CN116349886A - Probiotic packaging material, packaged probiotic and preparation method thereof - Google Patents
Probiotic packaging material, packaged probiotic and preparation method thereof Download PDFInfo
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- CN116349886A CN116349886A CN202310286037.4A CN202310286037A CN116349886A CN 116349886 A CN116349886 A CN 116349886A CN 202310286037 A CN202310286037 A CN 202310286037A CN 116349886 A CN116349886 A CN 116349886A
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- probiotics
- probiotic
- protective layer
- encapsulated
- procyanidine
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Abstract
The invention belongs to the technical field of biological agents, and particularly relates to a probiotic packaging material, a packaged probiotic and a preparation method thereof. The probiotics encapsulating material provided by the invention comprises a protective layer and a functional layer which are arranged in a lamination way from inside to outside; the protective layer is a procyanidine metal complex; the functional layer is a functional active molecule which is physically adsorbed or chemically combined with the procyanidine group in the protective layer, the chemical combination is that the functional active molecule is chemically combined with phenolic hydroxyl in the procyanidine group, and the functional active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof. The probiotic packaging material provided by the invention improves the quantity of the probiotics reaching the colon part, and simultaneously remarkably improves the adhesion, colonization and survival rate of the probiotics in a complex gastrointestinal tract environment, thereby improving the oral availability and in vivo survival rate of the probiotics.
Description
Technical Field
The invention belongs to the technical field of biological agents, and particularly relates to a probiotic packaging material, a packaged probiotic and a preparation method thereof.
Background
Humans are "superliving organisms" composed of human cells and symbiotic microflora, and there is a complex interaction process between the human body and the flora, so that various physiological processes of the human body need to consider the influence of symbiotic microorganisms. Intestinal flora is an extremely complex symbiotic microbial ecosystem in humans, in numbers up to 10 13 . The anaerobic microorganism in the intestinal tract has more than 1000 species, and the encoding of about 330 ten thousand specific genes is 150 times of the encoding base factor of human genome. The intestinal flora of the human body has various physiological functions due to the diversity, and is in relatively stable balance under normal conditions, however, in the process of co-evolution of the intestinal flora and the host, the balance is broken under certain conditions, so that diseases occur. Intestinal flora is closely related to physiological functions such as metabolism, nutrition, immunity and the like and host health, and intestinal flora disorder can cause occurrence of a plurality of host diseases such as obesity, diabetes, enteritis and even malignant tumors, so that intestinal microbial homeostasis plays an extremely important role in the immunoregulation and health maintenance of the host.
The intestinal flora has good plasticity, and personalized accurate regulation and control can positively influence the treatment of various diseases. Numerous basic research and clinical trial results demonstrate that probiotics play an important role in regulating intestinal flora and maintaining flora homeostasis. Faecal fungus transplantation is an effective means of preventing and treating diseases caused by dysbacteriosis, but such interventional therapy inevitably reduces patient compliance and may also present gastrointestinal irritation and potential complications.
Oral administration is a conventional and most acceptable mode of administration, and is an ideal route for supplementing probiotics. Thus, modulation of intestinal microorganisms by oral intervention with specific probiotics may be a new approach to the treatment of a variety of diseases including metabolic-related diseases, cardiovascular diseases, and even tumors. The oral probiotics can obviously inhibit pathogenic bacteria from colonising, regulate intestinal flora and enhance intestinal immunity. However, oral probiotics also suffer from a number of problems and limitations, complex gastrointestinal environments and sustained intestinal motility leading to lower oral availability and limited intestinal colonisation rates. Firstly, the too low pH value in gastric juice, a large amount of digestive enzymes and bile acid existing in intestinal tracts can lead to inactivation of probiotics, so that the oral availability and the treatment effect of the probiotics are reduced; secondly, the rapid peristalsis of the gastrointestinal tract reduces the residence time of the probiotics in the intestinal tract, thereby reducing the adhesion and colonization of the probiotics in the intestinal tract. Furthermore, the integrity of the intestinal barrier function also affects colonisation of the intestinal tract by oral probiotics. These problems have limited the clinical use and market conversion of oral probiotics to a great extent.
Therefore, the accurate control of the oral probiotics and the relevant regulation of the biological behaviors of the probiotics are urgently needed, so that the oral availability and the in-vivo survival rate of the probiotics are improved. The existing means comprise the steps of carrying out genetic modification on probiotics by utilizing methods such as synthesis biology and the like, so that the genetically engineered bacteria with stronger stress resistance function are generated, and meanwhile, the genetically engineered bacteria have some characteristics which are not possessed by the original probiotics. However, the transformation method is based on gene recombination, is an irreversible process, can bring about some potential gene mutation, and has certain potential safety hazard in application to human bodies. In recent years, researchers have attempted to improve the in vivo survival rate of oral probiotics by means of enteric capsules, hydrogels, dry powders, and the like, but no very desirable effect has been achieved so far. In addition, complex preparation processes and the encapsulating materials used also affect the activity of probiotics, thereby further increasing the difficulty of clinical transformation.
With the development of technology, surface modification of bacteria has become a simple and effective strategy, the surface of bacteria is extremely important for maintaining biological behaviors, and the surface of bacteria has a great number of inherent antigens, adhesion factors, and 'sports organs' such as flagella, which play a very critical role in physiological functions of bacteria, such as adhesion and colonization of bacteria in intestinal tracts. Therefore, on the basis of not affecting the activity of the living probiotics, the development of a material chemical modification method of the living organism surface interface protects the probiotics from being damaged by gastrointestinal tracts and endows the probiotics with additional pharmaceutical activity has profound clinical significance and great market value.
Disclosure of Invention
The invention aims to provide a probiotic packaging material, an encapsulated probiotic and a preparation method thereof, and the probiotic packaging material provided by the invention improves the number of the probiotic reaching colon parts, and remarkably improves the adhesion, colonization and survival rate of the probiotic in complex gastrointestinal tract environments, so that the oral availability and in-vivo survival rate of the probiotic are improved.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a probiotic packaging material, which comprises a protective layer and a functional layer which are arranged in a lamination way from inside to outside; the protective layer is a procyanidine metal complex; the functional layer is a functional active molecule which is physically adsorbed or chemically combined with the procyanidine group in the protective layer, the chemical combination is that sulfhydryl or amino in the functional active molecule is chemically combined with phenolic hydroxyl in the procyanidine group through Michael addition or Schiff base reaction, and the functional active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof.
Preferably, the metal ion in the procyanidin metal complex comprises Fe 3+ 、Zn 2+ 、Ca 2+ 、Mg 2+ 、Ti 4+ 、Ce 4 + 、Cu 2+ And Mn of 2+ One or more of the following.
The invention provides a preparation method of encapsulated probiotics, which comprises the following steps of;
mixing procyanidine, water-soluble metal salt and water, and performing coordination chelation reaction on the obtained mixed solution to obtain a protective layer;
mixing the protective layer with the probiotic solution for first encapsulation to obtain probiotics encapsulated by the protective layer;
and mixing the probiotics encapsulated by the protective layer with functional active molecule solution to perform physical adsorption or chemical combination for second encapsulation, and forming a functional layer on the outer surface of the protective layer to obtain the encapsulated probiotics.
Preferably, in the mixed solution, the mass concentration of the procyanidine is 0.5-50 mg/mL, and the mass concentration of the metal ion is 0.1-5 mg/mL.
Preferably, the pH value of the mixed solution ranges from 4 to 10.
Preferably, the concentration of probiotics in the probiotics solution is 1×10 5 ～1×10 9 CFU/mL。
Preferably, the probiotic bacteria include one or more of enterococcus, streptococcus, bifidobacterium, lactobacillus, propionibacterium, bacillus, yeast and escherichia coli.
Preferably, the mass concentration of the functional active molecule solution is 0.5-50 mg/mL.
Preferably, the temperature of the coordination chelation reaction is 25-37 ℃, and the time of the coordination chelation reaction is 1-2 h;
the temperature of the first encapsulation is 25-37 ℃ and the heat preservation time is 1-2 h;
the temperature of the second encapsulation is 1-4 ℃, and the heat preservation time is 1-2 h.
The invention provides the encapsulated probiotics prepared by the preparation method of the technical scheme, which comprises probiotics and an encapsulating material for encapsulating the probiotics, wherein the encapsulating material is the probiotics encapsulating material of the technical scheme, and a protective layer in the encapsulating material is in contact with the probiotics.
The invention provides a probiotic packaging material, which comprises a protective layer and a functional layer which are arranged in a lamination way from inside to outside; the protective layer is a procyanidine metal complex; the functional layer is a functional active molecule which is physically adsorbed or chemically combined with the procyanidine group in the protective layer, the chemical combination is that sulfhydryl or amino in the functional active molecule is chemically combined with phenolic hydroxyl in the procyanidine group through Michael addition or Schiff base reaction, and the functional active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof. According to the invention, the procyanidine metal complex with a three-dimensional network structure formed by coordination chelation reaction of procyanidine and metal ions is used as a protective layer of the packaging material of the probiotics, so that the survival rate of the probiotics in complex gastrointestinal tract environments (such as strong acid conditions, antibiotics, strong oxidation and the like) can be remarkably improved, and the number of the probiotics reaching colon parts is increased; meanwhile, one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof are used as functional active molecules to react with procyanidine groups in procyanidine metal complexes to form a functional layer, the formed functional layer has anionic surface charge and mucous membrane adhesiveness, and can also interact with inflammatory receptors, so that adhesion and colonization of probiotics in intestinal tracts can be remarkably improved, non-targeted immunosuppression can be avoided, the probiotics can be effectively adhered to cationic inflammatory surfaces, the concentration of local inflammatory area probiotics drugs is enhanced, and site-specific delivery of probiotics in intestinal tract inflammatory areas is realized. Therefore, the probiotic packaging material provided by the invention improves the quantity of the probiotics reaching the colon part, and obviously improves the adhesion, colonization and survival rate of the probiotics in a complex gastrointestinal tract environment, thereby improving the oral availability and in-vivo survival rate of the probiotics. The results of the examples show that the presence of the functional layer of the probiotic encapsulating material provided by the invention can effectively adhere the encapsulated probiotic to the inflammatory area and enhance the concentration of the probiotic drug in the inflammatory area in comparison to healthy mice in a mouse model of colitis. However, the physical mixing of the functional molecules with the probiotics only with the protective layer does not significantly increase the adhesion of the probiotics in the inflammation area, and the probiotic encapsulating material provided by the invention encapsulates the probiotics to significantly inhibit the secretion of the gastroenteritis factor. The results of the above examples show that the probiotic packaging material provided by the invention can effectively adhere to the surface of cationic inflammation, enhance the concentration of probiotic medicines in local inflammation areas and realize the site-specific delivery of probiotics in intestinal inflammation areas.
The invention provides a preparation method of encapsulated probiotics, which comprises the following steps of; mixing procyanidine, water-soluble metal salt and water, and carrying out coordination chelation reaction on the obtained mixed solution to obtain a protective layer; mixing the protective layer with the probiotic solution for first encapsulation to obtain probiotics encapsulated by the protective layer; and mixing the probiotics encapsulated by the protective layer, the aqueous solution of the functional active molecules and the buffer solution to perform physical adsorption or chemical combination for second encapsulation, and forming a functional layer on the outer surface of the protective layer to obtain the encapsulated probiotics. The encapsulation method provided by the invention is simple, convenient, quick and safe, and is suitable for industrial production.
Drawings
FIG. 1 is a graph showing the growth of probiotics, ecN@PC-Fe and EcN@PC-Fe/HA according to the invention in example 1 when exposed to Simulated Gastric Fluid (SGF) containing pepsin and bile acid (pH=2), respectively;
FIG. 2 is a live-dead staining of probiotics, ecN@PC-Fe and EcN@PC-Fe/HA according to the invention of detection example 1, respectively, exposed to Simulated Gastric Fluid (SGF) containing pepsin and bile acid (pH=2);
FIG. 3 shows the live and dead staining of the invention in detection example 2 by exposure to Simulated Gastric Fluid (SGF) containing pepsin and bile acid (pH=2), respectively, of LaC, lac@PC-Ca and Lac@PC-Ca/Alg;
FIG. 4 is a graph showing the growth of probiotics, ecN@PC-Fe and EcN@PC-Fe/HA according to the invention in example 1 when exposed to Simulated Intestinal Fluid (SiF) containing trypsin (pH=6.8), respectively;
FIG. 5 is a morphological characterization of probiotics, ecN@PC-Fe and EcN@PC-Fe/HA under TEM in example 1 of the present invention;
FIG. 6 is a graph showing the in vitro growth curves of probiotics, ecN@PC-Fe and EcN@PC-Fe/HA according to the invention of example 1;
FIG. 7 shows in vivo distribution of EcN@PC-Fe, ecN@PC-Fe/HA and unwrapped EcN prepared in example 1 at various time points after intragastric administration into mice in accordance with an embodiment of the present invention;
FIG. 8 shows the distribution of the encapsulated EcN and unwrapped EcN prepared in example 1 into the intestinal tract of a mice over 120 hours in accordance with an embodiment of the present invention;
FIG. 9 shows the survival rate of the EcN@PC-Fe, ecN@PC-Fe/HA and unwrapped EcN prepared in example 1 of the present invention at different parts of the intestinal tract after being gavaged into the body of a mouse for 120 hours;
fig. 10 is a graph showing changes in body weight and disease activity index of a mouse model of colitis treated with encapsulated EcN and unwrapped EcN gavage prepared in example 1 in examples of the present invention;
FIG. 11 is an evaluation of colon histopathological damage using the results of the ecl@PC-Fe, ecl@PC-Fe/HA and unwrapped EcN lavage treatment of colitis mice intestinal hematoxylin and eosin staining prepared in example 1 in examples of the present invention;
FIG. 12 shows distribution and fluorescence intensity statistics of the encapsulated EcN prepared in example 1 of the present invention in the colon after intragastric administration to healthy mice and colitis mice;
FIG. 13 shows the changes in intestinal inflammatory factor of mice model treated with the present invention using EcN@PC-Fe, ecN@PC-Fe/HA and unwrapped EcN lavage prepared in example 1;
FIG. 14 is a TEM characterization of EcN@PC-Fe/HA prepared in example 1 of the present invention.
Detailed Description
The invention provides a probiotic packaging material, which comprises a protective layer and a functional layer which are arranged in a lamination way from inside to outside; the protective layer is a procyanidine metal complex; the functional layer is a functional active molecule which is physically adsorbed or chemically combined with the procyanidine group in the protective layer, the chemical combination is that sulfhydryl or amino in the functional active molecule is chemically combined with phenolic hydroxyl in the procyanidine group through Michael addition or Schiff base reaction, and the functional active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof.
In the present invention, all preparation materials/components are commercially available products well known to those skilled in the art unless specified otherwise.
In the present invention, the metal ion in the procyanidin metal complex preferably includes Fe 3+ 、Zn 2+ 、Ca 2+ 、Mg 2 + 、Ti 4+ 、Ce 4+ 、Cu 2+ And Mn of 2+ One or more of them, more preferably Fe 3+ 、Ca 2+ Or Mg (Mg) 2+ 。
In the present invention, the functional active molecule includes one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof, preferably hyaluronic acid, sodium alginate, chondroitin sulfate or heparan sulfate.
In the present invention, the molecular weight of the sodium hyaluronate is preferably 30 to 500kDa, more preferably 360kDa.
In the present invention, the ratio of the thicknesses of the protective layer and the functional layer is preferably (1 to 2): 3.
the invention provides a preparation method of encapsulated probiotics, which comprises the following steps of;
mixing procyanidine, water-soluble metal salt and water, and performing coordination chelation reaction on the obtained mixed solution to obtain a protective layer;
mixing the protective layer with the probiotic solution for first encapsulation to obtain probiotics encapsulated by the protective layer;
and mixing the probiotics encapsulated by the protective layer with functional active molecule solution to perform physical adsorption or chemical combination for second encapsulation, and forming a functional layer on the outer surface of the protective layer to obtain the encapsulated probiotics.
The invention mixes procyanidine, water-soluble metal salt and water (hereinafter referred to as first mixing), and the obtained mixed solution undergoes coordination chelation reaction to obtain a protective layer.
In the present invention, the first mixing of the procyanidins is preferably performed in the form of a procyanidin solution.
In the present invention, the solvent in the procyanidin solution is preferably Tris-HCl buffer. In the present invention, the pH of the Tris-HCl buffer is preferably 8.5, and the molar concentration of the Tris-HCl buffer is preferably 10mmol/L.
In the present invention, the water-soluble metal salt is preferably one or more of a water-soluble trivalent iron salt, a water-soluble zinc salt, a water-soluble calcium salt, a water-soluble magnesium salt, a water-soluble titanium salt, a water-soluble cerium salt, a water-soluble copper salt, and a water-soluble manganese salt, more preferably a water-soluble trivalent iron salt, a water-soluble zinc salt, a water-soluble calcium salt, or a water-soluble magnesium salt, particularly preferably a water-soluble trivalent iron salt and a water-soluble calcium salt.
In a specific embodiment of the present invention, the water-soluble metal salt is particularly preferably ferric chloride and calcium dichloride.
In the present invention, the first mixing preferably includes the steps of: dissolving the water-soluble metal salt in water to obtain a metal salt solution; mixing the procyanidine solution and the metal salt solution. In the present invention, the mixing of the procyanidin solution and the metal salt solution is preferably performed in a vortex shaker, and the mixing time in the vortex shaker is preferably 20s.
In the present invention, the procyanidine mass concentration in the mixed solution is preferably 0.5 to 50mg/mL, more preferably 1 to 45mg/mL. In the present invention, the mass concentration of the metal ion in the mixed solution is 0.1 to 5mg/mL, more preferably 0.2 to 4.5mg/mL.
In the present invention, the pH of the mixed solution is in the range of 4 to 10, preferably 5 to 8.
In the present invention, the temperature of the coordination chelate reaction is preferably 25 to 37 ℃, more preferably 37 ℃; the time of the coordination chelation reaction is preferably 1 to 2 hours, more preferably 1 hour; the coordination chelate reaction is preferably carried out under stirring, and the stirring speed is preferably 150rpm.
In the invention, in the mixed solution, the procyanidine and metal ions undergo coordination chelation reaction to generate the procyanidine metal complex with a three-dimensional reticular macromolecular structure.
In the present invention, the coordination chelate reaction is followed by a protective layer solution, and the present invention preferably directly encapsulates the protective layer solution.
After the protective layer is obtained, the protective layer and the probiotic solution are mixed (hereinafter referred to as second mixing) for first encapsulation, so that the probiotics encapsulated by the protective layer are obtained.
In the present invention, the concentration of the probiotics in the probiotics solution is preferably 1×10 5 ～1×10 9 CFU/mL, more preferably 2X 10 5 ～0.5×10 9 CFU/mL。
In the present invention, the probiotic solution is preferably a PBS buffer solution of probiotics.
The invention has no special requirements on the source or the preparation method of the probiotic solution, and the probiotic solution is obtained by adopting a resin method of a person skilled in the art.
In the present invention, the probiotics preferably include one or more of enterococcus, streptococcus, bifidobacterium, lactobacillus, propionibacterium, bacillus, yeast and escherichia coli, more preferably one or more of escherichia coli, propionibacterium, bacillus and lactobacillus.
In the present invention, the second mixing is preferably performed in a vortex oscillator, and the time of the second mixing is preferably 30s,
In the present invention, the temperature of the first encapsulation is preferably 25 to 37 ℃, more preferably 37 ℃; the reaction time of the encapsulation is preferably 1 to 2 hours, more preferably 1 hour. The first encapsulation is preferably carried out under stirring, preferably at a speed of 150rpm.
In the invention, during the first encapsulation, the probiotics are encapsulated by the three-dimensional netlike nano film formed by the protective layer, so as to obtain the probiotics encapsulated by the protective layer.
In the present invention, the first encapsulation reaction solution is obtained after the first encapsulation, and the present invention preferably performs a post-treatment on the first encapsulation reaction solution to obtain the probiotics encapsulated by the protective layer. In the present invention, the post-treatment preferably includes the steps of: carrying out solid-liquid separation on the first encapsulation reaction liquid to obtain a solid product; and washing the solid product to obtain the probiotics encapsulated by the protective layer. In the present invention, the solid-liquid separation is preferably centrifugation, the rotational speed of the centrifugation is preferably 1000 to 5000rpm, more preferably 1500 to 3500rpm, and the time of the centrifugation is preferably 5 minutes. The washing medium is preferably a sodium chloride solution, and the molar concentration of the sodium chloride solution is preferably 0.15mol/L. The number of times of the washing is preferably 3, and the unreacted raw materials are preferably washed by the washing.
After the probiotics encapsulated by the protective layer are obtained, the probiotics encapsulated by the protective layer and the functional active solution are mixed to be subjected to physical adsorption or chemical combination for second encapsulation, and a functional layer is formed on the outer surface of the protective layer, so that the encapsulated probiotics are obtained.
In the present invention, the solvent in the functional active solution is preferably Tris-HCl buffer. In the present invention, the pH of the Tris-HCl buffer is preferably 8.5, and the molar concentration of the Tris-HCl buffer is preferably 10mmol/L.
In the present invention, the mass concentration of the functional active molecule solution is preferably 0.5 to 50mg/mL, more preferably 1 to 45mg/mL.
In the present invention, the temperature of the second encapsulation is preferably 1 to 4 ℃, more preferably 4 ℃; the incubation time of the second encapsulation is preferably 1 to 2 hours, more preferably 1 hour, and the second encapsulation is preferably performed under stirring.
In the present invention, during the second encapsulation, the functional active molecule and the procyanidin group in the protective layer undergo a physical adsorption or chemical combination reaction, so as to form a functional layer on the surface of the protective layer.
In the invention, the second encapsulation reaction liquid is obtained after the second encapsulation, and the invention preferably carries out aftertreatment on the second encapsulation reaction liquid to obtain the encapsulated probiotics. In the present invention, the post-treatment preferably includes the steps of: carrying out solid-liquid separation on the second encapsulation reaction liquid to obtain a solid product; washing the solid product to obtain the encapsulated probiotic. In the present invention, the solid-liquid separation is preferably centrifugation, the rotational speed of the centrifugation is preferably 1000 to 5000rpm, more preferably 1500 to 3500rpm, and the time of the centrifugation is preferably 5 minutes. The wash medium is preferably a sterile PBS solution, preferably at a molar concentration of 0.01M. The number of washes is preferably 3, after which the invention preferably is centrifuged to collect the solid product to yield the encapsulated probiotic. The rotational speed of the centrifugation is preferably 1000 to 5000rpm, more preferably 1500 to 3500rpm, and the time of the centrifugation is preferably 5 minutes.
The invention provides an encapsulated probiotic, which comprises a probiotic and an encapsulating material for encapsulating the probiotic, wherein the encapsulating material is the probiotic encapsulating material according to the technical scheme, and a protective layer in the encapsulating material is in contact with the probiotic.
In the present invention, the encapsulated probiotics are preferably prepared according to the encapsulation method described in the technical scheme.
The invention provides an encapsulated probiotic comprising a nanocoating film for encapsulating the probiotic; the nano coating comprises two layers, wherein the first layer is a protective layer, a three-dimensional net-shaped nano film is formed by coordination and chelation of procyanidine and metal ions, and the second layer is a functional layer, and the functional layer is formed by physical adsorption or chemical combination reaction of functional molecules and procyanidine groups. The preparation process is simple, convenient, quick and safe, and the formed nano coating can remarkably improve the survival rate of probiotics in complex gastrointestinal tract environments and remarkably increase the adhesion and colonization of the probiotics in intestinal tracts.
The technical solutions provided by the present invention are described in detail below with reference to the drawings and examples for further illustrating the present invention, but they should not be construed as limiting the scope of the present invention.
Example 1
In this example, E.coli Nissle 1917 (hereinafter EcN) was encapsulated and characterized. The specific encapsulation method comprises the following steps:
1mL of 1X 10 8 EcN of CFU number or EcN carrying pBBR1MCS2-Tac-mCherry, centrifuging at 3000rpm for 3min, collecting supernatant, repeating the above centrifugation and supernatant collection operation for 3 times, adding 0.01mol/LPBS to adjust total volume to 200 μl, and mixing thoroughly to obtain EcN bacterial liquid for use.
15mL of procyanidine solution with the concentration of 1mg/mL is prepared by using a Tris-HCl buffer solution with the pH of 8.5 and 10mM, then 10 mu L of ferric trichloride aqueous solution with the concentration of 225mg/mL is added, vortex oscillation is carried out for 20s, the procyanidine solution and the Fe solution are fully mixed, and the procyanidine solution and the Fe solution react for 1h at the temperature of 37 ℃ and the rpm of 150rpm 3+ Coordination forms a three-dimensional network macromolecule. 200. Mu.L of EcN bacteria solution was then added thereto, followed by vortexing for 30s, and then allowing the mixture to react at 37℃and 150rpm for 1 hour. After the reaction was completed, the solid product was centrifuged at 4000rpm for 5min, and the unreacted raw materials were removed by washing the solid product 3 times with 0.15M NaCl solution, to obtain a probiotic bacteria encapsulated by a protective layer, which was designated as EcN@PC-Fe.
Subsequently 10mL of Tris-HCl buffer containing 1mg/mL of sodium hyaluronate with a molecular weight of 360kDa ph=8.5, 10mM was added for resuspension, the reaction was continued at 4 ℃,150rpm for 1h,4000rpm centrifugation was performed for 5min to obtain whole encapsulated thalli, 3 times with sterile 0.01M PBS, 4000rpm centrifugation was performed for 5min, and the precipitated product was collected to obtain encapsulated probiotics, designated ecn@pc-Fe/HA.
Example 2
In this example, lactobacillus crispatus Lactobacillus crispatus (hereinafter LaC) was encapsulated and characterized. The specific encapsulation method comprises the following steps:
1mL of 1X 10 6 And (5) centrifuging the mixture at 3500rpm for 5min at LaC of CFU number, taking supernatant, repeating the centrifugation and supernatant taking operations for 3 times, adding 0.01mol/LPBS to adjust the total volume to 200 mu L, and fully mixing to obtain EcN bacterial liquid for later use.
15mL of procyanidine solution at a concentration of 2mg/mL was prepared with Tris-HCl buffer at pH=8.5, 10mM, followed by addition of 10. Mu.L of aqueous solution of calcium dichloride at a concentration of 300mg/mL, vortexing for 20s, and shaking bothMixing them thoroughly, reacting at 37deg.C and 150rpm for 1 hr to obtain procyanidine and Ca 2+ Coordination forms a three-dimensional network macromolecule. Then 200. Mu.LLaC bacterial solution was added thereto, vortexed and shaken for 30s, and the mixture was allowed to react at 37℃and 150rpm for 1 hour. After the reaction, the solid product was washed 3 times with 0.15M NaCl solution and the unreacted raw material was removed by centrifugation at 4000rpm for 5min to obtain the probiotic bacteria encapsulated by the protective layer, designated as LaC@PC-Ca.
10mL of Tris-HCl buffer solution containing 5mg/mL sodium alginate and having pH=8.5 was then added for resuspension, the reaction was continued at 4℃and 150rpm for 1h, centrifugation was performed at 4000rpm for 5min to obtain whole encapsulated cells, washing was performed 3 times with sterile 0.01M PBS, centrifugation was performed at 4000rpm for 5min, and the precipitated product was collected to obtain encapsulated probiotics, which was designated as LaC@PC-Ca/Alg.
Test example 1
In vitro gastric juice stress resistance detection
Equivalent amount of probiotic bacteria (1X 10) in example 1 8 CFU) EcN, ecn@pc-Fe and ecn@pc-Fe/HA were resuspended in 1mL of artificial simulated gastric fluid SGF (ph=2.0, containing 20mg nacl, 3mg bile acid, 32mg pepsin and 70 μl hydrochloric acid per 100mL SGF), incubated in a 37 ℃ incubator at 120rpm for 2h, 50 μl of each sample was removed, washed 3 times with PBS, and finally the samples were diluted with PBS and spread evenly on LB agar plates. Colonies were counted after incubation at 37℃for 24 hours (as shown in FIG. 1). Then using bacterial live-dead fluorescent Probes (Molecular Probes'
BacLight TM Bacteria were fluorescent stained with the bacterial Viabailitykit and bacterial mortality was observed and counted for the different treatment groups using CLSM (see FIG. 2). The results show a significant increase in the tolerance of ecn@pc-Fe and ecn@pc-Fe/HA to SGF compared to the unencapsulated EcN, wherein ecn@pc-Fe/HA shows the best protective effect.
Equivalent amount of (1X 10) in example 2 6 CFU) LaC, lac@pc-Ca and lac@pc-Ca/Alg were resuspended in 1mL of artificial simulated gastric fluid SGF (ph=2.0, 20mg nacl, 3mg bile acid, 32mg pepsin and 70 μl hydrochloric acid per 100mL SGF), respectively, and placed at 37 °cIncubation was carried out in an incubator at 120rpm for 2h, 50. Mu.L of each sample was taken out, washed 3 times with PBS, and after dilution of the samples, bacterial live-dead fluorescent Probes (Molecular Probes'
BacLight TM Bacteria were fluorescent stained with the bacterial Viabailitykit and bacterial mortality was observed and counted for the different treatment groups using CLSM (see FIG. 3). The test results showed a significant increase in the resistance of the encapsulated LaC to SGF compared to the unencapsulated LaC, with lac@pc-Ca/Alg showing the best protective effect.
Test example 2
In vitro intestinal juice stress resistance detection
Equivalent amount of (1X 10) in example 1 8 CFU) EcN, ecn@pc-Fe and ecn@pc-Fe/HA were resuspended to 1mL of artificial simulated intestinal fluid SIF (ph=6.8, 0.68g KH per 100mL SIF 2 PO 4 15mL of 0.2M NaOH and 1g of trypsin), incubated at 120rpm in an incubator at 37℃for 2 hours, 50. Mu.L of each sample was taken out, washed 3 times with PBS, and finally the samples were diluted with PBS and spread evenly on LB agar plates. Colony counts were performed after incubation at 37℃for 24 hours (as shown in FIG. 4). The results show a significant increase in the resistance of the encapsulation EcN to SIF compared to the unencapsulated EcN, with ecn@pc-Fe/HA exhibiting the best protective effect.
Test example 3
Coating probiotic characterization analysis
The cell morphology of EcN, ecN@PC-Fe and EcN@PC-Fe/HA in example 1 was observed by TEM. The TEM specifically operates as follows: and (3) dripping 10 mu L of diluted sample on a 200-mesh carbon film copper net, standing for 5min, and sucking residual liquid by using water absorption paper. Then washed 3 times with PBS, and the copper mesh was dried in a constant temperature incubator at 37℃for 12 hours. The observation was performed using a transmission electron microscope (Hitachi H7500), and the detection voltage was set to 80kV. The transmission electron microscope image showed that the ecn@pc-Fe surface had a translucent film-like coating with a thickness of about 50nm, and the modification of the functional molecule HA increased the layer thickness of the surface of the bacterial body to about 200nm (e.g., a in fig. 5 is EcN without coating, b in fig. 5 is ecn@pc-Fe, c in fig. 5 and 14 is ecn@pc-Fe/HA). As can be seen from FIG. 5, the ecN@PC-Fe/HA encapsulating layer prepared in example 1 of the present invention is significantly thicker than ecN@PC-Fe, which indicates that the ecN@PC-Fe/HA encapsulating layer prepared in example 1 of the present invention HAs two layers, the first layer is a protective layer, a three-dimensional network nano-film is formed by coordination chelation of procyanidins and metal ions, and the second layer is a functional layer, and functional molecules and procyanidin groups are formed by physical adsorption or chemical combination reaction.
Test example 4
Growth curve monitoring of coated probiotics
To examine whether the encapsulated coating would affect the growth of the cells, we diluted EcN, ecn@pc-Fe and ecn@pc-Fe/HA of example 1 with fresh LB medium to an optical density (OD 600) of 0.2, then incubated in a 37 ℃ incubator at 200rpm constant temperature, readings were recorded at OD600 nm every 30 minutes with a microplate spectrophotometer, thereby recording the growth curves of the encapsulated probiotics, as shown in fig. 6, the log growth periods of ecn@pc-Fe and ecn@pc-Fe/HA exhibited a hysteresis of 1-2h compared to EcN, indicating that the probiotic had a time to break through the coating barrier, resulting in a relatively retarded log growth period, which was related to the coating thickness, but the presence of the coating did not affect the viability of the encapsulated probiotic.
Test example 5
Evaluation of retention and survival of encapsulated probiotics in mice
The experimental animals are C57BL/6J mice with 6 weeks of age, all the mice are adaptively kept for one week in SPF-grade animal houses with constant temperature (22+/-1 ℃) and constant humidity (50% -60%), the light and dark circulation is carried out for 12 hours, and the animals drink water freely. Will be 1X 10 8 CFU coated probiotics and uncoated probiotics with pBBR1MCS2-Tac-mCherry plasmid intervene in mice by gastric lavage, and at time points of 0.5h,4h,12h,24h,48h and 72h, mice were anesthetized with 2% isoflurane and oxygen mixed gas, and imaged in a small animal in vivo imager (Perkinelmer, waltham, mass., USA). As shown in FIG. 7, after 72 hours, ecN@PC-Fe and EcN@PC-F were compared to EcNThe fluorescence intensity of the e/HA gastrointestinal tract is significantly increased. After 120h, the mice were euthanized, the digestive tracts of the mice were taken for in vitro fluorescence detection and fluorescence signal intensity was recorded, and as shown in fig. 7, the fluorescence of the EcN group intestinal tract was almost disappeared, while the fluorescence in the ecn@pc-Fe/HA group intestinal tract was strongest compared with the ecn@pc-Fe group.
The contents of the colon and ceca of the mice were then scraped, resuspended in PBS, diluted, and plated on LB agar plates containing 50. Mu.g/mL kanamycin, incubated at 37℃for 24h, and subjected to colony counting to evaluate the retention and survival of the encapsulated probiotics in the mice. The results are shown in figure 9, where the probiotics were mainly distributed in the cecum and colon after 120 h. The number of encapsulated probiotics was significantly increased in the cecum and colon of mice compared to EcN, with the number of viable bacteria of the ecn@pc-Fe/HA group being the greatest. In the colon, the number of viable bacteria was 150 times and 1.5 times that of the EcN group and the EcN@PC-Fe group, respectively. The above results are consistent with the in vitro stress-resistant results, and the coated probiotics show significantly increased survival and colonization rates in the gastrointestinal tract, particularly the cecum and colon.
Test example 6
Evaluation of treatment effect of encapsulated probiotics on ulcerative colitis in mice
The method for establishing a model by selecting the most classical method for freely drinking Dextran Sodium Sulfate (DSS) aqueous solution for 7 days comprises the following specific operations: DSS was dissolved in distilled water to prepare 2.5% (w/w) solution, and the mice were freely consumed 2.5% DSS water for 7d every 2d, and daily intake, water intake, body weight, fecal occult blood were recorded. And disease activity index (Disease activity index, DAI) was calculated from the body weight loss and fecal consistency and occult blood status (table 1).
TABLE1 mouse disease Activity index Table1-1 Disease activity index ofmice
DSS-induced colitis mice were given 1X 10 daily by gastric lavage intervention 8 CFU encapsulated probiotics, administered for 5 days, for smallThe body weight change and disease activity index of the mice are characterized; to evaluate the ameliorating effect of the encapsulated probiotics on ulcerative colitis in mice. As shown in FIG. 10, the EcN@PC-Fe and EcN@PC-Fe/HA significantly improved the weight loss and decreased the disease activity index in colitis mice compared to mice given PBS and EcN, with EcN@PC-Fe/HA exhibiting the best effect. The mice were euthanized and dissected, the distal colon tissue was rapidly removed about 1cm, rinsed 3 times with pre-chilled saline, then placed in 4% paraformaldehyde fixing solution to allow adequate fixation of the tissue, and the remaining colon tissue placed in a sterile EP tube and stored in a-80 ℃ refrigerator. The fixed distal colon tissue was dehydrated with gradient ethanol, paraffin embedded and the slice thickness was 5 μm. The colon pathological tissue injury is evaluated by hematoxylin and eosin staining results, and the results in FIG. 11 show that the healthy mice have complete colon structure and no inflammation and edema, and glandular cells are orderly arranged; the colon epithelial layer of the DSS model mouse is incomplete, the crypt, the gland and the goblet cells disappear, a large amount of inflammatory infiltration occurs in the colon, and the tissue is damaged and serious. After the intervention of probiotics, the colon injury degree is obviously improved, and the inflammatory infiltration is relieved. Among them, ecN@PC-Fe/HA shows the best improvement effect.
Test example 7
Site-specific adhesion of probiotic encapsulated intestinal inflammatory regions
DSS-induced colitis mice and healthy mice were fasted for 12h, then 1 x 10 8 CFU ecn@pc-Fe/HA with pBBR1MCS2-Tac-mCherry plasmid the colon of the mice were euthanized after 8h by intervention in the colon of the colon in a small animal living imager (PerkinElmer, waltham, MA, USA). The results of fig. 12 show that compared to healthy mice, the adhesion of ecn@pc-Fe/HA in colon tissue of enteritis mice is significantly increased, resulting in a significant increase in the probiotic concentration in the local inflammatory area, enabling site-specific delivery of probiotics in the intestinal inflammatory area.
Test example 8
Effect of encapsulated probiotics on expression of inflammatory factors in colon tissue of mice
Taking a 50mg colon tissue sample, adding 1mL of precooled TRIzon reagent, fully lysing the colon tissue sample by using a tissue homogenizer, standing on ice for 10min, adding 200 mu L of chloroform, shaking for 15s, and standing for 3min at room temperature; centrifuging at 12000rpm for 15min at 4deg.C, collecting 500 μl of colorless aqueous phase (upper layer), and transferring to EP tube of new RNase-free; adding 500 mu L of isopropanol, uniformly mixing and standing for 10min; centrifuging at 12000rpm for 10min at 4deg.C, and discarding supernatant; 1mL of 75% ethanol was added to wash the precipitate (with 4 ℃ C. DEPC water); centrifuging at 2000rpm for 3min at 4deg.C, and discarding supernatant; standing at room temperature for 5-8min, adding 30 μL DEPC water, and dissolving completely; and carrying out reverse transcription on the extracted RNA to obtain cDNA, and measuring the expression quantity of inflammatory factors TNF-alpha, IL-6 and IL-1 beta in colon tissues of the mice by adopting real-time fluorescence quantitative PCR. The results in FIG. 13 show that the expression levels of TNF- α, IL-6 and IL-1β were all significantly increased in colon tissue of mice in the model group compared to normal mice, indicating that DSS induced severe intestinal inflammation. Unencapsulated probiotic EcN HAs poor effect of improving intestinal inflammation, however, the coated probiotic intervention can significantly inhibit the expression of inflammatory factors, wherein ecn@pc-Fe/HA shows the best effect of inhibiting inflammation.
Although the foregoing embodiments have been described in some, but not all embodiments of the invention, other embodiments may be obtained according to the present embodiments without departing from the scope of the invention.
Claims (10)
1. The probiotic packaging material is characterized by comprising a protective layer and a functional layer which are arranged in a lamination way from inside to outside; the protective layer is a procyanidine metal complex; the functional layer is a functional active molecule which is physically adsorbed or chemically combined with the procyanidine group in the protective layer, the chemical combination is that sulfhydryl or amino in the functional active molecule is chemically combined with phenolic hydroxyl in the procyanidine group through Michael addition or Schiff base reaction, and the functional active molecule comprises one or more of hyaluronic acid, sodium alginate, chondroitin sulfate, heparan sulfate, dermatan sulfate and derivatives thereof.
2. The probiotic encapsulating material of claim 1 wherein the metal ion in the procyanidin metal complex comprises Fe 3+ 、Zn 2+ 、Ca 2+ 、Mg 2+ 、Ti 4+ 、Ce 4+ 、Cu 2+ And Mn of 2+ One or more of the following.
3. A method of preparing an encapsulated probiotic comprising the steps of;
mixing procyanidine, water-soluble metal salt and water, and performing coordination chelation reaction on the obtained mixed solution to obtain a protective layer;
mixing the protective layer with the probiotic solution for first encapsulation to obtain probiotics encapsulated by the protective layer;
and mixing the probiotics encapsulated by the protective layer with functional active molecule solution, and performing secondary encapsulation through physical adsorption or chemical combination to form a functional layer on the outer surface of the protective layer, thereby obtaining the encapsulated probiotics.
4. The method according to claim 3, wherein the procyanidin is present in the mixed solution in a mass concentration of 0.5 to 50mg/mL and the metal ion is present in a mass concentration of 0.1 to 5mg/mL.
5. The method according to claim 3 or 4, wherein the pH of the mixed solution is in the range of 4 to 10.
6. A method of preparing according to claim 3, wherein the concentration of probiotics in the probiotic solution is 1 x 10 5 ～1×10 9 CFU/mL。
7. The encapsulation method of claim 6, wherein the probiotic bacteria comprise one or more of enterococci, streptococci, bifidobacteria, lactobacilli, propionibacteria, bacilli, yeasts, and escherichia coli.
8. The method according to claim 3, wherein the functional active molecule solution has a mass concentration of 0.5 to 50mg/mL.
9. The method according to claim 3, wherein the temperature of the coordination and chelation reaction is 25 to 37 ℃ and the time of the coordination and chelation reaction is 1 to 2 hours;
the temperature of the first encapsulation is 25-37 ℃ and the heat preservation time is 1-2 h;
the temperature of the second encapsulation is 1-4 ℃, and the heat preservation time is 1-2 h.
10. The encapsulated probiotic bacteria prepared by the preparation method according to any one of claims 3 to 9, comprising probiotic bacteria and an encapsulating material for encapsulating the probiotic bacteria, wherein the encapsulating material is the probiotic bacteria encapsulating material according to claim 1 or 2, and the protective layer in the encapsulating material is in contact with the probiotic bacteria.
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| CN108079914A (en) * | 2017-12-13 | 2018-05-29 | 温州生物材料与工程研究所 | The method that one-step method prepares poly aminated compounds microcapsules |
| CN113499325A (en) * | 2021-07-08 | 2021-10-15 | 成都邦家乐君生物科技有限公司 | Biomass-based encapsulating material for probiotic activity protection and encapsulating method |
| WO2022172649A1 (en) * | 2021-02-09 | 2022-08-18 | 国立研究開発法人農業・食品産業技術総合研究機構 | Polyphenol-iron complex capsule, hydrogen peroxide capsule, fenton reaction kit, and method for breeding fish and shellfish or treating diseases of fish and shellfish |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108079914A (en) * | 2017-12-13 | 2018-05-29 | 温州生物材料与工程研究所 | The method that one-step method prepares poly aminated compounds microcapsules |
| WO2022172649A1 (en) * | 2021-02-09 | 2022-08-18 | 国立研究開発法人農業・食品産業技術総合研究機構 | Polyphenol-iron complex capsule, hydrogen peroxide capsule, fenton reaction kit, and method for breeding fish and shellfish or treating diseases of fish and shellfish |
| CN113499325A (en) * | 2021-07-08 | 2021-10-15 | 成都邦家乐君生物科技有限公司 | Biomass-based encapsulating material for probiotic activity protection and encapsulating method |
| US20220000793A1 (en) * | 2021-07-08 | 2022-01-06 | Chengdu bangjialejun Biotechnology Co., Ltd | Biomass-based encapsulating material for protection of probiotic activity and an encapsulating method |
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