocyanin is lower, structure changes vulnerable to such environmental effects, in dietary supplements
It will receive limitation in the application such as food and drink.
Layer-by-layer is a kind of benefit as a kind of novel loading bioactive ingredients micro-nano ball technology of preparing
With the technology of the spontaneous deposition film forming of the active force between high-molecular compound, wherein electrostatic self-assembled technology is to utilize the opposite electricity of band
Polyelectrolyte between lotus adsorbs deposition mutually based on the effect of electrostatic force to form multilayer film, easy to operate, production film
Layer uniform, controllable and performance stabilization, are widely recognized in various fields.
Chitosan is the marine polysaccharide of a kind of polycation amino obtained after chemistry or enzyme modification by chitin,
And pectin is a kind of natural less toxic polyanionic polysaccharide extracted from the most plants cell wall such as apple, citrus, pears, is passed through
Electrostatic interaction forms compound polyelectrolyte (PEC), can be used for loading bioactive substance anthocyanin.
Summary of the invention
The carrier that the object of the present invention is to provide cowberry anthocyanin contents is high, stability is good, and keep its functional activity at
Divide and be not easily susceptible to lose, proposes a kind of chitosan-pectin compound system preparation method for enhancing cowberry anthocyanin stability
And its application, the system preparation process is simple, and has various functions health-care effect, and compared with anthocyanin, anthocyanin is steady
The activity of state compound system is higher: for the oxidative stress of Caenorhabditis elegans, heat shock, ultraviolet damage and acrylamide poison
Property there is different degrees of protective effect, and lead to the inhibiting effect of normal rat kidney (NRK) Apoptosis to acrylamide.
In order to achieve the above object, it is compound to provide a kind of chitosan-pectin for enhancing cowberry anthocyanin stability by the present invention
The preparation method of system, includes the following steps:
S1, it prepares mixed solution: using chitosan-pectin as electrostatic self-assembled carrier load anthocyanin, taking concentration
Cowberry anthocyanin solid powder is slowly added into aqueous pectin solution by the aqueous pectin solution of 0.01~0.1mg/ml, is mixed, then
It is slowly added to the chitosan solution that concentration is 0.02~0.12mg/ml, stirring is obtained to being uniformly mixed containing chitosan-pectin-flower
The mixed solution A of color glycosides, on the basis of the pectin weight in the aqueous pectin solution, the weight ratio of the anthocyanin and pectin is
2:3~2:5；The volume ratio of the chitosan solution and aqueous pectin solution is 1:1~1:2
S2, loading: under the conditions of 4~25 DEG C, passing through homogenate, ultrasonic disperse and homogeneous for mixed solution A described in step S1,
Obtain mixed solution B；
S3, freeze-drying: carrying out vacuum freeze drying for mixed solution B described in step S2, obtain chitosan-pectin compound system,
Vacuum sealing preservation.
Under preferred embodiment, the deacetylation of chitosan described in step S1 is 95%~100%；The system of the chitosan solution
Preparation Method are as follows: dissolve chitosan in the acetic acid solution of 1%~2% (V acetic acid/(V water+V acetic acid)).
Under preferred embodiment, loaded described in step S2 specifically: mixed solution A described in step S1 is placed in 1000~
3500rpm 10~20min of homogenate, in 200~500W of power, 4~25 DEG C, 5~15min of ultrasound, using 20~70MPa homogeneous
Obtain mixed solution B.
Under preferred embodiment, step S3 specifically: by mixed solution B described in step S2 be placed in -20 DEG C~-80 DEG C pre-cooling 2~
4h, -50 DEG C, 24~48h of vacuum freeze drying under the conditions of vacuum degree 40Pa, drying process are protected from light, and obtain chitosan-pectin complex
System, vacuum sealing are placed in 4~25 DEG C of preservations.
Under preferred embodiment, chitosan-pectin compound system preparation method of the enhancing cowberry anthocyanin stability, packet
Include step:
S1, it prepares mixed solution: being specifically electrostatic self-assembled carrier load anthocyanin using chitosan-pectin, take dense
Degree is 0.04mg/mL aqueous pectin solution 3mL, and 0.08mg cowberry anthocyanin solid powder is slowly added to the aqueous pectin solution
In, it is uniform using magnetic stirrer, it is slow added into 3mL 0.04mg/mL chitosan solution, is uniformly mixed, must mix molten
Liquid A；Wherein, the preparation method of the chitosan solution is to dissolve chitosan in 2% (V acetic acid/V (water+acetic acid)=2%)
Acetic acid solution in；
S2, loading: by mixed solution A described in step S1 with adjustable high-speed refiner in 3000rpm speed conditions backspin
Turn 10min, is ultrasonically treated, ultrasonic power 200W, time 15min, to ensure anthocyanin encapsulation completely, finally using equal
Matter machine carries out homogeneous, and homogenization pressure 40MPa keeps anthocyanin evenly dispersed, obtains mixed solution B, whole operation process is at 20 DEG C
Cryogenic conditions under carry out；
S3, by mixed solution B homogeneous described in step S2 it is complete after gently pour into culture dish, -80 DEG C of pre-cooling 2h, -50
DEG C, carry out vacuum freeze drying processing for 24 hours under the conditions of vacuum degree 40Pa, obtain chitosan-pectin compound system, sufficiently after freeze-drying,
Sealing is vacuumized, is refrigerated under the conditions of 4 DEG C spare.
It is a further object of the invention to provide chitosan-pectin complexs of the enhancing cowberry anthocyanin stability
The application of system, for protecting oxidative stress, heat shock, ultraviolet damage and the inhibition acrylamide toxicity of Caenorhabditis elegans, with
And for inhibiting acrylamide to lead to the apoptosis of normal rat kidney (NRK) cell.
It is a kind of for inhibiting the drug or food of acrylamide toxicity, the shell that above method preparation is added in the preparation is poly-
Sugar-pectin compound system；A kind of shell that for inhibiting the drug or food of oxidative damage, in the preparation prepared by the addition above method
Glycan-pectin compound system；Above method preparation is added in a kind of drug or food for reducing ultraviolet damage in the preparation
Chitosan-pectin compound system；It is a kind of for resisting the drug or food of heat shock, in the preparation be added the above method preparation
Chitosan-pectin compound system.
The beneficial effects of the present invention are:
1, the present invention uses chitosan and pectin as complex carrier raw material, though this raw material is it has been reported that utilize this
Raw material prepares carrier load cowberry anthocyanin, and further carries out the invention of functional activity evaluation still using caenorhabditis elegan and cell
Belong to the first.
2, the storage-stable of bioactive ingredients cowberry anthocyanin can be improved in the present invention, reduces because of conditions such as environmental factors
Cause the destruction of anthocyanin molecular structure.
3, the present invention has various functions health-care effect: for the oxidative stress of Caenorhabditis elegans, heat shock, ultraviolet damage
Acrylamide toxicity is injured with different degrees of protective effect；Normal rat kidney (NRK) Apoptosis is caused to acrylamide
With certain inhibiting effect.
Detailed description of the invention
Fig. 1 is atomic force microscope (AFM) 3 dimensional drawing of cowberry anthocyanin.
Fig. 2 is atomic force microscope (AFM) 2 d plane picture of cowberry anthocyanin.
Fig. 3 is chitosan-pectin compound system atomic force microscope (AFM) 3 dimensional drawing prepared in embodiment 1.
Fig. 4 is chitosan-pectin compound system atomic force microscope (AFM) 2 d plane picture prepared in embodiment 1.
Fig. 5 is the atomic force microscope (AFM) that balling-up is encapsulated in the chitosan-pectin compound system prepared in embodiment 1
2 d plane picture.
Fig. 6 is the particle size that balling-up is encapsulated in the chitosan-pectin compound system prepared in embodiment 1.
Fig. 7 is chitosan-pectin compound system cold field emission scanning electron microscope (SEM) figure prepared in comparative example 1.
Fig. 8 is chitosan-pectin compound system cold field emission scanning electron microscope (SEM) figure prepared in embodiment 1.
Fig. 9 is chitosan-pectin compound system Fourier Transform Infrared Spectroscopy (FT-IR) figure prepared in embodiment 1；
Wherein ANC indicates that cowberry anthocyanin solid particle, PC are expressed as jelly powder, and CS is expressed as chitosan powder, and CS-PC ANC indicates real
Apply glycosides chitosan-pectin compound system of the preparation of example 1.
Figure 10 is the cowberry anthocyanin stable state compound system for preparing in cowberry anthocyanin and embodiment 1 under the conditions of being protected from light
Storage-stable；Wherein ANC indicates that cowberry anthocyanin solid particle, CS-PC ANC indicate chitosan-prepared by embodiment 1
Pectin compound system.
Figure 11 is chitosan-storage of the pectin compound system under illumination condition prepared in cowberry anthocyanin and embodiment 1
Hide stability；Wherein ANC indicates that cowberry anthocyanin solid particle, CS-PC ANC indicate chitosan-pectin prepared by embodiment 1
Compound system.
Figure 12 is that cowberry anthocyanin acts on caenorhabditis elegan oxidative stress.
Figure 13 is that the chitosan-pectin compound system prepared in embodiment 1 acts on caenorhabditis elegan oxidative stress.
Figure 14 is that cowberry anthocyanin acts on caenorhabditis elegan heat shock.
Figure 15 is that the chitosan-pectin compound system prepared in embodiment 1 acts on caenorhabditis elegan heat shock.
Figure 16 is cowberry anthocyanin damaging action ultraviolet for caenorhabditis elegan.
Figure 17 is the chitosan-pectin compound system damaging action ultraviolet for caenorhabditis elegan prepared in embodiment 1.
Figure 18 is that cowberry anthocyanin inhibits the effect of caenorhabditis elegan acrylamide toxicity.
Figure 19 is that the chitosan-pectin compound system prepared in embodiment 1 inhibits the effect of caenorhabditis elegan acrylamide toxicity.
Figure 20 is the microscope photo of the morphological observation of normal rat kidney (NRK) cell.
Figure 21 is morphological observation of the acrylamide (10mmol/L) for normal rat kidney (NRK) cytotoxicity.
Figure 22 is the morphological observation that cowberry anthocyanin (5 μ g/mL) inhibits acrylamide toxicity.
Figure 23 is the morphological observation that cowberry anthocyanin (10 μ g/mL) inhibits acrylamide toxicity.
Figure 24 is the morphological observation that cowberry anthocyanin (20 μ g/mL) inhibits acrylamide toxicity.
Figure 25 is the morphological observation that cowberry anthocyanin (40 μ g/mL) inhibits acrylamide toxicity.
Figure 26 is that the chitosan-pectin compound system (5 μ g/mL) prepared in embodiment 1 inhibits the thin of acrylamide toxicity
Born of the same parents' morphological observation.
Figure 27 is that the chitosan-pectin compound system (10 μ g/mL) prepared in embodiment 1 inhibits the thin of acrylamide toxicity
Born of the same parents' morphological observation.
Figure 28 is that the chitosan-pectin compound system (20 μ g/mL) prepared in embodiment 1 inhibits the thin of acrylamide toxicity
Born of the same parents' morphological observation.
Figure 29 is that the chitosan-pectin compound system (40 μ g/mL) prepared in embodiment 1 inhibits the thin of acrylamide toxicity
Born of the same parents' morphological observation.
Figure 30 is the attached cell fluorescence in situ detection figure of normal rat kidney (NRK) cell.
Figure 31 is attached cell fluorescence in situ of the acrylamide (10mmol/L) for normal rat kidney (NRK) cytotoxicity
Detection figure.
Figure 32 is the attached cell fluorescence in situ detection figure that cowberry anthocyanin (20 μ g/mL) inhibits acrylamide toxicity.
Figure 33 is the attached cell fluorescence in situ detection figure that cowberry anthocyanin (40 μ g/mL) inhibits acrylamide toxicity.
Figure 34 is that the chitosan-pectin compound system (20 μ g/mL) prepared in embodiment 1 inhibits the patch of acrylamide toxicity
Parietal cell fluorescence in situ detection figure.
Figure 35 is that the chitosan-pectin compound system (40 μ g/mL) prepared in embodiment 1 inhibits the patch of acrylamide toxicity
Parietal cell fluorescence in situ detection figure.
Figure 36 is that the chitosan-pectin compound system prepared in cowberry anthocyanin and embodiment 1 inhibits acrylamide toxicity
MTT measurement wherein ANC indicates cowberry anthocyanin, AA indicates that acrylamide, the shell that CS-PC ANC indicates prepared by embodiment 1 are poly-
Sugar-pectin compound system；Obtained by Figure 20~Figure 36 is observed under 10 times of visuals field of fluorescence inverted microscope, scale bar size is equal
It is 250 μm.
Specific embodiment
In order to make those skilled in the art better understand the present invention, With reference to embodiment to the present invention into
One step explanation.
A kind of chitosan-pectin compound system building and application enhancing cowberry anthocyanin stability, using with phase
The chitosan and pectin of counter charges form compound polyelectrolyte (PEC) by electrostatic interaction, and it is living to act on loading biology
Property substance anthocyanin to keep its functional active components not suffer a loss there is protection for Caenorhabditis elegans and cellular damage
Effect.Include the following steps:
S1, preparation cowberry anthocyanin chitosan-pectin compound system: the system using chitosan-pectin as electrostatic oneself
Carrier load anthocyanin is assembled, aqueous pectin solution is taken, anthocyanin solid powder is slowly added into aqueous pectin solution, is mixed,
It is slow added into chitosan solution, stirring obtains cowberry anthocyanin chitosan-pectin compound system, the pectin to being uniformly mixed
Weight ratio 2:3~2:5 of 0.01~0.1mg/ml of concentration of aqueous solution, the anthocyanin and pectin solution, the chitosan are molten
The concentration of liquid is 0.02~0.12mg/ml；
S2, loading: under the conditions of 4~25 DEG C, cowberry anthocyanin chitosan described in step S1-pectin compound system is passed through
Homogenate, ultrasonic disperse and homogeneous, obtain mixed solution；
S3, freeze-drying: mixed solution described in step S2 is subjected to vacuum freeze drying, obtains cowberry anthocyanin chitosan-pectin
Compound system, vacuum sealing preservation.
Under preferred embodiment, stirring described in step S1 is low-medium speed magnetic agitation, 15~30min of stirring；The chitosan
Deacetylation is 95%~100%；The anthocyanin is cowberry anthocyanin；The chitosan solution the preparation method comprises the following steps: by shell
Glycan is dissolved in the acetic acid solution of 1%~2% (V acetic acid/V water).
Under preferred embodiment, embedded described in step S2 specifically: cowberry anthocyanin chitosan-pectin described in step S1 is compound
1000~3500rpm of system homogenate, 10~20min, 200~500W of power, 4~25 DEG C, 5~15min of ultrasound, using 20~
70MPa homogeneous obtains mixed solution.
Under preferred embodiment, vacuum freeze drying described in step S3, parameter are as follows: -20 DEG C~-80 DEG C pre-cooling 2~4h, -50 DEG C,
24~48h of vacuum freeze drying, drying process are protected from light under the conditions of vacuum degree 40Pa；The temperature of the preservation is 4~25 DEG C.
The cowberry anthocyanin chitosan-pectin compound system for the oxidative stress of Caenorhabditis elegans, heat shock,
Ultraviolet damage and acrylamide toxicity have different degrees of protective effect, and lead to normal rat kidney to acrylamide
(NRK) inhibiting effect of Apoptosis.
The cowberry anthocyanin that following each examples use, manufacturer: Jianfeng Natural Product R&D Development Co., Ltd., Tianjin,
No. CAS: 84082-34-8；Chitosan, manufacturer: aladdin Aladdin, article No.: C105799-100g, No. CAS: 9012-
76-4；Pectin, manufacturer: aladdin Aladdin, article No.: P112756-100g, No. CAS: 900-69-5.
Embodiment 1: chitosan-pectin compound system is prepared
S1, it prepares mixed solution: being specifically electrostatic self-assembled carrier load anthocyanin using chitosan-pectin, prepare
Concentration is 0.04mg/mL aqueous pectin solution 3mL, and 0.08mg cowberry anthocyanin solid powder is slowly added to aqueous pectin solution
In, it is uniform using magnetic stirrer, it is slow added into 3mL 0.04mg/mL chitosan solution, is uniformly mixed, must mix molten
Liquid A is stand-by；Wherein, the preparation method of the chitosan solution be dissolve chitosan in 2% (V acetic acid/V (water+acetic acid)=
2%) in acetic acid solution；
S2, loading: by mixed solution A described in step S1 with adjustable high-speed refiner in 3000rpm speed conditions backspin
Turn 10min, is ultrasonically treated, ultrasonic power 200W, time 15min, to ensure anthocyanin encapsulation completely, finally using equal
Matter machine carries out homogeneous, and homogenization pressure 40MPa keeps anthocyanin evenly dispersed, and whole operation process is under 20 DEG C of cryogenic conditions
It carries out, obtains mixed solution B；
S3, mixed solution B described in step S2 is gently poured into culture dish, -80 DEG C of pre-cooling 2h, in -50 DEG C, vacuum degree
Vacuum freeze drying processing is carried out under the conditions of 40Pa for 24 hours, obtains chitosan-pectin compound system, sufficiently after freeze-drying, vacuum-pumping density
Envelope, refrigerates spare under the conditions of 4 DEG C.
Embodiment 2: chitosan-pectin compound system morphology characterization, size and storage-stable
Chitosan-pectin compound system pattern: Fig. 1, Fig. 2 are the atomic force microscope (AFM) of cowberry anthocyanin respectively
3 D stereo and 2 d plane picture, Fig. 3, Fig. 4 are the chitosan-pectin compound system atomic force prepared in embodiment 1 respectively
The 3 D stereo and 2 d plane picture of microscope (AFM), Fig. 5, Fig. 6 are that chitosan-pectin for preparing in embodiment 1 is compound respectively
Atomic force microscope (AFM) 2 d plane picture and particle size of system.The results show that compared to cowberry anthocyanin, chitosan-
The chain of mountains shape that pectin compound system is evenly distributed, is deposited on mica sheet, shows chitosan-pectin compound system particle size
In 160nm or so, it is individually present and is evenly distributed in the solution；
Chitosan-pectin compound system microstructure: Fig. 7 is chitosan-pectin compound system prepared by comparative example 1
Cold field emission scanning electron microscope (SEM) picture, Fig. 8 are chitosan-pectin compound system cold field emission scanning prepared by embodiment 1
Electronic Speculum (SEM) picture, compared to for comparative example 1, it can be observed that in chitosan-pectin compound system prepared by embodiment 1
Cowberry anthocyanin surrounding molecules are wrapped to form subcircular sugar-coat shell, illustrate that cowberry anthocyanin is successfully loaded to sugar-coat shell
In, preferably protected；
Chitosan-pectin compound system fourier-transform infrared (FT-IR) spectrum: chitosan (CS)-pectin in Fig. 9
(PC) cowberry anthocyanin (ANC) infrared spectroscopy is in 1500~1700cm-1The variation at place discloses CS and PC and interacts, and
And 1727cm-1And 1631cm-1Peak value respectively with the carboxyl (COO in PC-) and amide-I and amide-II group in CS vibration
Move it is related, these the result shows that CS ionization amino (NH3 +) with the ionization carboxyl (COO of PC-) between there are electrostatic interactions；
Figure 10, Figure 11 are that chitosan-pectin compound system prepared by embodiment 1 is being protected from light and the storage-stable under illumination condition respectively
Property: the results show that being protected from light under illumination condition, the storage-stable of anthocyanin is can be improved in chitosan-pectin compound system.
Embodiment 3: the protective effect that chitosan-pectin compound system damages Caenorhabditis elegans
Chitosan-pectin the compound system prepared to embodiment 1 detects the protective effect of Caenorhabditis elegans；It will
Wild type N2 synchronizing of nematode, when cultivating nematode to L4 period, respectively feeding contain final concentration of 0 (control),
40, (OD570 is about by the cowberry anthocyanin of 80,160,320 μ g/mL or chitosan-pectin compound system Escherichia coli OP50
0.55~0.6)；The cowberry anthocyanin for taking 200 μ L to contain above-mentioned various concentration respectively or chitosan-pectin compound system large intestine
Bacillus OP50 is placed in nematode growth medium (Nematode Growth Media, NGM) culture medium and cultivates, and culture dish diameter is
35mm, the nematode in 20 L4 periods of picking in every group of NGM culture medium, 3 groups are parallel, and cultivation temperature is 20 DEG C, feeding for 24 hours after, will
Nematode picking adds the incentive condition that damage is caused for nematode into 96 orifice plates respectively:
(1), oxidative stress
The hydrogenperoxide steam generator (M9 buffer) of 200 μ L 15mM is added in every hole in 96 orifice plates, counts line every 1h
The survival rate of worm, until nematode is all dead, nematode body is stiff, and i.e. confirmation reactionless to platinum wire stimulation is dead.
(2), heat shock
The above-mentioned NGM culture medium containing nematode is placed in 35 DEG C of incubators, every the survival rate of 1h statistics nematode, arrives line
Worm is all dead, and nematode body is stiff, and i.e. confirmation reactionless to platinum wire stimulation is dead.
(3), ultraviolet irradiation
By in the camera bellows of the above-mentioned ultraviolet light irradiation of NGM culture medium containing nematode, the ultraviolet lamp tube for the use of wavelength being 365nm
Irradiation, it is reactionless to platinum wire stimulation to confirm death until the whole death of nematode every the survival rate of 1h statistics nematode.
(4), acrylamide toxicological experiment
200 μ L 10mM acrylamide solutions (M9 buffer) are added in every hole in 96 orifice plates, count nematode every 1h
Survival rate, until nematode is all dead, nematode body is stiff, it is reactionless to platinum wire stimulation confirm it is dead.
Above-mentioned experiment is repeated 3 times, and is damaged with studying cowberry anthocyanin and chitosan-pectin compound system for caenorhabditis elegan
Protective effect, at interval of 1h observe a nematode survival rate.To nematode death judgment criteria: without mobile and swallowing act,
Platinum wire touch after still without any reaction；Figure 12, Figure 13 are cowberry anthocyanin and chitosan-pectin compound system respectively for show
Habronemic oxidative stress effect；Figure 14, Figure 15 are cowberry anthocyanin and chitosan-pectin compound system respectively for beautiful line
The effect of worm heat shock；Figure 16, Figure 17 are cowberry anthocyanin and chitosan-pectin compound system damage ultraviolet for caenorhabditis elegan respectively
Wound effect；Figure 18, Figure 19 are cowberry anthocyanin and chitosan-pectin compound system respectively for caenorhabditis elegan acrylamide toxicity
Effect；The results show that passing through feeding chitosan-pectin compound system caenorhabditis elegan time-to-live compared with cowberry anthocyanin
It is longer, and for oxidative stress, heat shock, the toxicity of ultraviolet light irradiation and acrylamide, the preferable energy for resisting damage is presented
Power.
Embodiment 4: inhibiting effect of the chitosan-pectin compound system for cell acrylamide (AA) toxicity
Measure inhibiting effect of the chitosan-pectin compound system to cell acrylamide toxicity of the preparation of embodiment 1；It selects
The DMEM culture medium of NRK cell and the fetal calf serum containing 10% (V fetal calf serum/culture medium=1/9 V DMEM).By cell with
5×104The density in/hole is inoculated in 96 orifice plates, the CO for being 5% in volume fraction212h is incubated in incubator, then in the medium
The cowberry anthocyanin and chitosan-pectin compound system for adding different final concentrations (0,5,10,20,40 μ g/mL) are further cultured for 12h
Afterwards, the original position that the reaction of 50 μ L acrylamides (10mmol/L) carries out morphological observation and attached cell afterwards for 24 hours is added in every hole
Fluorescence detection, every hole is added 20 μ L of MTT (0.5mg/mL) solution and carries out 4h incubation later, detects cell viability.
Morphological observation: Figure 20 is the cell of normal rat kidney (NRK) cell (cell culture medium culture is only added)
The microscope photo of morphological observation, is shown as the normal cell form of shuttle shape, Figure 21~Figure 25 be respectively various concentration (0,5,
10,20,40 μ g/mL) cowberry anthocyanin inhibit acrylamide toxicity morphological observation, be added acrylamide effect after
Round cell form, be shown as Apoptosis or necrosis (Figure 21), and the cowberry anthocyanin under the concentration of 40 μ g/mL (Figure 25)
It is inhibited for acrylamide toxicity；Figure 26~Figure 29 is that various concentration (0,5,10,20,40 μ g/mL) shell is poly- respectively
Sugar-pectin compound system inhibits the morphological observation of acrylamide toxicity, in 20 μ g/mL (Figure 28) and 40 μ g/mL (figure
29) chitosan-pectin compound system has better inhibiting effect for the acrylamide toxicity of cell.
The fluorescence in situ of attached cell detects: using Annexin V-FITC and propidium iodide (PI) dyeing liquor for cell
It is dyed, figure Green fluorescence is Annexin V-FITC staining positive cells (cytoplasm), and red fluorescence is propidium iodide
Staining positive cells (nucleus).It is only apoptotic cell by green fluorescence dyeing (having at bright spot), by green and red fluorescence
Double dyes (bright spot is compared with many places) are non-viable non-apoptotic cells, are not normal cell by fluorescent staining (no bright spot).Figure 30 is normal rat
The attached cell fluorescence in situ of kidney (NRK) cell (cell culture medium culture is only added) detects figure；Figure 31~Figure 33 is not respectively
Same final concentration (0,20,40 μ g/mL) cowberry anthocyanin inhibits the attached cell fluorescence in situ of acrylamide toxicity to detect figure, figure
34, Figure 35 is that different final concentration (20,40 μ g/mL) chitosans-pectin compound system inhibits the adherent of acrylamide toxicity respectively
Cell in-situ fluorescence detection figure, normal rat kidney (NRK) cell (Figure 30) will not be colored (no bright spot) as the result is shown, not plus more
The cell of tangerine anthocyanin is influenced to be shown as apoptosis or necrosis (all bright spots), normal chitosan-by acrylamide toxicity
Pectin compound system has better protective effect for cell.
The MTT of cell is measured: Figure 36 normal rat kidney (NRK) cell (cell culture medium culture is only added) as the result is shown
Cell viability is fine (control), after acrylamide is added, since the toxic effect of acrylamide declines cell viability, very
To apoptosis or necrosis；And during the cultivation process, due to cowberry anthocyanin, the addition of chitosan-pectin compound system, for propylene
The toxicity of amide has certain resistant function, and is added to chitosan-pectin compound system cell resistance acrylamide damage
The effect of wound is more preferable.Obtained by all of above cell picture is observed under 10 times of visuals field of fluorescence inverted microscope, scale bar is big
Small is 250 μm.
Comparative example 1:
S1, it prepares mixed solution: being specifically electrostatic self-assembled carrier load anthocyanin using chitosan-pectin, prepare
Concentration is 0.04mg/mL aqueous pectin solution 3mL, and 0.12mg cowberry anthocyanin solid powder is slowly added to aqueous pectin solution
In, it is uniform using magnetic stirrer, it is slow added into 3mL 0.04mg/mL chitosan solution, is uniformly mixed, must mix molten
Liquid A；Wherein, the preparation method of the chitosan solution is to dissolve chitosan in 2% (V acetic acid/V (water+acetic acid)=2%)
Acetic acid solution in；
S2, loading: by mixed liquor A described in step S1 with adjustable high-speed refiner in 3000rpm speed conditions backspin
Turn 10min, is ultrasonically treated, ultrasonic power 200W, time 15min, to ensure anthocyanin encapsulation completely, finally using equal
Matter machine carries out homogeneous, and homogenization pressure 40MPa keeps anthocyanin evenly dispersed, and whole operation process is under 20 DEG C of cryogenic conditions
It carries out, obtains mixed solution B；
S3, mixed solution B described in step S2 is gently poured into culture dish, -80 DEG C of pre-cooling 2h, in -50 DEG C, vacuum degree
Vacuum freeze drying processing is carried out under the conditions of 40Pa for 24 hours, obtains chitosan-pectin compound system, sufficiently after freeze-drying, vacuum-pumping density
Envelope, refrigerates spare under the conditions of 4 DEG C.
The foregoing is only a preferred embodiment of the present invention, but scope of protection of the present invention is not limited thereto,
Anyone skilled in the art within the technical scope of the present disclosure, according to the technique and scheme of the present invention and its
Inventive concept is subject to equivalent substitution or change, should be covered by the protection scope of the present invention.
Claims (14)
1. a kind of chitosan-pectin compound system preparation method for enhancing cowberry anthocyanin stability, which is characterized in that including
Step:
S1, prepare mixed solution: taking concentration is the aqueous pectin solution of 0.01~0.1mg/ml, and cowberry anthocyanin is added to pectin
It in aqueous solution, mixes, adding concentration is 0.02~0.12mg/ml chitosan solution, and stirring obtains poly- containing shell to being uniformly mixed
Sugar-pectin-anthocyanin mixed solution A；On the basis of the total weight of pectin in the aqueous pectin solution, the cowberry anthocyanin
Weight ratio with pectin is 2:3~2:5；The volume ratio of the chitosan solution and aqueous pectin solution is 1:1~1:2；
S2, loading: it under the conditions of 4~25 DEG C, by mixed solution A described in step S1 by homogenate, ultrasonic disperse and homogeneous, obtains mixed
Close solution B；
S3, freeze-drying: mixed solution B described in step S2 is subjected to vacuum freeze drying, obtains chitosan-pectin compound system, vacuum
Sealing preservation.
2. enhance chitosan-pectin compound system preparation method of cowberry anthocyanin stability according to claim 1,
Be characterized in that, chitosan solution described in step S1 the preparation method comprises the following steps: dissolving chitosan in volume fraction is 1%~2%
In acetic acid solution.
3. enhance chitosan-pectin compound system preparation method of cowberry anthocyanin stability according to claim 1,
Be characterized in that, described in step S2 load specifically: by mixed solution A described in step S1 be placed in 1000~3500rpm homogenate 10~
20min obtains mixed solution B using 20~70MPa homogeneous in 200~500W of power, 4~25 DEG C, 5~15min of ultrasound.
4. enhance chitosan-pectin compound system preparation method of cowberry anthocyanin stability according to claim 1,
It is characterized in that, step S3 specifically: mixed solution B described in step S2 is placed in -20 DEG C~-80 DEG C pre-cooling 2~4h, -50 DEG C, true
Reciprocal of duty cycle 40Pa, dry 24~48h, obtain chitosan-pectin compound system, vacuum sealing is placed in 4~25 DEG C of preservations.
5. enhance chitosan-pectin compound system preparation method of cowberry anthocyanin stability according to claim 1,
It is characterized in that, comprising steps of
S1, prepare mixed solution: taking concentration is 0.04mg/mL aqueous pectin solution 3mL, and 0.08mg cowberry anthocyanin is added to institute
It states in aqueous pectin solution, it is uniform using magnetic stirrer, 3mL0.04mg/mL chitosan solution is added, is uniformly mixed,
Obtain mixed solution A；Wherein, the preparation method of the chitosan solution be dissolve chitosan in volume fraction 2% acetic acid it is molten
In liquid；
S2, loading: by mixed solution A described in step S1 with adjustable high-speed refiner in 3000rpm homogenate 10min；It carries out again
Ultrasonic treatment, ultrasonic power 200W, time 15min；Homogeneous finally is carried out using homogenizer, homogenization pressure 40MPa must be mixed
Solution B；Whole operation process carries out under conditions of 20 DEG C；
S3, mixed solution B described in step S2 is poured into culture dish, -80 DEG C of pre-cooling 2h, in -50 DEG C, vacuum degree 40Pa, drying
For 24 hours, chitosan-pectin compound system is obtained, sealing is vacuumized, in 4 DEG C of preservations.
6. a kind of for inhibiting the drug or food of acrylamide toxicity, which is characterized in that claim 1-5 is added in the preparation
Chitosan-pectin compound system of either method preparation.
7. a kind of for inhibiting the drug or food of oxidative damage, which is characterized in that it is any that claim 1-5 is added in the preparation
Chitosan-pectin compound system of method preparation.
8. a kind of drug or food for reducing ultraviolet damage, which is characterized in that it is any that claim 1-5 is added in the preparation
Chitosan-pectin compound system of method preparation.
9. a kind of for resisting the drug or food of heat shock, which is characterized in that in the preparation be added claim 1-5 either one
Chitosan-pectin compound system of method preparation.
10. a kind of chitosan-pectin compound system application for enhancing cowberry anthocyanin stability, which is characterized in that by right
It is required that chitosan-pectin compound system of 1-5 either method preparation is used to protect the oxidative damage of Caenorhabditis elegans.
11. a kind of chitosan-pectin compound system application for enhancing cowberry anthocyanin stability, which is characterized in that by right
It is required that chitosan-pectin compound system of 1-5 either method preparation is used to protect the heat shock of Caenorhabditis elegans.
12. a kind of chitosan-pectin compound system application for enhancing cowberry anthocyanin stability, which is characterized in that by right
It is required that chitosan-pectin compound system of 1-5 either method preparation is used to protect the ultraviolet damage of Caenorhabditis elegans.
13. a kind of chitosan-pectin compound system application for enhancing cowberry anthocyanin stability, which is characterized in that by right
It is required that chitosan-pectin compound system of 1-5 either method preparation is for inhibiting acrylamide to the toxicity of Caenorhabditis elegans.
14. a kind of chitosan-pectin compound system application for enhancing cowberry anthocyanin stability, which is characterized in that by right
It is required that chitosan-pectin compound system of 1-5 either method preparation is for inhibiting acrylamide to lead to withering for Rat renal NRK cell
It dies.
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