CN117568180A - Bacteria-carrying microalgae, preparation method and application thereof - Google Patents
Bacteria-carrying microalgae, preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a bacteria-carrying microalgae, a preparation method and application thereof. The bacteria-carrying microalgae takes spirulina platensis SP as a carrier, and probiotics are directly loaded and delivered to the colon for the treatment of ulcerative colitis. The bacteria-carrying microalgae is a bacteria-algae symbiotic system, and has the following advantages in the aspect of delivering probiotics: 1. the bacteria-carrying microalgae can be used as a prebiotic to promote the proliferation of probiotics; 2. in the gastric acid environment, the bacteria-carrying microalgae can combine more probiotics to help the bacteria-carrying microalgae pass through the stomach rapidly; 3. the bacteria-carrying microalgae with the spiral structure can entrap probiotics and is beneficial to intestinal tract colonisation. The oral administration of the bacteria-carrying microalgae can effectively relieve inflammatory reaction of ulcerative colitis and maintain balance of intestinal flora. The invention has wide application prospect in oral delivery of probiotics for treating intestinal diseases.
Description
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a bacteria-carrying microalgae, a preparation method and application thereof.
Background
The intestinal microbiota is a large complex population, often considered an important organ acquired by the human body and called the "second brain" of the human body. The composition, structure and proportion of the intestinal flora are closely related to human health, and once the microecological balance of the intestinal flora is broken, various gastrointestinal and systemic diseases can be caused. The plasticity of the intestinal microbiota makes it possible to reshape the intestinal microbial structure by the manual manipulation of external influencing factors. The oral delivery of probiotics to the intestinal microbiome has received great attention as a non-invasive mode of administration, which has the beneficial effects of inhibiting the colonization of the intestinal tract by pathogens, protecting the intestinal mucosal barrier, etc., and is an effective strategy for actively regulating the balance of the intestinal microbiome. However, the oral probiotics have low availability and limited intestinal implantation due to complex gastrointestinal tract environment and strong liquidity. Therefore, how to design the oral administration carrier of the probiotics and improve the functional characteristics, the survival rate and the stability of the probiotics in the gastrointestinal tract is a problem to be solved by utilizing the probiotics to balance the intestinal microbiome.
Based on the above, the probiotic delivery system constructed by natural biological materials has great application prospect for oral treatment of intestinal diseases. Microalgae are a common class of single-cell photosynthetic organisms, which are mainly found in marine or freshwater lake environments. In recent years, microalgae have the characteristics of good biocompatibility, low cost, large surface area and active surface, phototaxis, high propulsive force and the like, and have great advantages in the aspects of biological imaging, drug delivery, anaerobic tumor treatment, wound healing and the like. Among them, spirulina platensis SP used in the present invention is one of large-scale industrialized microalgae, has extremely high nutritional components such as phycocyanin, carotenoid and polysaccharide, etc., and has been recommended by many organizations for medical, pharmaceutical and nutritional dietary supplements. The spirulina platensis SP has biodegradability in a physiological environment, and provides a certain biological safety for the spirulina platensis SP applied to the biomedical field. In addition, spirulina platensis SP can also modulate intestinal microbiota by increasing the abundance of probiotics. Thus, microalgae-based probiotic delivery systems may provide an innovative approach to oral administration of probiotics. The search of the design of all oral probiotics administration systems at home and abroad and the results of patent and literature for treating intestinal diseases show that: no report of probiotics-spirulina platensis oral preparation and application thereof is found at present.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides the preparation and the application of the microalgae mediated probiotic delivery system, and the bacteria-carrying microalgae prepared by the method can be combined with more probiotics in a stomach strong acid environment to help the probiotics to pass through the stomach quickly, after entering the intestinal tract, the spiral structure of the spirulina platensis can intercept the probiotics to help the probiotics to colonize the intestinal tract, and the bacteria-carrying microalgae can also be used as a prebiotic to promote the proliferation of the probiotics, so that the oral availability of the probiotics is obviously improved, and a new idea and method are provided for treating intestinal diseases by oral administration of the probiotics.
The technical scheme adopted by the invention is as follows:
a bacteria-loaded microalgae consisting of spirulina platensis SP and probiotics loaded on spirulina platensis.
The preparation method of the bacteria-carrying microalgae comprises the following steps:
activating and transferring probiotics in LB culture medium for multiple times, culturing to the middle and late stages of logarithmic growth, centrifuging to obtain thalli, washing with sterile PBS for multiple times, and re-suspending; centrifuging to obtain spirulina platensis SP, washing with sterile PBS for multiple times, and re-suspending; and adding the re-suspended probiotics into the re-suspended spirulina to obtain the bacteria-carrying microalgae.
Further, the probiotics are escherichia coli.
Further, the probiotics content is 10 9 CFU/mL, spirulina content was 2mg/mL.
The application of the bacteria-carrying microalgae comprises the following steps: preparing the probiotic-microalgae preparation for oral treatment of ulcerative colitis.
The bacteria-carrying microalgae greatly improves the oral availability of probiotics, is beneficial to the colonization of probiotics in intestinal tracts, has high biological safety, and can be used as an oral administration system of the probiotics for treating intestinal diseases.
Furthermore, the bacteria-carrying microalgae can be prepared by selecting proper probiotics according to different disease models. The concentration of the probiotic used in the probiotic-spirulina platensis system as a therapeutic means can be routinely determined by the clinician. The dosage regimen will depend on various factors such as the type of bowel disorder, the health of the patient, sex and age, etc. By referring to the probiotic dosing regimen, one skilled in the art can determine the optimal concentration and formulation of the probiotic and spirulina platensis of the present invention.
Compared with the prior art, the invention has the beneficial effects that the spirulina platensis is used as the carrier of probiotics, and has great potential for commercialization and clinical transformation. The method for preparing the bacteria-carrying microalgae by using the one-step method is simple to operate, easy to obtain raw materials and environment-friendly.
In the aspect of oral treatment application, the spirulina platensis SP can be used as a prebiotic to promote proliferation of probiotics EcN. The spirulina platensis and the probiotics can be combined more tightly in the stomach acidic environment, which is beneficial to the rapid gastric passing of the probiotics. In addition, the spiral structure of the spirulina platensis is easy to be captured by intestinal villi, so that probiotics can be trapped, long-time retention of intestinal tract parts is facilitated, and the colonization of the probiotics in the intestinal tract is facilitated. The bacteria-carrying microalgae has good anti-inflammatory and intestinal flora regulating functions in the process of treating ulcerative colitis.
Drawings
FIG. 1 optical microscope and Scanning Electron Microscope (SEM) pictures under Bright field (Bright-field) and fluorescent field (Fluorescence) of Spirulina platensis SP and probiotic EcN, wherein probiotic EcN is observed under Bright field after gram staining. The spirulina platensis SP bright field, fluorescence field and scanning electron microscope picture scale = 20 μm; the probiotic EcN light field picture scale=20 μm, the fluorescent field picture scale=10 μm, the scanning electron microscope picture scale=2 μm.
FIG. 2 statistical graphs of colony counts at various time points for probiotics EcN cultured alone and co-cultured with spirulina platensis SP and its fragments. P < 0.05, p < 0.01, p < 0.001).
Fig. 3 scanning electron microscope pictures of the binding of bacteria and algae at different pH values, scale = 100 μm.
FIG. 4 in vitro fluorescence images of the gastrointestinal tract and major organs (brain, heart, liver, spleen, lung, kidney and bladder) were collected at different time points after oral administration of probiotic EcN and the mycotic symbiotic EcN-SP.
Figure 5 staining of frozen sections of ileum and colon collected after oral administration of probiotic EcN and mycosis symbiotic system EcN-SP, scale = 100 μm.
FIG. 6 comparison of colon length (5 replicates) after treatment of DSS-induced ulcerative colitis with probiotic EcN, spirulina platensis SP, and mycosis symbiotic system EcN-SP.
FIG. 7 is a graph comparing the therapeutic effects of probiotic EcN, spirulina platensis SP, and mycosis symbiotic system EcN-SP on DSS-induced ulcerative colitis (tumor necrosis factor- α).
FIG. 8 is a graph comparing the effects of probiotic EcN, spirulina platensis SP, and mycosis symbiotic system EcN-SP on beneficial and deleterious bacteria in the intestinal flora of DSS-induced ulcerative colitis mice.
FIG. 9 is a graph comparing the effect of probiotic EcN, spirulina platensis SP, and mycophyta symbiotic EcN-SP on the ratio of Bacteroides/Thick-walled bacteria in the intestinal flora of DSS-induced ulcerative colitis mice. (ns, p value > 0.05; p value < 0.01; p value < 0.001).
FIG. 10 is a cluster heat map of intestinal flora at the target level for mice of different treatment groups.
FIG. 11 is a graph comparing blood normals (WBC, white blood cells, RBC, red blood cells, HGB, hemoglobin, MCH, mean red blood cell hemoglobin, MCHC, mean red blood cell hemoglobin concentration, MCV, mean cell volume, PLT, platelets, HCT, hematocrit) and blood biochemical indicators (ALT, alanine transferase, AST, aspartate transferase, BUN, urea nitrogen, CREA, creatinine) after 30 days of continuous oral probiotic EcN, spirulina platensis SP, and mycosis symbiotic system EcN-SP.
Figure 12 schematic of weight monitoring during consecutive days of oral probiotic EcN, spirulina platensis SP, and mycosis symbiotic EcN-SP 30.
Detailed Description
The present invention will be further described with reference to the following drawings and examples, but the present invention is not limited to the following examples.
EXAMPLE 1 Synthesis of bacterial-carrying microalgae EcN-SP
The probiotic (E.coli EcN) stored at-80℃was transferred to LB medium, cultured at 37℃and 200rpm for 1 day, transferred to new LB medium, and the probiotic was continuously transferred to three times for activation. Activated probiotic EcN was cultured to mid-late logarithmic growth, and cells were harvested by centrifugation at 6,500×g at 4 ℃ for 10 min and resuspended after three washes with sterile Phosphate Buffer (PBS). Spirulina Platensis (SP) cultured in Zarrouk medium was centrifuged at 3,260×g at 4 ℃ for 10 min, washed three times with sterile PBS and resuspended. Adding the re-suspended probiotic EcN into the re-suspended spirulina platensis SP to obtain a fungus-algae symbiotic system-bacteria-carrying microalgae EcN-SP, so that the final probiotic content is 10 9 CFU/mL, spirulina content was 2mg/mL. The microscope and scanning electron microscope pictures were taken, and it was seen that spirulina platensis SP was in a 3D spiral shape and had red fluorescence imaging properties, and probiotics EcN were in a rod shape and had green fluorescence imaging properties (fig. 1).
EXAMPLE 2 promotion of proliferation of probiotics by Spirulina platensis
Activated probiotic EcN was washed three times with sterile PBS and resuspended so that the final OD 600 =2; the spirulina platensis SP was resuspended after three washes with sterile PBS such that the final OD 680 =2. Then, the two were mixed in equal volumes and incubated at 37℃and 200rpm, and the probiotic EcN was used as a control with equal volumes of sterile PBS, sampled every 2 hours, and plated for gradient dilution. Results referring to fig. 2, spirulina platensis SP can promote the proliferation of probiotics EcN, demonstrating that spirulina platensis SP has the characteristics of prebiotics and can promote the proliferation of probiotics.
Example 3 bacterial algae binding at different pH
The bacteria-carrying microalgae EcN-SP prepared in example 1 was added to sterile water at ph=2.0 and ph=7.0, respectively, and after culturing at 37 ℃ for 30 minutes at 200rpm, the bacteria-carrying microalgae mixture was collected for scanning electron microscope sample preparation. The sample was first resuspended in 2.5vol% glutaraldehyde solution and fixed overnight at 4 ℃; centrifuging at 4500rpm for 10 min to collect thallus, pouring glutaraldehyde solution, sucking concentrated bacterial liquid with phosphate buffer solution, placing on square filter paper, folding into nail cover, and placing in centrifuge tube; washing with PBS three times to remove the fixing solution, fixing with 1vol% osmium acid solution for 2 hours, centrifuging at 4500rpm for 10 minutes, washing with PBS three times to remove osmium acid; dehydrating with gradient concentration ethanol solution (30 vol%, 50vol%, 70vol%, 90vol%, 95vol% and 100 vol%); and (5) drying the critical point, and observing the morphology by using a scanning electron microscope after coating. As a result, referring to fig. 3, it can be seen that in an acidic environment with ph=2.0, the bacteria and algae are more tightly bound, and more probiotics EcN are bound to the surface of spirulina platensis SP. It shows that in the acid stomach environment, more probiotics are combined with the spirulina, so that the probiotics can pass the stomach rapidly, and the harm of gastric acid to the probiotics is reduced.
EXAMPLE 4 Probiotics EcN and Spirulina platensis SP distribution in mice
The fluorescence imaging of the gastrointestinal tract and major organs (brain, heart, liver, spleen, lung, kidney and bladder) of mice after gavage administration via the probiotic alone-probiotic spirulina platensis symbiotic system was tested using a small animal biopsy instrument. 200. Mu.L of probiotic (10) 9 CFU/mL) and probiotic-spirulina platensis symbiotic system (EcN =10) 9 CFU/mL, sp=2 mg/mL) was injected into Balb/c white mice (6 weeks old, females) by intragastric administration. Mice were sacrificed after various times and signal patterns of the gastrointestinal tract and major organs of the mice were obtained with a small animal in vivo imager. Wherein the chlorophyll channel of the SP is channel Cy5.5, the excitation wavelength is 605nm, and the emission wavelength is 615-665nm; ecN, the GFP channel, excitation wavelength 488nm, emission wavelength 505-550nm. Results referring to FIG. 4, the probiotic EcN remained in the intestine for a longer period of time than the probiotic alone gastric lavage group, and the probiotic-spirulina platensis SP remained in the intestine for a stronger fluorescence signal detected at the intestinal site after 24 hours, indicating that the probiotic-spirulina platensis symbiotic system had better intestinal retention capacity than in the acidic environmentThe lower probiotics are closely combined with the spirulina and the spiral structure of the spirulina platensis, and the spiral spirulina platensis can retain the probiotics, so that the probiotics can stay in the intestinal tract for a longer time.
EXAMPLE 5 intestinal distribution of Spirulina platensis SP and Probiotics EcN after oral administration
To study differences in retention of probiotic EcN alone and in the intestinal tract of mice following oral administration with spirulina platensis SP, balb/c mice (6 week old, female) were fasted overnight, respectively gavaged with 200 μl of probiotic (10 9 CFU/mL) and probiotic-spirulina platensis symbiotic system (EcN =10) 9 CFU/mL, sp=2 mg/mL). Mice were euthanized 1 hour after and 4 hours after gavage, ileum and colon tissues of mice of the gavage probiotic group and gavage algae group alone were collected, respectively, frozen sections and DAPI staining were performed, and intestinal tissues and contents were observed with an inverted microscope. As a result, referring to fig. 5, after 1 hour of separate gastric lavage of the probiotic bacteria, the probiotic bacteria were washed out and not retained in the intestinal tract due to the mobility of the intestinal tract, but were lavaged together with spirulina platensis SP and probiotic bacteria EcN were observed in the ileum and colon, indicating that co-delivery of spirulina platensis could help retain the probiotic bacteria in the intestinal tract for a longer period of time, facilitating colonization of the intestinal tract by the probiotic bacteria.
EXAMPLE 6 treatment of ulcerative colitis Properties
C57BL/6J female mice were fed with 3vol% aqueous DSS for 7 days, DSS-induced ulcerative colitis mice models were constructed, 200. Mu.L of probiotics (10 9 CFU/mL), 200 μl of spirulina platensis (2 mg/mL), and probiotic-spirulina platensis symbiotic system (EcN =10) 9 CFU/mL, sp=2 mg/mL) was injected into C57BL/6J female mice by intragastric administration. Mice were euthanized on day 15, the colon was removed for measurement and sections were stained for immunohistochemistry (tumor necrosis factor- α). Results referring to fig. 6 and 7, mice treated with the probiotic-spirulina platensis symbiotic system maintained colon length similar to normal groups, significantly superior to other treated groups; the immunohistochemical staining result shows that the mice treated by the probiotic-spirulina platensis symbiotic system have inflammatory responseSignificantly eases. The probiotic-spirulina platensis symbiotic system has good anti-inflammatory capability and can effectively treat the colitis induced by the DSS.
Example 7 ability to modulate intestinal flora
Before euthanizing mice on day 15, fresh feces of different groups of mice were taken, flora DNA was extracted, and the V3-V4 region of the 16SrRNA gene in the genome was sequenced. Mice treated with the probiotic-spirulina platensis symbiotic system had significantly increased relative abundance of beneficial bacteria compared to DSS groups, significantly decreased relative abundance of harmful bacteria (fig. 8), and significantly decreased bacteroides/firmicutes ratio (fig. 9). The results of the clustering analysis at the mesh level showed that the bacterial algae group and the PBS group clustered together, indicating that the bacterial algae group was more similar to the intestinal flora of healthy mice (fig. 10). The results show that the probiotic-spirulina platensis symbiotic system can regulate the intestinal flora by increasing the number of probiotics and inhibiting the growth of harmful bacteria, so that the structure of the intestinal flora is restored to a level more similar to that of healthy mice, and the health of the intestinal flora is maintained.
EXAMPLE 8 oral safety
200. Mu.L of probiotic (10 9 CFU/mL), 200 μl of spirulina platensis (2 mg/mL), and probiotic-spirulina platensis symbiotic system (EcN =10) 9 CFU/mL, sp=2 mg/mL) was injected into Balb/c female mice by intragastric administration and intragastric administration once a day. After 30 days of continuous administration, mice were bled for routine blood and biochemical blood tests during which weight changes were monitored. Results referring to fig. 11 and 12, after administration of the probiotic-spirulina platensis symbiotic system, the main blood routine and blood biochemical indexes of mice were in the normal range, and the weight of mice in each experimental group was not significantly different from that in the normal group, indicating that the spirulina platensis SP, the probiotic EcN and the probiotic-spirulina platensis symbiotic system had good oral safety.
Claims (5)
1. The bacteria-carrying microalgae is characterized by comprising spirulina platensis SP and probiotics loaded on the spirulina platensis.
2. The bacteria-loaded microalgae of claim 1, wherein the probiotics are escherichia coli.
3. The bacteria-carrying microalgae of claim 1, wherein the probiotic is present in an amount of 10 9 CFU/mL, spirulina content was 2mg/mL.
4. A method for preparing a bacteria-carrying microalgae according to any of claims 1 to 3, characterized in that it comprises the following steps: activating and transferring probiotics in LB culture medium for multiple times, culturing to the middle and late stages of logarithmic growth, centrifuging to obtain thalli, washing with sterile PBS for multiple times, and re-suspending; centrifuging to obtain spirulina platensis SP, washing with sterile PBS for multiple times, and re-suspending; and adding the re-suspended probiotics into the re-suspended spirulina to obtain the bacteria-carrying microalgae.
5. Use of a bacteria-laden microalgae according to any of claims 1 to 3, characterized in that it comprises: preparing the probiotic-microalgae preparation for oral treatment of ulcerative colitis.
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