CN117286058A - Lactobacillus plantarum ZJUIDS19, and preparation and application thereof - Google Patents
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- CN117286058A CN117286058A CN202311157886.6A CN202311157886A CN117286058A CN 117286058 A CN117286058 A CN 117286058A CN 202311157886 A CN202311157886 A CN 202311157886A CN 117286058 A CN117286058 A CN 117286058A
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- lactobacillus plantarum
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- lactalbumin
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- XOSXWYQMOYSSKB-LDKJGXKFSA-L water blue Chemical compound CC1=CC(/C(\C(C=C2)=CC=C2NC(C=C2)=CC=C2S([O-])(=O)=O)=C(\C=C2)/C=C/C\2=N\C(C=C2)=CC=C2S([O-])(=O)=O)=CC(S(O)(=O)=O)=C1N.[Na+].[Na+] XOSXWYQMOYSSKB-LDKJGXKFSA-L 0.000 description 1
- 235000021119 whey protein Nutrition 0.000 description 1
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Abstract
The invention provides lactobacillus plantarum ZJUIDS19, and preparation and application thereof. The classification of this strain was named: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC No.28018. The 16S rDNA complete sequence of the lactobacillus plantarum ZJUIDS19 is shown as SEQ ID No. 1. The invention is based on probiotics separated from inner Mongolian acid dairy products, and plant lactobacillus with antibacterial and antioxidant effects is selected from the probiotics, and its metayuan is extracted and compounded with modified lactalbumin to form a novel hydrogel material. The material has good antibacterial property, strong antioxidant capacity and biocompatibility, and can play a role in promoting wound healing as a wound dressing.
Description
Technical Field
The invention belongs to biomedical materials, in particular to lactobacillus plantarum ZJUIDS19, preparation and application thereof, and relates to lactobacillus plantarum ZJUIDS19, preparation of antibacterial lactalbumin hydrogel loaded with metazoan and application thereof in biomedical materials.
Background
Skin acts as an important interface between the body and the surrounding environment and plays an extremely important role in preventing water loss and preventing invasion of harmful substances and pathogenic microorganisms, where various microflora coexist, including bacteria, fungi and viruses. However, once the skin is damaged, it provides an opportunity for harmful bacteria to invade living tissue, resulting in wound infection and even serious tissue damage. In order to solve bacterial infection of skin wounds, new antibiotics, antibacterial nanoparticles, cationic polymer compounds and antibacterial peptides have become hot spots of research in recent years.
Live bacterial therapies have been used to treat a variety of inflammatory and immunopathogenic diseases through bacterial interference and immunomodulation, and have received great attention over the past few years. Certain beneficial bacteria can create unique local microenvironments by secreting large amounts of metabolites and antimicrobial agents that are suitable for their own survival, but which inhibit the growth of competing microorganisms. Accordingly, beneficial bacteria that secrete biologically active substances with antibacterial, anticancer or immunosuppressive capabilities have been widely used for diagnosis and treatment. However, bacterial use is largely limited due to the inherent properties of bacteria, including rapid proliferation and colonization. Metazoans are safer, more controllable and easier to store as bacterial metabolites than or bacteria, and therefore, extraction and use of metazoans is becoming a hotspot.
Hydrogels are popular biomedical polymers with 3D molecular networks that have been widely used in the fields of drug delivery, implantation and tissue engineering. Hydrogels are used to promote tissue repair and regeneration due to the high biocompatibility and moist healing environment. Lactalbumin (α -LA) is a small globular protein that is found in whey of all mammals. Lactalbumin is the second highest protein content in bovine whey, and bovine and human lactalbumin have 74% sequence identity, similar biological activity and similar amino acid content. These characteristics make it a viable biomaterial, including low immunogenicity, low risk of disease transmission, and biological activity associated with anti-tumor, antibacterial, antioxidant and antihypertensive effects. However, there is little research on the use of lactalbumin hydrogels as medical materials.
Disclosure of Invention
The first object of the present invention is to provide a lactobacillus plantarum ZJUIDS19 strain which has been deposited in the general microbiological center of the chinese microbiological bacterial deposit management committee at month 7 of 2023, classified under the name: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC NO.28018, the preservation address is: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute. The full sequence of the 16S rDNA of the lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS19 is shown as SEQ ID No. 1.
The second object of the present invention is to provide a method for preparing an antibacterial lactalbumin hydrogel loaded with metazoan, which is realized by the following steps:
(1) Sterile fermentation supernatant
Inoculating lactobacillus plantarum ZJUIDS19 into an MRS liquid culture medium by an inoculating loop for culture, transferring the inoculated lactobacillus plantarum ZJUIDS19 into the sterilized MRS liquid culture medium for culture according to the inoculum size of 2 percent (volume percent), obtaining fermentation liquor after culture, centrifugally collecting thalli, suspending the thalli in distilled water, centrifugally collecting supernatant, adjusting the pH value to 6.0, and filtering to obtain sterile fermentation supernatant;
wherein, the MRS liquid culture medium is prepared by dissolving the following components of 10g of peptone, 10g of beef extract, 5g of yeast extract, 2 g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80 ml of Tween, 0.5g of magnesium sulfate and 15 g of manganese sulfate 0.25 agar powder in each liter of distilled water;
(2) Preparation of Lactobacillus plantarum ZJUIDS19 metagen
Vacuum freeze concentrating the aseptic fermentation supernatant, pre-freezing the supernatant at ultralow temperature, freeze drying, vacuumizing, completely freeze drying, decompressing, taking out the metaplasia, and placing in a refrigerator at 4 ℃ to obtain the metaplasia of the lactobacillus plantarum ZJUIDS 19;
(3) Adding the extracted metagen into the modified lactalbumin solution at room temperature, and curing the metagen into gel under ultraviolet irradiation to form the antibacterial lactalbumin hydrogel loaded with the metagen.
The culture time in the step (1) is 18 hours, the centrifugation condition is 10000r/min for 20min, and the pH value is regulated by 2M NaOH.
The ultralow temperature pre-freezing condition of the step (2) is that the step is placed in an ultralow temperature refrigerator at the temperature of minus 80 ℃ to be pre-frozen for at least 2 hours.
The ultraviolet irradiation condition in the step (3) is that the ultraviolet wavelength is 380-405 nm, the irradiation time is 10-60 s, and the ultraviolet intensity is 30mW/cm 2 。
A third object of the present invention is to provide the use of the metaloaded antibacterial milk protein hydrogel in the preparation of biomedical materials. The biomedical material comprises lactobacillus bacteriocin, antibacterial wound dressing, antibacterial and antioxidant live bacteria preparation and antibacterial and antioxidant bacteria powder.
The lactobacillus bacteriocin can be used as a preservative for meat products, milk products, alcoholic beverages, or can be combined with other preservative technologies to be used as a barrier technology in food processing and storage.
The culture solution of the lactobacillus plantarum (Lactobacillus plantarum) ZJUIDS19 provided by the invention has no antibiotic resistance, which indicates that the strain does not carry a drug resistance gene. The antibacterial protein hydrogel loaded with the postsource provided by the invention has the effects of bacteriostasis, anti-inflammatory and antioxidative properties, no antibiotic resistance of culture solution, promotion of skin wound healing and the like. Therefore, the lactobacillus plantarum ZJUIDS19 can be widely applied to the fields of biomedicine and the like.
The invention is based on probiotics separated from inner Mongolian acid dairy products, and plant lactobacillus with antibacterial and antioxidant effects is selected from the probiotics, and its metayuan is extracted and compounded with modified lactalbumin to form a novel hydrogel material. The material has good antibacterial property, strong antioxidant capacity and biocompatibility, and can play a role in promoting wound healing as a wound dressing.
Drawings
FIG. 1 is a colony morphology of Lactobacillus plantarum ZJUIDS19 according to the invention.
FIG. 2 is a diagram showing the morphology of gram-stained cells of Lactobacillus plantarum ZJUIDS19 according to the present invention.
FIG. 3 is an electrophoretically identified graph of 16S rDNA of Lactobacillus plantarum ZJUIDS19 according to the present invention.
FIG. 4 is a graph of a loaded metachromatic lactalbumin hydrogel of the present invention.
Fig. 5 is a graph showing rheological properties of the prepared metachromatic lactalbumin hydrogels.
FIG. 6 shows the microstructure of the experimental loaded metachromatic lactalbumin hydrogel.
FIG. 7 is a graph of the in vitro antibacterial results of metachromatic lactalbumin hydrogels.
FIG. 8 is a graph showing the in vivo antibacterial results of metachromatic lactalbumin hydrogels.
Fig. 9 is a graph showing wound healing morphology of each group of mice tested.
FIG. 10 is a graph of HE staining of wounds from mice of each group according to the invention.
FIG. 11 is a map of Masson staining of wounds from mice of each group according to the invention.
Detailed Description
The invention will be further described with reference to the drawings and the specific embodiments, but the scope of the invention is not limited thereto.
Example 1 screening, identification and preparation of metazoan of Lactobacillus plantarum ZJUIDS19
1. Screening of Lactobacillus plantarum ZJUIDS19
1.1 sample Source
The strain used in the invention is derived from an inner Mongolian acid emulsion sample. Samples were collected in 20 total.
1.2 isolation and purification of Strain
About 5g of the sample was collected in a sterile tube and immediately sent to a laboratory for strain isolation and purification. 1g of sample is taken and placed into 9mL of MRS liquid culture medium during separation, and after vortex mixing, enrichment culture is carried out for 48 hours at 37 ℃; then 1mL of culture solution is sucked in an ultra-clean bench, ten-time gradient dilution is carried out by using sterile normal saline, and 10 is selected -5 、10 -6 、10 -7 Three dilution gradients, 100. Mu.L of each bacterial suspension was applied to MRS solid medium and incubated at 37℃for 48h. After the culture is finished, selecting a plate with 30-300 single colonies from the MRS solid culture medium, picking typical colonies, carrying out repeated streak separation on the MRS agar plate until the colony morphology on the whole plate is consistent, and picking single colonies to the MRS liquid culture medium for expansion culture. The resulting strain was finally stored frozen in a-80℃refrigerator containing 40% (w/v) glycerol in MRS liquid medium.
The liquid MRS culture medium is prepared by dissolving 10g of peptone, 10g of beef extract, 5g of yeast extract, 2 g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80 ml of Tween, 0.5g of magnesium sulfate and 0.25 g of manganese sulfate in each liter of distilled water.
The solid MRS culture medium is prepared by dissolving 10g of peptone, 10g of beef extract, 5g of yeast extract, 2 g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80 ml of Tween, 0.5g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder in each liter of distilled water.
2. Identification of Lactobacillus plantarum ZJUIDS19
2.1 colony characterization
After the lactobacillus plantarum ZJUIDS19 is cultured in an MRS solid culture medium for 48 hours, the surface edges are smooth, the shape is regular and circular, and the milk white is shown in figure 1.
2.2 morphology under microscope
Lactobacillus plantarum ZJUIDS19 colony smear: gram staining was positive, sporulation was absent, rod-like, see figure 2.
2.3 16S rDNA identification
Extracting genome DNA of a target strain by using an Ezup column type bacterial genome DNA extraction kit, taking the extracted lactobacillus genome DNA as a template for PCR amplification, carrying out a PCR experiment of 16S rDNA by using bacterial universal primers 27F and 1492R, and taking a PCR product to carry out agarose gel detection and photographing after the PCR reaction amplification is finished, wherein the length of an amplified fragment is about 1500bp, as shown in figure 3. The PCR product was sent to the biological engineering (Shanghai) Inc. for sequencing, and the results were shown as SEQ ID NO.1, and BLAST sequence alignment was performed on the NCBI website, which showed that the sequence had over 99% homology with the identified 16SrDNA sequence of Lactobacillus plantarum.
The lactobacillus plantarum ZJUIDS19 is preserved in the China general microbiological culture Collection center of the China Committee for culture Collection of microorganisms at the age of 25 in 2023, and is classified and named as: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCCNO.28018, the preservation address is: beijing, chaoyang area, north Chen Xili No.1, 3, china academy of sciences, microbiological institute. The 16S rDNA complete sequence of the lactobacillus plantarum ZJUIDS19 is shown as SEQ ID No. 1.
3. Preparation of Lactobacillus plantarum ZJUIDS19 metagen
3.1 preparation of Lactobacillus plantarum ZJUIDS19 fermentation supernatant
Lactobacillus plantarum ZJUIDS19 was inoculated with 100mL of MRS liquid medium using an inoculating loop, and placed in an incubator at 37℃for 18h. Then transferred to 250mL MRS broth after sterilization at an inoculum size of 2% (vol%). Placed in a bacterial incubator at 37℃and incubated for 18h. The obtained fermentation broth was centrifuged at 10000r/min for 20min, and the cells were collected. The cells were suspended in distilled water, and the supernatant was collected by centrifugation at 10000r/min for 20 min. The pH value of the sample is regulated to 6.0 by 2M NaOH, and the sample is filtered by a 0.45 mu M filter membrane, so that the aseptic fermentation supernatant can be obtained and stored in a refrigerator at the temperature of 4 ℃ for standby.
3.2 freeze concentration
The sterile fermentation supernatant was concentrated using vacuum freeze. After the aseptic fermentation supernatant is pre-frozen for at least 2 hours in an ultralow temperature refrigerator at the temperature of minus 80 ℃, the aseptic fermentation supernatant is quickly taken out and put into a vacuum freeze dryer for vacuumizing treatment, and the vacuum degree is maintained to be about 0.1P. And after the complete freeze drying, decompressing and taking out the metaplasia, and putting the metaplasia into a refrigerator at the temperature of 4 ℃ to obtain the metaplasia of the lactobacillus plantarum ZJUIDS 19.
Example 3 antioxidant Capacity verification of Lactobacillus plantarum ZJUIDS19
1. Sample preparation
Strains stored in glycerol tubes were streaked on MRS solid medium and incubated for 48h at 37℃in an inverted position. And (3) picking single colonies by using an inoculating loop, inoculating the single colonies into a sterilized MRS liquid culture medium, and standing and culturing for 18-24 hours at 37 ℃ to obtain a culture solution. The culture solution was adjusted to a lactic acid bacteria concentration of 10 with distilled water 10 CFU/mL,4 ℃, 8000 Xg centrifugal 20min, collecting the supernatant, namely fermentation supernatant. The centrifuged cell pellet was resuspended and washed with 0.02M PBS buffer (ph=7.4), and centrifuged at 8000×g for 20min at 4 ℃ for 3 replicates. Resuspending the washed cells in PBS buffer and adjusting the cell concentration to 10 10 CFU/mL to obtain the bacterial suspension.
2. Total antioxidant capacity assay
The total antioxidant capacity (FRAP method) was determined by adding 150. Mu.L of TPTZ working solution (0.3M acetic acid-sodium acetate buffer, 20mM ferric chloride solution, 10mM TPTZ buffer, and V: V=10:1:1, as-is) and 20. Mu.L of sample to the ELISA plate, shaking and mixing, reacting at 37℃for 10min, and measuring the absorbance of the solution at 593 nm. The absorbance measured for the sample was substituted into the ferrous sulfate standard curve and the antioxidant capacity of the sample was expressed as ferrous sulfate equivalent (mu mol FeSO4/mL sample). Each sample was run in 3 replicates and the average was calculated.
Ferrous sulfate standard curve: ferrous sulfate solutions with different mass concentrations (0 mu M, 50 mu M, 100 mu M, 200 mu M, 400 mu M, 600 mu M and 800 mu M) were prepared, the ferrous sulfate solutions with different molar concentrations, 10mM TPTZ buffer and 0.3M acetate buffer were mixed according to V: V=1:1:10, 170 mu L of the mixed solution was added to an ELISA plate, the reaction was carried out at 37℃for 10min, and the absorbance of the solution at 593nm was measured. And drawing a standard curve by taking the mass concentration of ferrous sulfate as an abscissa and the absorbance as an ordinate.
3. Reduction ability measurement
1mL of the sample was placed in a centrifuge tube, and 0.2M PBS solution (pH 6.6) and 1mL of each of 1% (w/v) potassium ferricyanide solution were added and mixed well. Water bath at 50 ℃ for 20min, and cooling in ice bath. Then 10% (w/v) trichloroacetic acid 1mL is added, 6000 Xg is centrifugated for 5min, 1mL of supernatant is taken, 0.1% (w/v) ferric trichloride 1mL and distilled water 1mL are added, the mixture is fully and uniformly mixed, the mixture is stood for reaction for 10min, and the absorbance of the sample at 700nm is measured. The samples were replaced with PBS buffer or MRS broth as blank. Each sample was run in 3 replicates and the average was calculated.
Reduction ability (%) = [ (a) s -A b )/A b ]*100% of the formula: a is that s -sample group absorbance; a is that b Blank absorbance.
DPPH radical scavenging Capacity assay
Preparing V with different concentration gradients C Solution (0-30. Mu.g/ml). 100 mu L of the sample to be tested (or V) is added into the ELISA plate C Standard solution) and 100 mu L of 0.2mM DPPH ethanol solution (prepared by absolute ethanol, stored at 4 ℃ in dark place and used at present) are shaken uniformly and kept at room temperature in dark place for 30min, and the absorbance of the solution at 517nm is measured; 100 mu L of absolute ethyl alcohol is used to replace 100 mu L of DPPH ethanol solution to form a blank group; 100. Mu.L of PBS buffer (or MRS liquid culture medium) is used for replacing 100. Mu.L of the sample to be tested as a control group, and 100. Mu.L of PBS buffer (or MRS liquid culture medium) and absolute ethanol mixed solution are used for blank zeroing. The average was calculated by repeating 3 replicates for each sample. DPPH radical scavenging ability (%) = [1- (a) s -A b )/A c ]*100% of the formula: a is that s -sample group absorbance; a is that b -blank absorbance; a is that c Control absorbance.
TABLE 1 in vitro antioxidant properties of Lactobacillus plantarum ZJUIDS19
| Index (I) | Thallus suspension | Fermentation supernatant |
| Total antioxidant capacity/Fe 2 SO 4 Equivalent mu mol/mL -1 | 0.27±0.21 | 0.93±0.09 |
| Reducing power/% | 12.02±1.52 | 53.05±3.05 |
| DPPH radical scavenging/% | 42.72±3.86 | 85.29±1.41 |
EXAMPLE 5 confirmation of antibiotic sensitivity of Lactobacillus plantarum ZJUIDS19
The antibiotic susceptibility of lactic acid bacteria strains was measured by the paper diffusion method, which is referred to the technical guidelines of the Clinical and Laboratory Standards Institute (CLSI). Preparing MRS solid culture medium with wide-mouth conical flask, sterilizing, placing in 55deg.C water bath, maintaining temperature, taking out after temperature is reduced, placing in sterilized super clean bench, adding lactobacillus suspension (10 8 CFU/mL) was inoculated into a conical flask in an amount of 1%, shaken until the mixture was uniform, and after the lactobacillus suspension was mixed with MRS solid medium, poured into a sterile dish to prepare a 15 mL/dish LB plate. After the MRS plates were solidified, two antibiotic paper sheets were gently attached to each plate with forceps uniformly. Plates with antibiotic paper were incubated at 37℃for 24h. After the cultivation is finished, the diameter of the inhibition zone is measured by a vernier caliper and recorded.
The diameter of the antibiotic susceptibility zone of lactobacillus plantarum ZJUIDS19 is shown in Table 5. The lactobacillus plantarum ZJUIDS19 shows extremely high sensitivity to ampicillin, cefazolin, ciprofloxacin, erythromycin and chloramphenicol and medium sensitivity to compound neonomine as can be obtained by referring to the CLSI (2017) drug sensitivity test standard. Experimental results show that the lactobacillus plantarum ZJUIDS19 is sensitive to common antibiotics.
TABLE 2 results of sensitivity of Lactobacillus plantarum ZJUIDS19 to antibiotics
Note that: s, sensitivity; i, intermediation; r, drug resistance
With the wide application of antibiotics in clinical treatment, the drug resistance of lactobacillus is also more and more serious, and long-term application of the drug-resistant lactobacillus can bring great difficulty to clinical treatment. The lactobacillus plantarum ZJUIDS19 provided by the invention is sensitive to common antibiotics and cannot cause harm to human health.
Example 6 confirmation of pathogenic bacteria inhibitory Capacity of Lactobacillus plantarum ZJUIDS19
The antibacterial activity of the lactic acid bacteria is measured by an international agar diffusion method. The four frozen indicator strains (escherichia coli, salmonella, staphylococcus aureus and listeria monocytogenes) were activated 2-3 times on LB solid medium. And respectively picking the activated single colonies into LB culture medium, and culturing at 37 ℃ for 18 hours. Collecting bacterial cells by centrifugation, and re-suspending in brine to a concentration of 10 8 CFU/mL. The indicator fungus suspension is added into sterilized LB solid medium cooled to about 55 ℃ according to the amount of 1% (v/v), and the mixture is poured into a culture dish (15 mL/dish) after being uniformly mixed, and a sterile oxford cup placed in advance is pulled out after condensation. After the activated Lactobacillus plantarum ZJUIDS19 was grown in MRS medium for 18 hours, the supernatant was collected by centrifugation (8000 rpm,5min,4 ℃) and the cell pellet was discarded. The metabolic supernatant of lactobacillus plantarum ZJUIDS19 was adjusted to ph=6.2 with 5M NaOH solution, and the pH-adjusted supernatant was added to the cup well (200 μl/well) and non-inoculated MRS medium (pH 6.2) was used as a blank. After incubation at 37℃for 24 hours, the diameter of the zone of inhibition was measured. The strains with obvious inhibition zones around the small holes are selected, the diameters of the inhibition zones are measured, and each is repeated three times.
As shown in Table 6, the metabolites of Lactobacillus plantarum ZJUIDS19 have better inhibitory effects on Staphylococcus aureus, escherichia coli, salmonella typhimurium and Listeria monocytogenes. The metabolite of the strain has antibacterial property.
TABLE 3 results of the inhibitory Capacity of Lactobacillus plantarum ZJUIDS19 against pathogenic bacteria
Staphylococcus aureus is the most common pathogen in suppurative infection of humans, some escherichia coli can cause severe diarrhea and septicemia, and some salmonella species can also cause food poisoning in humans. Bacteriocin, organic acid, hydrogen peroxide and other antibacterial products generated by lactic acid bacteria metabolism can inhibit the growth of pathogenic bacteria singly or jointly. The metabolite of the lactobacillus plantarum ZJUIDS19 provided by the invention has a certain antagonism to the four pathogenic bacteria, which plays an important role in resisting wound bacterial infection.
EXAMPLE 7 preparation method of metachromatic Albumin hydrogel
The milk white protein powder is fully dissolved in PBS buffer solution (pH 7-8, namely 1 XPBS buffer solution) at room temperature to prepare milk white protein powder stock solution with the concentration of 1-10 g/100 ml. Firstly, adding methacrylic anhydride into the milk white protein powder stock solution, wherein the dosage ratio (v/w) of the methacrylic anhydride to the milk white protein powder in the step 1) is 1ml/20 g-2 ml/1g; then the pH is regulated to 6.0-10.0, and the reaction is continuously stirred for 3-24 h at the temperature of 10-25 ℃.
And (3) diluting the solution obtained in the step (2) with deionized water, dialyzing for 48-72 h, and freeze-drying the dialyzed trapped fluid to obtain freeze-dried powder (methacryloylated whey protein powder). Adding the freeze-dried powder into deionized water, and stirring until the freeze-dried powder is dissolved, thus obtaining a freeze-dried powder solution with the concentration of the freeze-dried powder of 10-20 g/100 ml. Dissolving 0.1-0.4 g/ml of metazoan obtained in experimental example 1 inAnd (3) adding the photoinitiator into the freeze-dried powder solution obtained in the step (4) under the light-shielding condition until the final concentration of the photoinitiator is 0.05-0.5 g/100ml (namely, 0.05-0.5%, w/v), and uniformly mixing. Irradiating the mixed solution obtained in the step 5) with ultraviolet rays (380-405 nm) for 10-60 s to obtain hydrogel, wherein the ultraviolet intensity is 30mW/cm 2 . The morphology of the resulting metacrylic-loaded methacryloyl lactalbumin (α -LAMA-P) hydrogel is shown in fig. 4.
Example 8 rheological Properties of metachromatic Albumin hydrogels
The ultraviolet intensity obtained in example 7 was changed from 30mW/cm 2 Changed to 12mW/cm 2 . The irradiation time is from 0s to 200s; the remainder being identical to example 7; thereby verifying the rheological properties of metacrylic lactalbumin hydrogels loaded with metagens.
The change in storage modulus (G ') and loss modulus (G') of the metacrylic acid-containing lactalbumin hydrogel loaded with the metacrylic acid-containing hydrogel with a stress of 1% and a frequency of 50Hz was measured by a conventional rheological measurement method using an MCR302 rheometer, and is shown in FIG. 5.
As can be seen from fig. 5, the metaloading of the metacrylic aqueous solution of lactalbumin in this example had a gel time of only about 42s (the critical point time for the solution to change into gel), and the gel speed was relatively fast compared to other aqueous solutions of milk proteins; in addition, the hydrogel in the example has excellent characteristics and good application prospect.
EXAMPLE 9 microstructure of metachromatic Albumin hydrogel
The metalactalbumin working solution obtained in example 7 was subjected to photocuring (photocuring conditions: ultraviolet irradiation at a light intensity of 30mW/cm2 for 30 s) to prepare a hydrogel having a diameter of 8mm and a thickness of 6 mm. And pre-freezing the prepared loaded metalactalbumin hydrogel at-80 ℃ for 6 hours, and then freeze-drying at-80 ℃ for 48 hours. The freeze-dried loaded metalactalbumin hydrogel is sliced, and a microstructure is observed through scanning electron microscope imaging, as shown in fig. 6.
The cross-sectional morphology and microstructure of the hydrogel prove that the loaded metalactalbumin hydrogel has a compact and uniform porous structure, which indicates that the loaded metalactalbumin hydrogel has more crosslinking sites and higher mechanical strength.
EXAMPLE 10 confirmation of antibacterial Properties of metaloaded Albumin hydrogel
1. In vitro bacteriostasis test of metachromatic lactalbumin hydrogel
Staphylococcus aureus (CMCC 26003) and escherichia coli (ATCC 25922) were pre-amplified overnight in LB liquid medium (37 ℃ thermostat shaker, 180 rpm). The ZJUIDS19 metazoans prepared in example 1 were thoroughly dissolved with deionized water to form polymer solutions having concentrations of 0.20, 0.30 and 0.40g/mL, diluted in sequence. The bacterial solution was centrifuged at 4000rpm for 10min, and after discarding the supernatant, it was diluted to a final concentration of 10 using PBS 6 CFU/mL。
The loaded metalactalbumin solution prepared in example 7 was poured evenly into 48 well plates, 400 μl each. By ultraviolet light (intensity: 30 mW/cm) 2 ) Irradiating for 60s to form hydrogel. Then 10. Mu.L of diluted bacterial solution was added to the surface of the hydrogel, and incubated in a constant temperature incubator at 37℃for 6 hours. The control group contained no hydrogel and only 10. Mu.L of diluted bacterial liquid was added. After the incubation, 1mL of PBS was added to each well, and after the well was rinsed, 100. Mu.L of the solution was spread on LB solid medium and incubated in a 37℃incubator for 24 hours. Finally, the bacteria growth is observed by photographing and counted. The calculated antibacterial rate is shown in table 4.
TABLE 4 antibacterial Rate of metaplasia-loaded Albumin hydrogels
With the increase of the metagen concentration, the antibacterial property of the hydrogel is gradually improved, and the antibacterial rate of the metagen concentration to staphylococcus aureus and escherichia coli reaches more than 90% when the metagen concentration is 0.28 and 0.4g/mL, so that the metagen has good antibacterial property.
2. In vivo bacteriostasis test of metachromatic lactalbumin hydrogel
Under the conventional isoflurane+oxygen flow anesthesia, the back of the mouse is prepared, disinfected, the back skin of the mouse is gently lifted, the forced traction deformation is avoided, the circular skin defect with the diameter of 6mm is formed on the back of the mouse, the depth reaches the full dermis, and the consistency of the wound size is confirmed. 20 mu L of 10 8 CFU staphylococcus aureus was added evenly to the wound.
The wound surface was covered with different test materials and then a tergaderm film was applied.
The metalactalbumin working solution and the lactalbumin solution obtained in example 7 are respectively used as the test materials; thereby forming a loaded metaplasia lactalbumin group (α -LAMA-P) and a lactalbumin group (α -LAMA), respectively; untreated was used as a blank (blank).
After 2 days of wound infection in mice, 10. Mu.L of exudate was taken from the wound, diluted 100-fold with sterile PBS, and 100. Mu.L of the diluted solution was spread on LB solid medium. The medium was incubated in a constant temperature incubator at 37℃for 24 hours. Finally, the bacteria growth is observed by photographing and counted. The number of bacteria infected by the wound is shown in Table 5.
TABLE 5 bacterial count of infected wounds in mice
| Control group | alpha-LAMA group | alpha-LAMA-P group | |
| Bacterial count (. Times.10) 5 CFU) | 8.76 a | 8.21 a | 1.48 b |
The milk white protein gel water loaded with metazoan can be used as a wound dressing to effectively reduce bacterial infection at the wound of a mouse, and has good antibacterial effect.
Example 11 confirmation of skin repair Properties of metaloaded Albumin hydrogel
Under the conventional isoflurane+oxygen flow anesthesia, the back of the mouse is prepared, disinfected, the back skin of the mouse is gently lifted, the forced traction deformation is avoided, the circular skin defect with the diameter of 6mm is formed on the back of the mouse, the depth reaches the full dermis, and the consistency of the wound size is confirmed. 20 mu L of 10 8 CFU staphylococcus aureus was added evenly to the wound.
The wound surface was covered with different test materials and then a tergaderm film was applied. The general healing morphology was recorded using split-scope imaging at days 0,2,4,7, 10 (fig. 9 and table 6), and a portion of the wound base and surrounding healthy skin tissue was cut at 10 days while the specimens were cut at the wound margin. The specimens were fixed in formalin.
The metalactalbumin working solution and the lactalbumin solution obtained in example 7 are respectively used as the test materials; thereby forming a loaded metaplasia lactalbumin group (α -LAMA-P) and a lactalbumin group (α -LAMA), respectively; untreated was used as a blank (blank). The specific procedures for paraffin sections HE and Masson staining photographs are as follows:
(1) Tissue dehydration: the tissue of the specimen is dehydrated by 75% alcohol (4 h) -85% alcohol (2 h) -90% alcohol (1.5 h) -95% alcohol (1 h) -absolute alcohol I (0.5 h) -absolute alcohol II (0.5 h) in sequence.
(2) Tissue transparency: dehydrated tissues are sequentially subjected to absolute ethyl alcohol: xylene (1:1) (10 min) -xylene I (10 min) -xylene II (7 min) completes the tissue transparency process.
(3) Wax dipping: the transparent tissue blocks are sequentially subjected to wax dipping by 3-cylinder paraffin (60 ℃). Paraffin i (60 ℃) (1 h) -paraffin ii (60 ℃) (1 h) -paraffin iii (60 ℃) (1 h);
all the steps are completed in the biological tissue dehydrator.
(4) Embedding: the temperature of the embedding wax is slightly higher than the wax dipping temperature, so that the tissue block and the embedding wax are completely fused together, and the tissue block soaked with the wax is wrapped in the paraffin block.
(5) Slicing and baking: before slicing, the section of the wax block is frozen on a freezing table for a plurality of minutes, and a target embedding block is fixed by a specimen clamp, so that the external section of the embedding block is parallel to the section of the specimen clamp, and the embedding block protrudes slightly. After the cutter table is pushed to the outer edge, the spiral of the blade clamp is loosened, the blade is arranged, an included angle of about 15 degrees is formed between the plane of the slicing cutter and the section of the tissue, and the upper edge and the lower edge of the embedding block are parallel to the knife edge. The desired thickness (4 μm) of the slice was adjusted on the micro-motion device, the knife block was moved to a position near the specimen block, and the knife edge was brought into slight contact with the tissue section to begin slicing. The right hand rotates the rotating wheel uniformly and uniformly, the left hand holds the brush pen to cut the chip slightly under the knife edge, and holds the cut wax ribbon, after the wax ribbon forms a certain length, the right hand stops rotating, holds the other brush pen to gently pick up the wax ribbon, and places the wax ribbon in a water bath chip-spreading pot at about 42 ℃. The cut sections need to be flattened and then attached to a glass slide. The specific process of the drag-out method comprises the following steps: firstly, putting a series of slices into a warm water bath slice spreading pot at about 42 ℃, naturally flattening the slices due to the action of surface tension after the slices float on the water surface, separating the slices by tweezers, then obliquely inserting an APES or polylysine treated anti-drop glass slice into the water surface to scoop the slices, attaching the slices at a proper position of the glass slice, and baking the slices in a 60 ℃ oven for 3 hours after the sticking is finished.
(6) Slice dewaxing: paraffin sections were sequentially put into xylene i (10 min) -xylene ii (10 min) -absolute ethanol i (5 min) -absolute ethanol ii (5 min) -95% alcohol (3 min) -90% alcohol (3 min) -80% alcohol (2 min) -70% alcohol (2 min), and then rinsed with distilled water for 2min.
(7) Dyeing:
HE staining: the Harris hematoxylin dye solution is dyed for 5-7min, and the washing is carried out by water to turn blue. Slicing, differentiating with 1% hydrochloric acid alcohol for 2-5s, washing with tap water, and washing to get blue. The sections were stained with 1% water-soluble eosin dye solution for 2min, and rinsed with tap water for 30s. Slicing into absolute ethyl alcohol, dehydrating, making xylene transparent, air-drying and sealing with neutral resin.
Masson staining: the mixture was stained with the prepared Weibert iron hematoxylin staining solution for 8 minutes. The acidic ethanol differentiated solution was differentiated for 15 seconds and washed with water. The Masson bluing solution returns to blue for 5 minutes and is washed with water. Distilled water was washed for 1 minute. Ponceau staining solution was stained for 5 minutes. Washing with weak acid working solution for 1 min. The phosphomolybdic acid solution was washed for 1 minute and the weak acid working solution was washed for 1 minute. The aniline blue staining solution is stained for 2 minutes and washed with weak acid for 1 minute. The 95% ethanol is dehydrated rapidly for 2-3 seconds, the absolute ethanol is dehydrated for 3 times, each time for 5-10 seconds, the dimethylbenzene is transparent for 3 times, each time for 1-2 minutes, and the neutral gum is sealed.
(8) Photographing under a lens: after completion of the dyeing, the image was observed under an optical lens as shown in FIGS. 10 and 11.
The metazoan gel water loaded with metazoan has good antibacterial effect as wound dressing, is beneficial to wound healing, has healing speed higher than that of the metazoan gel water and blank control, has no toxic or side effect on wound tissues, and has good healing state.
TABLE 6 skin wound State Change after treatment with different gel materials
According to the results of table 6, the rate of healing of skin wound by the metaplasia-loaded lactalbumin hydrogel was significantly higher than that of the control group and the lactalbumin hydrogel group, and the healing rate reached 97.75% after 10 days, with excellent healing effect.
Claims (8)
1. Lactobacillus plantarum ZJUIDS19, characterized in that the classification of the strain is named: lactobacillus plantarum (Lactobacillus plantarum), accession number: CGMCC NO.28018, the preservation address is: the 16S rDNA complete sequence of the lactobacillus plantarum ZJUIDS19 is shown as SEQ ID No.1, wherein the 16S rDNA complete sequence is shown in North Xielu No.1, national institute of microbiology, china academy of sciences.
2. The preparation method of the metaloading antibacterial lactalbumin hydrogel is realized by the following steps:
(1) Sterile fermentation supernatant
Inoculating the lactobacillus plantarum ZJUIDS19 in the MRS liquid culture medium by an inoculating loop for culture, transferring the inoculating quantity with the volume ratio of 2% into the MRS liquid culture medium for culture to obtain fermentation liquor, centrifugally collecting thalli, suspending the thalli in distilled water, centrifugally collecting supernatant, adjusting the pH value to 6.0, and filtering to obtain aseptic fermentation supernatant;
(2) Preparation of Lactobacillus plantarum ZJUIDS19 metagen
Vacuum freeze concentrating the aseptic fermentation supernatant, pre-freezing the supernatant at ultralow temperature, freeze drying, vacuumizing, completely freeze drying, decompressing, taking out the metaplasia, and placing in a refrigerator at 4 ℃ to obtain the metaplasia of the lactobacillus plantarum ZJUIDS 19;
(3) Adding the extracted metagen into the modified lactalbumin solution at room temperature, and curing the metagen into gel under ultraviolet irradiation to form the antibacterial lactalbumin hydrogel loaded with the metagen.
3. The method according to claim 2, wherein the incubation time in step (1) is 18 hours, centrifugation is performed at 10000r/min for 20min, and pH is adjusted with 2M NaOH.
4. The preparation method of the MRS liquid culture medium according to claim 2, wherein the MRS liquid culture medium in the step (1) is prepared by dissolving 10g of peptone, 10g of beef extract, 5g of yeast extract, 2 g of diammonium citrate, 5g of sodium acetate, 20g of glucose, 80 ml of tween, 0.5g of magnesium sulfate and 0.25 g of manganese sulfate in each liter of distilled water.
5. The method according to claim 2, wherein the ultralow temperature pre-freezing condition of step (2) is that the step of pre-freezing is performed in an ultralow temperature refrigerator at-80 ℃ for not less than 2 hours.
6. The method according to claim 2, wherein the ultraviolet irradiation condition in the step (3) is that the ultraviolet wavelength is 380-405 nm, the irradiation time is 10-60 s, and the ultraviolet intensity is 30mW/cm 2 。
7. The application of the metazoan-loaded antibacterial milk protein hydrogel obtained by the preparation method of claim 2 in preparing biomedical materials, wherein the biomedical materials comprise lactobacillus bacteriocin, antibacterial wound dressing, antibacterial and antioxidant live bacteria preparation and antibacterial and antioxidant bacteria powder.
8. The use according to claim 6, wherein the lactic acid bacterial bacteriocin is used as a preservative for meat products, milk products, alcoholic beverages, or in combination with other preservation techniques, as a barrier technique in food processing and storage.
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