RU2740380C1 - Bioengineered structure based on bacterial alginate and probiotic bacteria and method for production thereof - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention relates to medicine and pharmaceutics, in particular to an article for treating and creating experimental models of gastrointestinal disorders, due to the therapeutic action of living probiotic bacteria cultivated in situ in a bioengineering structure. Disclosed is a bioengineering structure for probiotic bacteria of genera Lactobacillus and Bifidobacterium, which is a layer of porous microspheres in a hydrogel arranged on a porous substrate. Porous substrate is made from poly-3-oxybutyrate and/or its copolymer with 3-oxyvalerate with molecular weight from 1 × 10⁵ to 2 × 10⁶ g/mol and is characterized by the presence of through pores (or bonded pores), total porosity from 75 to 99 %, average pore diameter from 0.5 to 20 mcm. Porous microspheres are made from poly-3-oxybutyrate with molecular weight from 1 × 10⁴ to 1 × 10⁶ g/mol and are characterized by availability of through pores (or connected pores), total porosity from 75 to 99 %, average pore diameter from 0.5 to 20 mcm. Pores of substrate and microspheres are filled with hydrogel containing probiotic bacteria. Hydrogel is made from bacterial alginate with molecular weight from 10⁵ to 10⁶ g/mol, molar content of guluronic acid residues in polymer from 31 mol. % to 48 mol. % and acetylation level is from 10 mol. % to 46 mol. %. Besides, a method of producing the above bioengineered structure is proposed.

EFFECT: technical result is obtaining a hybrid bioengineering structure based on probiotic bacteria and bacterial polymers (poly-3-oxyalkanoate and alginate), providing at least 95 % survival rate of bacteria, as well as increasing their protection (increase in bacteria survival by at least 50 %) in an unfavorable medium while preserving biocompatibility (no cytotoxicity) of the construct in vitro.

18 cl, 2 dwg, 4 ex

Description

The technical field to which the invention relates

The present invention relates to the field of biodegradable, biocompatible and biologically active products for medicine and experimental biology, which have an additional therapeutic effect due to the biological activity of living probiotic bacteria cultivated in situ in a bioengineered construct placed in the area of damage to the wall of the gastrointestinal tract, for the treatment of certain chronic diseases of the gastrointestinal tract, such as peptic ulcer of the duodenum, ulcerative colitis, ischemic colitis, Crohn's disease, as well as surgical interventions on the intestine with local tissue removal and their complications, as well as to create experimental models of these diseases in order to develop new medicines and diagnostics for their treatment. The inventive method makes it possible to create a medical product for regenerative medicine of a new type, where probiotic bacteria serve as an active active principle, and can be used in biotechnology, tissue engineering, regenerative medicine, and can be used in biotechnology, tissue engineering, regenerative medicine

State of the art

Chronic inflammatory diseases of the large and small intestine, such as duodenal ulcer, ulcerative colitis, ischemic colitis, Crohn's disease, as well as surgical interventions on the intestine with local tissue removal and their complications can cause the formation of perforated ulcers and fistulas. Such damage to the intestinal wall is a very serious pathology and can lead to the development of peritonitis and death, therefore, they almost always require surgical treatment. Currently, when performing surgical operations on the small and large intestines, intestinal patches and endoprostheses (platy, stents) made of biodegradable polymers are increasingly used to close the defect in the intestinal wall tissues and regenerate them. However, the disadvantages of such medical devices are often insufficient compatibility with the surrounding tissues, the inability to biodegrade within a given time frame, and the absence of a long-term therapeutic effect on the tissue defect area in order to regenerate it, which is associated with the use of synthetic polymeric materials that have poor biocompatibility as with tissues. intestinal walls, and with probiotic bacteria, which have a therapeutic effect in tissue repair. In addition, such products often have a simplified structure that does not take into account the morphological and physiological characteristics of the intestinal mucosa, which worsens their therapeutic efficacy, or, on the contrary, an excessively complex design, which significantly complicates their production [Bonartsev A.P., Voinova V.V. ., Bonartseva G.A. Poly-3-hydroxybutyrate and human microbiota. Applied Biochemistry and Microbiology, 2018, 54 (6), 561-584].

Currently, a number of natural biopolymers are actively used in medicine, pharmaceuticals and tissue engineering: alginate, chitosan, dextran, hyaluronic acid, agarose, xanthan, chondroitin sulfate, fibrin, collagen (gelatin), lye fibroin, poly-L-lysine, polyglutamic acid, poly-3-hydroxyalkanoates [Bonartsev A. P., Voinova V. V., Bonartseva G. A. Poly-3-hydroxybutyrate and human microbiota. Applied Biochemistry and Microbiology, 2018, 54 (6), 561-584]. Thus, alginates that form biocompatible hydrogels are actively used in medicine, pharmaceuticals, cosmetology and food industry, in biotechnology, for example, for encapsulating cells of various mammalian cultures (fibroblasts, myoblasts, osteoblasts, MSCs, chondrocytes, etc.), as well as in tissue engineering for the manufacture of matrices for the growth of cells of various types [Lee KY, Mooney DJ Alginate: properties and biomedical applications. Prog. Polym. Sci. 2012,37 (1) 106-126]. However, the medical use of biopolymers often does not take into account their role and functions in nature. So, if in algae alginates perform mainly a structural and mechanical function, then in bacteria capable of synthesizing this polysaccharide of the genera Azotobacter and Pseudomonas, their functions are more diverse, but the main one is protective. The cells of these bacteria synthesize and secrete this exopolychasaccharide to form the outer mucous membrane of individual cells and their clusters - cysts or biofilms that protect bacterial cells from adverse external factors. Biofilm formation is the main cause of the pathogenesis of infectious diseases caused by Pseudomonas aeruginosa, which cause a dangerous infection of the lung epithelium. In addition, bacterial alginate is resistant to alginases of the intestinal microbiota, in contrast to, for example, alginate of algae. This is due to the fact that bacterial alginate differs from alginates isolated from algae by its higher molecular weight, higher molar content of hyaluronic acid residues in the polymer, and the level of acetylation of hydroxyl groups of mannuron residues in positions 2 and 3. It has been shown that these physicochemical properties bacterial alginates are critically important for the formation of biofilms by the bacteria Pseudomonas sp. Together, these properties of the biopolymer make it possible to create such a mucous membrane around the bacterial cell, which prevents: the cleavage of the biofilm by alginases, the penetration of other bacteria through it, phagocytosis of encapsulated bacteria by macrophages and neutrophils, the effect of cytotoxes on them and antibodies, reduces the effect on bacterial cells of toxins and antibacterial drugs. In addition, alginate hydrogel of biofilm provides protection against drying out, retention and accumulation of nutrients, retention and binding of cations, accumulation of extracellular bacterial virulence factors, utilization of metabolic products of other bacteria, stability of plasmids and efficiency of genetic exchange between bacteria, improved intercellular communication, creation and maintenance of microenvironment [Ghafoor A., Hay ID, Rehm V.N. Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture. Appl. Environ. Microbiol., 2011, 77 (15), 5238-5246]. Moreover, recent studies indicate that other bacteria use alginates synthesized by P. aeruginosa to form their own biofilm [Scoffield JA, Duan D., Zhu F., Wu H. A commensal streptococcus hijacks a Pseudomonas aeruginosa exopolysaccharide to promote biofilm formation. PLoS Pathog., 2017, 13 (4), e1006300]. It was also shown that the formation of biofilms by pathogenic bacteria plays an important role in the healing and chronic inflammation of epithelial damage. Certain representatives of the epithelial microflora, such as Staphylococcus epidermidis, Proteus mirabilis, Pseudomonas aeruginosa, Enterococcus faecalis (durans), Escherichia coli, Fusobacterium sp. Form biofilms, which are a thick mucous barrier that protects microorganisms from external threats, which Percival SL, McCarty SM, Lipsky B. Biofilms and Wounds: An Overview of the Evidence. Adv. Wound Care (New Rochelle) 2015, 4 (7): 373-381].

In recent years, more and more data indicate that the human microbiota plays an important role in the processes of epithelial regeneration, incl. the intestinal mucosa when it is damaged, and this role is not always negative. It has been shown that microbiota activates and maintains innate immunity, is important for the normal functioning of the immune system, and regulates inflammatory processes. It has been shown that changes in the composition of the intestinal microbiota are associated with the occurrence of various pathological conditions, including inflammatory bowel diseases, asthma, type I diabetes mellitus, obesity, cardiovascular diseases and psychiatric abnormalities [Aron-Wisnewsky J., Clement K. The gut microbiome, diet, and links to cardiometabolic and chronic disorders. Nat. Rev. Nephrol., 2016, 12 (3): 169-181]. Moreover, bacteria can not only resist the healing of epithelial tissues, in particular the intestinal mucosa, but also promote it [Uchida M., Shimizu K., Kurakazu K. Yogurt containing Lactobacillus gasseri OLL 2716 (LG21 yogurt) accelerated the healing of acetic acid -induced gastric ulcer in rats. Biosci. Biotechnol. Biochem., 2010, 74 (9), 1891-1894; Lam E.K., Yu L., Wong H.P., Wu W.K., Shin V.Y., Tai E.K., So W.H., Woo P.C., Cho C.H. Probiotic Lactobacillus rhamnosus GG enhances gastric ulcer healing in rats. Eur. J. Pharmacol., 2007,565 (1-3), 171-179]. Alginates isolated from algae were used to encapsulate probiotic bacteria of the intestinal microbiota, these studies have shown the preservation of the viability of bacteria and the positive effect of encapsulating bacteria for the treatment of intestinal diseases when used as probiotics [D'Orazio G., Di Gennaro P., Boccarusso M., Presti I., Bizzaro G., Giardina S., Michelotti A., Labra M., La Ferla B. Microencapsulation of new probiotic formulations for gastrointestinal delivery: in vitro study to assess viability and biological properties. Appl Microbiol Biotechnol. 2015, 99 (22), 9779-9789].

Thus, given the relevance of research on the development of medical materials for the restoration of defects in various tissues and studies of the therapeutic potential of probiotic bacteria for tissue regeneration, especially relevant technical solutions are those associated with the development of a structure based on bacterial polymers, which serves to ensure viability, growth and therapeutic activity of probiotic bacteria for the regeneration of intestinal wall tissues. This method makes it possible to create a medical device for regenerative medicine of a new type, where probiotic bacteria serve as an active active principle, and can be used in biotechnology, tissue engineering, and regenerative medicine.

A composition based on bacterial exopolysaccharides, namely gellan gum, is known from the prior art. This composition, mainly for oral administration, may also contain an agent that improves the condition of the gastrointestinal tract [RU2563137, 20.09.2015]. The invention is claimed to provide methods and compositions for the prevention or treatment of diarrhea in a mammal. The disadvantages of this composition and method can be attributed to the fact that the composition is not shown its ability to improve the viability of probiotic bacteria, and the method does not allow a local targeted therapeutic effect on damaged tissues of the wall of the digestive tract.

A method for producing a controlled release preparation capable of releasing 99% or more of a poorly soluble drug in the upper part of the small intestine is known from the prior art [US6274174B1, 14.08.2001]. It is claimed that the inventors have succeeded in establishing a method for producing a poorly soluble drug on aggregates of spherical microparticles of multivalent metal alginate, in which each of the secondary particles has a specific area of 1 to 280 m ² / g. The disadvantage of this composition is its inability to improve the viability of probiotic bacteria, and the method does not allow for local targeted therapeutic effects on damaged tissues of the wall of the digestive tract and is not intended for encapsulation as a bioactive agent of probiotic microorganisms.

A composition is known from the prior art, which consists of a hydrogel based on a collagen matrix in combination with a poloxamer or alginate and enclosed viral vectors [US6333194B1, 25.12.2001]. As stated, the present invention is directed to the targeted healing of various types of tissues or cells by the principle of direct delivery of the given composition to the site of injury, therefore, such a composition may belong to the field of pharmacology. The disadvantage of such a composition is that some types of viruses that can be delivered in combination, namely, lentiviruses, adenoviruses or herpes virus, can cause insertional mutagenesis due to accidental incorporation of host cells into the genome, they also have cytotoxicity and immunogenicity, which is also undesirable when therapy of living objects.

It is known from the prior art about a biopolymer material in the form of microspheres with high porosity based on poly-3-hydroxybutyrate, obtained by bacterial biosynthesis, possessing biocompatibility and biodegradation properties, possessing the ability to controlled sorption and subsequent prolonged release of a positively charged therapeutic protein when microspheres are introduced into various tissue, which can be used in regenerative medicine for long-term therapy of damage to various tissues. However, the disadvantage of this composition is its inability to improve the viability of probiotic bacteria [RU2692768, 29.12.2017].

It is known from the prior art about a polymer composition in the form of microcapsules from various biomaterials, including polysaccharides, including alginate, containing probiotic bacteria and placed in a gel. The disadvantages of this material are tough methods of encapsulating probiotic bacteria, which reduces their viability; the use of a large number of different materials, which can reduce the biocompatibility of the material; and a fast time of destruction at a temperature of 37 ± 1 ° C [RU2652277, 13.04.2017].

This composition can be selected as the closest analogue in terms of the number of common similar features.

Disclosure of invention

The technical objective (of the invention) is to obtain a bioengineering structure consisting of a porous matrix based on a bacterial poly-3-hydroxyalkanoate filled with a bacterial alginate hydrogel, in which live probiotic bacteria with antagonistic activity are placed, which ensures their growth in situ under aggressive conditions. the environment of the gastrointestinal tract in its various diseases and their replacement of pathogenic microflora in the area of tissue damage. It can be used to treat various diseases of the gastrointestinal tract due to the therapeutic functionality of probiotics.

The technical result is to obtain a hybrid bioengineering structure based on probiotic bacteria and bacterial polymers (poly-3-hydroxyalkanoate and alginate), providing at least 95% survival rate of bacteria, assessed by the standard method for determining the number of CFU in the material [GOST R 56139-2014 "Food Products functional. Methods for the determination and counting of probiotic microorganisms "], as well as an increase in their protection (increase in the survival of bacteria by at least 50%) in an unfavorable environment while maintaining the biocompatibility (absence of cytotoxicity) of the construct in vitro.

The technical result is achieved by a bioengineering structure for probiotic bacteria, which is a layer of porous microspheres in a hydrogel located on a porous substrate, while a porous substrate or a porous substrate with microspheres is made of poly-3-hydroxybutyrate and / or its copolymer with 3-hydroxyvalerate with a molecular weight from 1 × 10 ⁴ to 2 × 10 ⁶ g / mol and is characterized by the presence of through pores (or connected pores), a total porosity of 75 to 99%, an average pore diameter of 0.5 to 20 μm, where the pores are filled with a hydrogel containing probiotic bacteria, while the hydrogel is made of bacterial alginate with a molecular weight of 10 ⁵ to 10 ⁶ g / mol, a molar content of guluronic acid residues in the polymer from 31 mol% to 48 mol%, and a level of acetylation from 10 mol% to 46 mol%. In this case, the substrate is made with a thickness of 10 μm to 5 cm, and the diameter of the microspheres is from 2 to 2000 μm, the microspheres are arranged in several rows - from one to five. It is also possible to implement the design in the form of a tube obtained by rolling the substrate. Bacterial alginate is a biosynthetic product of bacteria of the genus Azotobacter. Lactobacillus and / or Bifidobacterium bacteria, or their genetic modifications, were used as probiotic bacteria, in a ratio of CFU from 1: 999 to 999: 1. Additionally, the bioengineering structure may contain at least one pharmacologically active substance having anti-inflammatory action and stimulating the regeneration of intestinal wall tissues, for example, simvastatin, curcumin, rosveratrol, while the pharmacologically active substance is directly mixed with poly-3-hydroxyalkanoate at a concentration from 0.01% to 50% (weight) of a drug substance in the obtained polymer product.

Also, the technical result is achieved by the method of obtaining the claimed bioengineering structure, which consists in the fact that it includes the following stages:

- microbiological biosynthesis of bacterial alginate using a producer strain of the genus Azotobacter,

- obtaining a porous support from poly-3-hydroxybutyrate and / or its copolymer with 3-hydroxyvalerate,

- growing probiotic bacteria of the genera Lactobacillus and Bifidobacterium in bacterial alginate,

- filling the porous substrate with alginate with bacteria,

- gelation of bacterial alginate in situ by adding an aqueous solution of calcium chloride.

In this case, obtaining a porous product from poly-3-hydroxyalkanoate is obtained by one of the following methods: lyophilization, drying, leaching, emulsification, electrospinning, rapid prototyping, laser cutting. Before filling the porous matrix with hydrogel, live probiotic bacteria were grown in the bacterial alginate, which forms its basis, to a content not less than 1 × 10 ⁷ CFU / ml. Additionally, at the stage of obtaining a porous matrix of poly-3-hydroxyalkanoate, at least one pharmacologically active substance can be introduced into it.

Biodegradable and biocompatible polymers were selected as a polymer material for the manufacture of a porous framework for a bioengineered structure: poly-3-hydroxybutyrate (PHB) and a copolymer of poly-3-hydroxybutyrate with 3-hydroxyvalerate (PHBV) of various molecular weights (from 1 × 10 ³ to 2 × 10 ⁶ ), and in the case of the copolymer - with different contents of 3-hydroxyvalerate monomers (from 0 to 30%). An efficient method has been developed for the controlled microbiological production of biocompatible and biodegradable polymers belonging to the class of poly-3-hydroxyalkanoates. The developed method of biosynthesis of PHB and its copolymers makes it possible to obtain a polymer with a given narrow molecular weight distribution and with a given monomer composition. For this, in the course of biosynthesis, the biopolymer-producing strains of the species Azotobacter chroococcum and Azotobacter vinelandii (for example, A. chroococcum 7B) are grown on a nutrient medium containing sucrose as a carbon source, mineral salts and an additional carbon source (a precursor of monomers in the PHB copolymer or a regulator molecular weight): sodium acetate, salts of carboxylic acids (propionic, valerian) at a certain concentration, as well as at different pH of the medium and at different levels of aeration [RU2194759, 20.12.2002; RU2201453, 27.03.2003; RU2307159, 27.09.2007; Bonartsev A.R., Zharkova II, Yakovlev SG, Myshkina VL, Mahina T.K., Voinova VV, Zemov AL, Zhuikov VA, Akoulina EA, Ivanova EV, Kuznetsova ES, Shaitan KV, Bonartseva GA Biosynthesis of poly (3- hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: a precursor feeding strategy. Preparative Biochemistry and Biotechnology, 2017, 47 (2), 173-184]. The use of these biopolymers for the development of new medicinal products seems promising, due to their unique properties: high biocompatibility with both tissues and organs and bacteria of the human microbiota, the ability to complete biodegradation at a controlled rate while at the same time relative resistance to the aggressive environment of the gastrointestinal tract, the ability stimulate the regeneration of various tissues and prevent the development of pathogenic microflora [Bonartsev A. P., Voinova V. V., Bonartseva G. A. Poly-3-hydroxybutyrate and human microbiota. Applied Biochemistry and Microbiology, 2018, 54 (6), 561-584].

Polymeric material of a hydrogel serving as a substrate for ensuring the viability and growth of probiotic bacteria and intended to fill a porous substrate and / or microspheres - bacterial alginate is obtained by the method of controlled microbiological biosynthesis also using the producer strain of this biopolymer Azotobacter chroococcum 7B [RU2.2002759; 20.12 Bonartseva G.A., Akulina E.A., Myshkina V.L., Voinova V.V., Makhina T.K., Bonartsev A.P. Biosynthesis of alginates by bacteria of the genus Azotobacter. Applied Biochemistry and Microbiology, 2017, 53 (1), 61-68]. To maintain the culture of bacteria, use Ashby's solid nutrient medium of the following composition (g / l): K ₂ HPO ₄ - 1.05; KH ₂ PO ₄ 0.2; MgSO ₄ 0.2; NaCl 0.2; Na ₂ MoO ₄ - 0.006; CaCO ₃ - 5.0; sucrose - 20, agar - 20. For the biosynthesis of alginate, the producer strain A. chroococcum 7B is cultivated in Burk's liquid medium of the following composition (g / l): KH ₂ PO ₄ - from 0.05 to 1.00; K ₂ HPO ₄ - from 0.05 to 1.00; MgSO ₄ 0.4; NaCl - 0.1; FeSO ₄ - 0.01; Na ₂ MoO ₄ - 0.06; CaCl ₂ - 0.1; sodium citrate - 0.5; sucrose - from 20 to 40. First, the inoculum is prepared, for which the producer strain A. chroococcum 7B is grown in Burk's liquid medium of the specified composition in 750 ml shaking flasks (from 100 ml of medium) at 250 rpm (initial pH of the medium - 7.2; cultivation temperature - 28 ° C) for 24 hours. Then biosynthesis of alginate is carried out by the producer strain A. chroococcum 7B, for which it is cultivated in the same medium after adding 4% (vol.) Of inoculum aged 24 hours in shaking flasks of 750 ml (volume of culture medium - 100 ml) on a microbiological shaker Innova 43 (New Brunswick Scientific, USA) at a stirring speed of 200 to 400 rpm for 72 hours. All procedures are performed under sterile conditions.

To isolate alginate, the biomass of the producer strain is treated with 0.9% NaCl and 50 mM EDTA, after which centrifugation is carried out at 15000 g for 30 minutes. The precipitate is separated and washed with water. Then a 3-fold volume of 96% ethyl alcohol is added to it. The precipitate is again separated and washed with water. After that, the resulting alginate is purified. For this, the alginate is again precipitated with a 3-fold volume of 96% ethanol and dialysis is performed against 0.5% NaCl for 24 h. The precipitate is again separated and dried using a freeze dryer. The resulting powder is stored at 4 ° C for no more than 3 months.

The developed method for the biosynthesis of alginate makes it possible to obtain a polymer with the following combination of parameters: molecular weight (determined by viscosimetry) from 10 ⁵ to 10 ⁶ g / mol, molar content of guluronic acid residues in the polymer from 31 mol% to 48 mol%, and acetylation level - from 10 mol% to 46 mol% (determined by IR spectroscopy). Such parameters of bacterial alginate are close to the properties of bacterial alginate, which forms a bacterial biofilm [Boyd A., Chakrabarty AM Pseudomonas aeruginosa biofilms: role of the alginate exopolysaccharide. J. Ind. Microbiol. 1995,15 (3) 162-168; Nivens DE, Ohman DE, Williams J., Franklin MJ Role of alginate and its O acetylation in formation of Pseudomonas aeruginosa microcolonies and biofilms. J. Bacteriol. 2001, 183 (3), 1047-1057; Tielen P., Strathmann M., Jaeger KE, Flemming HC, Wingender J. Alginate acetylation influences initial surface colonization by mucoid Pseudomonas aeruginosa. Microbiological Research 2005,160 (2) 165-176; Ghafoor A., Hay ID, Rehm BH Role of exopolysaccharides in Pseudomonas aeruginosa biofilm formation and architecture. Appl. Environ. Microbiol., 2011, 77 (15), 5238-5246].

After obtaining the bacterial alginate, various versions of the bioengineering structure are made in several stages.

At the 1st stage, porous polymer microspheres are made. For this, a solution of PHB or PHBV is prepared in chloroform or methylene chloride at a polymer concentration of 50 to 200 mg / ml (wt). Prepare a 5 ± 1% (mass.) Suspension of ammonium carbonate in water and homogenize it on a rotary homogenizer for 5 ± 1 minutes at 15000 ± 2000 rpm until a stable suspension is reached, after which it is transferred completely into a glass beaker with a PHB or PHBV solution in chloroform or methylene chloride. Then homogenized for another 20 ± 5 minutes at 15000 ± 2000 rpm until a stable emulsion is obtained. Then a 1 ± 0.5% (mass) solution of polyvinyl alcohol (PVA) in distilled water is prepared in a glass beaker, using an overhead stirrer with a propeller-type stirring element, the solution is stirred at a speed of 250 to 1000 rpm (which determines the diameter and the porosity of the formed microspheres) and with this constant stirring, the resulting complex colloidal mixture is added dropwise to it. Stirring is carried out until the chloroform has completely evaporated, approximately more than 3 ± 0.5 hours. Then move the beaker onto a magnetic stirrer with heating and stir for another 35 ± 5 min at a temperature of 35 ± 5 ° C for complete decomposition of the blowing agent and release of gases, which is expressed in the form of gas bubbles, as well as for additional distillation of chloroform; cessation of gas evolution can be tested using wet standard indicator paper (Khimmed, RF) for the absence of an alkaline reaction, which is produced by the formed ammonia. Then the liquid containing the formed polymer microspheres is centrifuged in a tabletop centrifuge at 500 ± 100 rpm to separate the microspheres from the PVA emulsifier solution, the supernatant is taken as much as possible. The resulting microspheres are washed from the emulsifier and other impurities 3-4 times with distilled water, the quality of washing is checked using wet standard indicator paper (Khimmed, RF) for the absence of an alkaline reaction. The washed microspheres are frozen in liquid nitrogen and lyophilized using a freeze dryer. The resulting microspheres are sieved through U1-ESL laboratory sieves with a mesh fabric in accordance with GOST 6613-86 with a mesh size of 40, 94 μm, 315 or 900 μm for additional more accurate separation of microspheres by their diameter.

Porous substrates are made using a modified leaching method [Bonartsev A.R. et al. Adhesion and growth of bone marrow mesenchymal stem cells on 3D scaffolds from poly (3-hydroxybutyrate) -poly (ethylene glycol) copolymer. Journal of Biomaterials and Tissue Engineering, 2016, 6 (1), 42-52]. This modification is based on thermal decomposition of the solid salt, while the standard method involves washing out the salt with a solvent. A PHBV solution with a molar content of 3-hydroxyvalerate from 6 to 30 mol% and a molecular weight from 1.5 × 10 ⁵ to 1.5 × 10 ⁶ g / mol in chloroform or methylene chloride at a concentration of 15 to 30 mg / ml was mixed with ammonium bicarbonate powder with linear the size of salt particles in the range of 90-300 microns in a ratio from 20: 1 to 5: 1 by weight and thoroughly mixed on a vortex for 3 ± 1 min. The mixture is placed in a flat-bottomed glass vessel (for example, a Petri dish) with a diameter of 40 to 90 mm and left at room temperature until the organic solvent has completely evaporated from 2 to 3 hours. After that, the mold is placed in heated distilled water (70 ± 5 ° C) until gassing has completely ceased, washed with distilled water 4-6 times to get rid of impurities, the quality of washing is checked using wet standard indicator paper (Khimmed, RF) for the absence of an alkaline reaction ... Then it is dried at 37 ± 1 ° C for 24-30 hours until the obtained porous polymer film of constant weight is reached.

Fischer A., Plickert R., Haag L.M., Otto В.,

Loddenkemper С.,

Heimesaat M.M. Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation. PLoS One, 2010, 5(12): e15099]. Для этого готовят раствор ЛВ в хлороформе или метиленхлориде в концентрации от 0,01 до 100 мг/мл и на стадии приготовления раствора ПОБВ в хлороформе добавляют в раствор полимера раствор ЛВ в объеме от 1 мкл до 100 мл для достижения массовой доле ЛВ в полученном полимерном изделии от 0,01% до 50% (вес.). Терапевтические свойства симвастатина для желудочно-кишечного тракта описаны в [Cote-Daigneault J., Mehandru S., Ungaro R., Atreja A., Colombel J.F. Potential Immunomodulatory Effects of Statins in Inflammatory Bowel Disease Inflammatory Bowel Diseases, 2016, 22(3), 724-732].In one embodiment of the invention, a medicinal substance (MD) is introduced into the porous support, which has an anti-inflammatory effect and stimulates the regeneration of tissues of the intestinal wall, for example, such as simvastatin, curcumin, rosveratrol [Bereswill S.,

Fischer A., Plickert R., Haag LM, Otto B.,

Loddenkemper S.,

Heimesaat MM Anti-inflammatory effects of resveratrol, curcumin and simvastatin in acute small intestinal inflammation. PLoS One, 2010, 5 (12): e15099]. For this, a drug solution is prepared in chloroform or methylene chloride at a concentration of 0.01 to 100 mg / ml, and at the stage of preparing a PHBV solution in chloroform, a drug solution is added to the polymer solution in a volume of 1 μl to 100 ml to achieve the drug mass fraction in the resulting polymer product from 0.01% to 50% (wt.). The therapeutic properties of simvastatin for the gastrointestinal tract are described in [Cote-Daigneault J., Mehandru S., Ungaro R., Atreja A., Colombel JF Potential Immunomodulatory Effects of Statins in Inflammatory Bowel Disease Inflammatory Bowel Diseases, 2016, 22 (3) , 724-732].

Strips from 1 to 400 mm long and 1 to 400 mm wide are cut from the obtained porous film. The resulting strips are dipped in chloroform or methylene chloride by full immersion in a solvent 3-4 times, then on the surface of the film, without waiting for drying, the previously obtained microspheres of various diameters from 10 to 1000 microns are poured in a continuous layer, evenly distributing them with a spatula and left to dry for 24-30 hours. In one embodiment of the invention, the resulting strip with microspheres deposited on it is rolled up with a slight overlap (from 5 to 10% of the length) into a tube with a diameter of 0.2 mm to 120 mm and a length of 1 to 400 mm. The edges of the film are glued using a solution of 2-3% PHB in chloroform, which is applied with a pipette to the edge of the film from the outside, after which the film is folded so that its other edge can be applied to it from the inside, gluing these edges and thus making a tube. The finished products are washed with distilled water 4-6 times to get rid of impurities, the quality of washing is checked using wet standard indicator paper (Khimmed, RF) for the absence of an alkaline reaction. Then it is dried at 37 ± 1 ° C for 24-30 hours until the obtained porous polymer film of constant weight is reached. Finished products can be cut into pieces of the required size.

Thus, a bioengineering structure is made of two types of bacterial polymers - poly-3-hydroxyalkanoate and alginate, obtained biotechnologically, so that a product made of one polymer plays a skeletal "rigid" role, and the second polymer would be a filler and a carrier in which would load probiotic bacteria or some other cells or substances as needed.

The surface and internal structure of microspheres is a complex system of branched through pores with a linear size of more than 4 microns, which is larger than the average size of bacteria. The creation of a layer of such porous microspheres can solve the problem of not only obtaining a certain framework that can be filled with an alginate hydrogel containing probiotic bacteria, but also allows to significantly increase the surface area and roughness of the structure, which is a very important characteristic of products for the regeneration of various tissues and facilitates penetration into such the design of nutrients and bioactive molecules, adhesion of cells to the product, its fusion with the surrounding living tissues, gradual biodegradation of the product with its replacement with regenerating tissue.

In the next step, probiotic bacteria are grown in microbiologically obtained alginate. First, cultures of bacteria of the genera Lactobacillus and Bifidobacterium are grown on a semi-liquid medium that ensures the growth of these bacteria. The culture medium is poured into the flasks, sealed with cotton-gauze stoppers and autoclaved. When cultivating bacteria of the genus Lactobacillus, the prepared medium is not stored. When cultivating bacteria of the genus Bifidobacterium, the prepared medium is stored for no more than 2 weeks in a household refrigerator at a temperature of 5-8 ° C. Sowing is carried out as soon as the temperature of the medium after autoclaving reaches 40 ° C. To do this, using a pipette, add 1.0 ± 0.1 ml of bacterial cell suspension to a semi-liquid nutrient medium and mix [Schillinger U., Holzapfel W.H. Chapter 8. Culture media for lactic acid bacteria, p. 127-140. In: Handbook of Culture Media for Food Microbiology. Edited by Janet E.L. Corry, G.D.W. Curtis, Rosamund M. Baird. V. 37, 2003].

In parallel, a 1 ± 0.1% (wt.) Aqueous solution of sodium alginate with a molecular weight of 1.0 × 10 ⁵ to 2 × 10 ⁶ g / mol, obtained microbiologically, is prepared in 150 ml of MRS-broth on a magnetic stirrer with heating at 25 ± 5 ° С and stirring speed 40-80 rpm.

For sowing a culture of bacteria of the genus Lactobacillus, prepare a liquid seed culture. For this, 3 ± 0.2 ml of MRS broth is added to a test tube with a culture growing on a slant MRS-agar. Cells are removed from the surface of the solid medium by repeated pipetting and the resulting suspension is transferred into a flask containing 150 ± 10 ml of a culture medium based on bacterial alginate in MRS broth. A flask with a liquid seed culture is incubated for 96 ± 6 hours under aerobic conditions on a microbiological shaker at 37 ± 1 ° C and a stirring speed of 120 ± 20 rpm (orbital diameter 1.9 cm).

To inoculate a culture of bacteria of the genus Bifidobacterium, first, subculture is carried out from a medium with agar-agar to a medium with alginate. For this purpose, 8.0 ± 0.2 ml is taken from a flask with a 96 ± 6 hour culture growing in a semi-liquid medium on agar-agar with a glass serological pipette and introduced into a flask with a freshly prepared semi-liquid medium based on bacterial alginate in MRS broth. The contents of the flask are mixed. To obtain a culture of bacteria of the genus Bifidobacterium in a semi-liquid medium, 4.0 ± 0.2 ml of a working culture is added to the bottom of the flask. The contents of the flask are not mixed. After inoculation, the flasks are sealed with cotton-gauze stoppers and placed in a microbiological shaker for thermostating. Incubate for 96 ± 6 hours at 37 ± 1 ° C without stirring.

In one embodiment of the proposed method, genetically modified strains of bacteria of the genera Lactobacillus and Bifidobacterium are also used. These strains can be obtained from natural strains of probiotic bacteria by conventional methods of genetic transformation using electrocorporation, described, for example, for bacteria from the genus Lactobacillus [Serror P., Sasaki T., Ehrlich SD, Maguin E. Electrotransformation of Lactobacillus delbrueckii subsp.bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids. Appl Environ Microbiol., 2002, 68 (1), 46-52] and for bacteria from the genus Bifidobacterium [Argnani A., Leer R.J., van Luijk N., Pouwels P.H. A convenient and reproducible method to genetically transform bacteria of the genus Bifidobacterium. Microbiology 1996, 142, 109-114].

Finally, at the last stage, the resulting structures are “impregnated” with a solution of the previously obtained bacterial capsular sodium alginate with the biomass of probiotic bacteria or the production of spheres from alginate with bacteria in the simplest version. For this procedure, use a 1 ± 0.1 wt% aqueous solution of sodium alginate containing at least 10 ⁷ CFU / g of bacteria of the genera Lactobacillus and Bifidobacterium grown in it. The structure is placed in this solution, and the container with it in a desiccator, where a negative pressure (0.7-0.8 atm.) Is created for 10 minutes to displace air from the pores of the microspheres and the substrate and replace it with an alginate solution and bacteria contained in it, at a temperature of 25 ± 3 ° C. Externally, the process of filling the pores of microspheres under these conditions looks like the formation of many microspheres of air around the microspheres, while the alginate solution fills the pores of the microspheres and the substrate. Then, the alginate is gelled, for which the structure impregnated with a sodium alginate solution is placed in a glass beaker with 100 ml of 50 ± 1 mM aqueous solution of calcium chloride for 10 ± 1 min.

In the simplest embodiment of the proposed method, granules are made from the previously obtained bacterial capsular sodium alginate with the biomass of probiotic bacteria grown therein. For this, an automatic pipette into a glass beaker with a 50 ± 1 mM aqueous solution of calcium chloride is added dropwise (10-30 μl) a solution of alginate containing bacteria, obtained by the previously described method. As a result, alginate granules with a diameter of 0.2 to 1 mm are formed.

In one of the embodiments of the proposed method, in addition to bacteria, biologically active substances are introduced into the alginate that have anti-inflammatory effects and stimulate the regeneration of tissues of the intestinal wall, for example, such as interleukin 10. For this, a solution of the bioactive substance in water is prepared at a concentration of 0.01 μg / l to 100 mg / ml, and at the stage of completion of the cultivation of bacteria in bacterial alginate, a drug solution in a volume of 1 μl to 100 ml is added to a solution of a polymer containing bacteria in a volume of 1 μl to 100 ml to achieve the mass fraction of drug in the obtained polymer product from 0.00001% to 50% (weight ). The therapeutic properties of various biologically active substances, incl. interleukin 10 for the gastrointestinal tract is described in [Takedatsu H., Mitsuyama K., Torimura T. Nanomedicine and drug delivery strategies for treatment of inflammatory bowel disease. World J. Gastroenterol. 2015, 21 (40), 11343-11352, doi: 10.3748 / wjg.v21.i40.11343].

The effectiveness of the obtained bioengineering constructs was assessed using model tests: resistance test at 37 ° C, resistance test in a simulated intestine environment, enzymatic resistance tests, resistance test in an alien bacteria environment, bacteria migration test from hydrogel, and in vitro cytotoxicity test. An encapsulated form of bacteria in algal alginate, made by dispersing the corresponding probiotic bacteria of the genera Lactobacillus and Bifidobacterium in a 2% (vol.) Solution of algal alginate, was used as a reference drug, followed by obtaining granules of alginate hydrogel with encapsulated bacteria in them using a calcium chloride solution that the content of bacteria in the obtained granules was the same as in the tested constructs.

Structural stability test at 37 ± 1 ° C: the resulting structures containing bacteria are placed on a glass surface of a Petri dish and placed in a thermostat maintained at 37 ± 1 ° C. It is kept for 10 ± 1 min, after which it is removed and the change in the shape and state of aggregation of the hydrogel is observed, recording the time of complete deformation from the moment it is placed on a glass surface.

Stability test in simulated intestine environment: the obtained bioengineering constructs in various forms (granules, microspheres, a support with a layer of microspheres in the form of a plate or tube) are placed in a glass flask with 100 ml of simulated intestinal environment of the following composition: 50 mM KH ₂ PO ₄ , 10 mg / ml of bile acids, pH = 7.5; incubation is carried out for 150 minutes. at 37 ° C. The resulting bioengineering structures should provide at least 50% increase in the survival rate of bacteria or at least 95% of their absolute survival rate, assessed by the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "].

Enzymatic resistance test using alginate lyase in a simulated intestinal environment: the resulting bioengineered constructs in various forms (granules, microspheres, a support with a layer of microspheres in the form of a plate or tube) are placed in a glass flask with 100 ml of simulated intestinal medium containing alginate lyase, of the following composition : 50 mM KH ₂ PO ₄ , 10 mg / ml bile acids, 1 mg / ml alginate lyase (Sigma-Aldrich, ≥10000 U / g), pH = 7.5; incubation is carried out for 150 minutes. at 37 ° C. The obtained bioengineering structures are authorized to increase the survival rate of bacteria by at least 50%, assessed by the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "].

Test of enzymatic resistance using lysozyme in a simulated intestinal environment: the obtained bioengineered constructs in various forms (granules, microspheres, a support with a layer of microspheres in the form of a plate or tube) are placed in a glass flask with 100 ml of simulated intestinal medium containing lysozyme, of the following composition: 50 mM KH ₂ PO ₄ , 10 mg / ml bile acids, 1 mg / ml lysozyme (Sigma-Aldrich, ≥40,000 units / mg), pH = 7.5; incubation is carried out for 150 minutes. at 37 ° C. The obtained bioengineering structures provide at least 50% increase in the survival rate of bacteria, assessed by the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "].

Test of resistance in the environment of foreign bacteria: the obtained bioengineering constructs in various forms (granules, microspheres, a substrate with a layer of microspheres in the form of a plate or a tube) under sterile conditions are placed in a 250 ml conical flask on a semi-liquid medium consisting of 200 ml of MRS broth and 0.3% agar-agar in the thickness of the medium, into which the E. coli BL21 culture (GE Healthcare, USA) was previously introduced, the contents of the flask are not stirred, after inoculation the flasks are sealed with cotton-gauze plugs and placed in a microbiological shaker for thermostating, after which incubated for 24 ± 4 hours at 37 ± 1 ° C without stirring. The obtained bioengineering constructs provide at least 50% increase in the resistance of cultures of bacteria of the genera Lactobacillus or Bifidobacterium to the penetration of alien bacteria E. coli BL21 into the structure or the presence in the structure of no more than 5% of foreign bacteria E. coli BL21 of the total number of bacteria, assessed by the standard method determination of the number of CFU in the design [GOST R 56139-2014 "Food functional products. Methods for the determination and counting of probiotic microorganisms "].

Bacteria migration test from a bioengineered construct: the resulting bioengineered constructs containing probiotic bacteria are sterilely placed in a 250 ml conical flask on a semi-liquid medium consisting of 200 ml MRS broth and 0.3% agar-agar into the medium, the contents of the flask do not mix, after inoculation, the flasks are sealed with cotton-gauze plugs and for thermostating placed in a microbiological shaker, after which they are incubated for 24 ± 4 hours at 37 ± 1 ° C without stirring. The resulting bioengineering structure should ensure the spread of bacteria of the genera Lactobacillus or Bifidobacterium in a semi-liquid medium, in which the structure is placed, at a distance of at least 10 mm from the hydrogel sample, assessed according to the standard method for determining the number of CFU in the medium [GOST R 56139-2014 "Functional food products ... Methods for the determination and counting of probiotic microorganisms "].

The in vitro cytotoxicity test was performed in accordance with GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effect of medical devices. Part 5. Testing for cytotoxicity: in vitro methods ”. As a cell culture, Balb / 3T3 mouse fibroblasts and direct contact assay were used. A qualitative assessment is carried out by points. The resulting bioengineering construct should not be cytotoxic (it should have a cytotoxicity score of no more than "0").

Manufacturing of a complex polymer structure, consisting of a porous framework filled with a hydrogel that contains probiotic bacteria, provides biological activity, viability and growth of probiotic bacteria acting as an active therapeutic agent. Such a structure of the bioengineering structure significantly increases the surface area and internal volume of the product both to ensure contact of probiotic bacteria with damaged tissues of the intestinal wall, and to protect them from pathogenic microflora in the treatment of certain chronic diseases of the gastrointestinal tract, such as duodenal ulcer, nonspecific ulcerative colitis, ischemic colitis, Crohn's disease, as well as bowel surgery with local tissue removal and their complications.

Brief Description of Drawings

The proposed invention is characterized by the following graphic materials.

FIG. 1 shows a diagram of the developed bioengineering structure. The structure consists of a porous polymer substrate (3), on the surface of which porous microspheres (1) are immobilized, which are filled with an alginate hydrogel (2) containing probiotic bacteria (4).

FIG. 2 shows the surface morphology of a bioengineered structure, wide-field light microscopy, × 400.

Implementation of the invention

Biodegradable and biocompatible polymers used to obtain a polymeric porous substrate and microspheres: PHB and PHBV were obtained by the method of controlled biosynthesis using the highly efficient producer strain Azotobacter chroococcum 7B [RU2194759, 20.12.2002; RU2201453, 27.03.2003; RU2307159, 27.09.2007; Bonartsev A.R., Zharkova I.I., Yakovlev S.G., Myshkina V.L., Mahina T.K., Voinova V.V., Zernov A.L., Zhuikov V.A., Akoulina E.A., Ivanova E.V., Kuznetsova E.S., Shaitan K.V., Bonartseva G.A. Biosynthesis of poly (3-hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: a precursor feeding strategy. Preparative Biochemistry and Biotechnology, 2017, 47 (2), 173-184]. For the biosynthesis of bacterial alginate also used the producer strain Azotobacter chroococcum 7B [Bonartseva GA, Akulina EA, Myshkina VL, Voinova VV, Makhina TK, Bonartsev A.P. Biosynthesis of alginates by bacteria of the genus Azotobacter. Applied Biochemistry and Microbiology, 2017, 53 (1), 61-68]. The optical density of the culture medium was monitored nephelometrically at 520 nm. The morphology of bacteria of the genus Azotobacter was monitored by light microscopy using a Biomed-1 microscope (Biomed, Russia) with a digital camera. Fuchsin staining was used to visualize cells and alginate. The purification efficiency of bacterial alginate was monitored by measuring the conductivity of a 1% (w / v) alginate solution using an S30 SevenEasyTM conductometer (Mettler Toledo, USA). The molecular weight of bacterial alginate is determined by viscometry; the chemical structure and the level of acetylation are determined by IR spectroscopy according to [GA Bonartseva, EA Akulina, VL Myshkin, VV Voinova, TK Makhina, AP Bonartsev. Biosynthesis of alginates by bacteria of the genus Azotobacter. Applied Biochemistry and Microbiology, 2017, 53 (1), 61-68].

All other used reagents and strains of probiotic bacteria of the genera Lactobacillus and Bifidobacterium are commercially available (OOO Sputnik-K), all procedures, unless otherwise stated, are carried out at room temperature or ambient temperature, that is, in the range from 18 to 25 ° С, and cultivation of probiotic bacteria - at a temperature of 37 ± 1 ° С. The microstructure of the bioengineered structure (total porosity, pore size, diameter of the obtained microspheres) was monitored by scanning electron microscopy using a JSM-6380LA electron microscope (Jeol, Japan) with preliminary gold deposition on the samples under study for 15 min using an IB-3 ( Giko, Japan) at a current of 15 mA and Image J image processing software. The total porosity was calculated as the ratio of the total volume of connected pores of the structure to its total volume. The growth of cultures is controlled by nephelometry, determining the optical density at a wavelength of 600 nm on a spectrophotometer (Ultrospec 1100 pro, Amersham Biosciences Limited, USA), morphological homogeneity is checked using light microscopy (microscope Biomed-1, "Biomed", RF, with a digital camera ). Determination of the number of bacteria in the obtained bioengineering structure is carried out according to the standard method of seeding bacteria on a dense agar nutrient medium, followed by counting the grown colonies; the results are expressed in colony-forming units (CFU) per ml (CFU / ml) according to [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "]. The terms “biologically active”, “bioengineered”, “probiotic bacteria”, “probiotics”, “cultivation”, “biocompatible”, “biodegradable”, “therapeutic” are used in accordance with their generally accepted definitions [Biocompatible materials: Study guide / Under ... ed. IN AND. Sevostyanova, M.P. Kirpichnikov. Moscow. Medical News Agency, 2011, 540 pp .; Probiotics and Prebiotics / World Gastroenterology Organization (WGO). Practical advice. 2008. http://www.gastroscan.ru/literature/authors/5634].

The present invention is illustrated by specific examples of execution, which are not the only possible, but clearly demonstrates the possibility of achieving the required technical result.

Example 1.

The manufacture of a bioengineered structure is carried out in several sequential stages.

At the first stage, bacterial alginate was also obtained by the method of controlled microbiological biosynthesis. To maintain the culture of bacteria used a solid nutrient medium Ashby, the following composition (g / l): K ₂ HPO ₄ - 1.05; KH ₂ PO ₄ 0.2; MgSO ₄ 0.2; NaCl 0.2; Na ₂ MoO ₄ - 0.006; CaCO ₃ - 5.0; sucrose - 20, agar - 20. For the biosynthesis of alginate, the producer strain A. chroococcum 7B [RF Patent No. 2194759, 20.12.2002; RF patent No. 2201453, 27.03.2003; RF patent No. 2307159, September 27, 2007; Bonartsev A.R., Zharkova II, Yakovlev SG, Myshkina VL, Mahina T.K., Voinova VV, Zernov AL, Zhuikov VA, Akoulina EA, Ivanova EV, Kuznetsova ES, Shaitan KV, Bonartseva GA Biosynthesis of poly (3- hydroxybutyrate) copolymers by Azotobacter chroococcum 7B: a precursor feeding strategy. Preparative Biochemistry and Biotechnology, 2017, 47 (2), 173-184] were cultured in Burk's liquid medium of the following composition (g / l): KH ₂ PO ₄ - 0.05; K ₂ HPO ₄ - 0.05; MgSO ₄ 0.4; NaCl - 0.1; FeSO ₄ - 0.01; Na ₂ MoO ₄ - 0.06; CaCl ₂ - 0.1; sodium citrate - 0.5; sucrose - 40. First, the inoculum was prepared, for which the producer strain A. chroococcum 7B was grown in Burk's liquid medium of the specified composition in 750 ml shaking flasks (with 100 ml of medium) at 250 rpm (initial pH of the medium - 7, 2; cultivation temperature - 28 ° C) for 24 hours. Then, biosynthesis of alginate was carried out by the producer strain A. chroococcum 7B, for which it was cultured in the same medium after adding 4% (vol.) Inoculum aged 24 hours in shaking flasks of 750 ml (the volume of the culture medium is 100 ml) on a microbiological shaker Innova 43 (New Brunswick Scientific, USA) at a stirring speed of 400 rpm for 72 hours. All procedures were performed under sterile conditions. To isolate alginate, the biomass of the producer strain was treated with 0.9% NaCl and 50 mM EDTA, after which centrifugation was performed at 15000 g for 30 min. The precipitate was separated and washed with water. Then a 3-fold volume of 96% ethyl alcohol was added thereto. The precipitate was again separated and washed with water. After that, the resulting alginate was purified. For this, the alginate was again precipitated with a 3-fold volume of 96% ethanol and dialysis was performed against 0.5% NaCl for 24 h. The precipitate was again separated and dried by freeze-drying. The resulting powder is stored at 4 ° C for no more than 3 months. The resulting alginate had the following combination of parameters: molecular weight (determined by viscosimetry) 1 × 10 ⁵ g / mol, molar content of guluronic acid residues in the polymer - 36%, and acetylation level - 23% (determined by IR spectroscopy).

At the 2nd stage, the probiotic bacteria Lactobacillus plantarum 8P-A3 (OOO Sputnik-K) were grown. On a semi-liquid medium, the culture of L. plantarum 8P-A3 was grown in 250 ml conical flasks containing 200 ml of MRS broth and 0.3% agar-agar. The culture medium was poured into the flasks, sealed with cotton-gauze stoppers and autoclaved. The finished medium is not stored. The inoculation was carried out as soon as the temperature of the medium after autoclaving reached 40 ° C.

A liquid seed culture is prepared for sowing. For this, 3 ml of MRS broth was added to a test tube with a culture growing on a slant MRS-arape. The cells were removed from the surface of the solid medium by repeated pipetting and the resulting suspension was transferred into a flask containing 150 ml of culture medium. A flask with a liquid seed culture was incubated for 96 hours under aerobic conditions on a microbiological shaker at 37 ° C and a stirring speed of 120 rpm (orbital diameter 1.9 cm). The growth of the culture was monitored by nephelometry, determining the optical density at a wavelength of 600 nm on a spectrophotometer (Ultrospec 1100 pro, Amersham Biosciences Limited, trademark of Microsoft Corporation, USA.), Morphological homogeneity was checked using light microscopy (microscope Biomed-1, "Biomed" , RF, with a digital camera).

In a 250 ml conical flask, a 1% (wt.) Solution of microbiologically obtained alginate was prepared in 150 ml of MRS broth on a magnetic stirrer with heating at 60 ° C and a stirring speed of 60 rpm. To obtain L. plantarum biomass on a liquid medium under aerobic conditions, 6 ml of a liquid seed culture (5 × 10 ⁸ CFU / g) was introduced into a conical flask containing the obtained 1% alginate solution in 150 ml of MRS broth, and incubated for 96 hours in a microbiological rocking chair at 37 ° C and stirring speed 120 rpm (orbital diameter 1.9 cm). After the end of the incubation, the biomass was precipitated by centrifugation for 15 min at 10000g on a Beckman J2-21 centrifuge. The supernatant was discarded. All procedures were performed under sterile conditions.

Granules were prepared from the previously obtained bacterial capsular sodium alginate with the biomass of probiotic bacteria grown in it. For this, a solution of alginate containing bacteria was added dropwise (10-30 μl) into a glass beaker with 100 ml of 50 mM calcium chloride solution with an automatic pipette of 100 μl.

The resulting granules had a diameter of 0.2 to 1 mm and contained probiotic bacteria at a concentration of 2.1 × 10 ⁸ CFU / g of L. plantarum 8P-A3 bacteria. Thus, there was an increase in the content of living bacteria in the granules against the initial (1.9 × 10 ⁷ CFU / g) 11.1 times compared to the drop in the content of bacteria in the composition described in the prototype patent, 11 times from 1 × 10 ⁸ CFU / g to 8.8 × 10 ⁶ CFU / g.

The stability test of the structure at a temperature of 37 ° C showed no deformation. In the resistance test in a simulated intestinal environment, the resulting construct ensured the retention of 2.1 × 10 ⁸ CFU / g of L. plantarum 8P-A3 bacteria, i.e. 98% bacterial survival, assessed by the standard method for determining the CFU number in comparison with the control bacterial preparation. encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using alginate lyase in a simulated intestinal environment, the resulting construct provided a 51% increase in bacterial survival, assessed by the standard method for determining the CFU number in comparison with the control preparation of bacteria encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using lysozyme in a simulated intestinal environment, the resulting constructs provided a 59% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of enzymatic resistance using lipase in a simulated intestinal environment, the resulting constructs provided a 61% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of resistance in the environment of foreign bacteria, the obtained bioengineering constructs ensured the presence of no more than 3% of foreign bacteria E. coli BL21. In the test for bacteria migration from the hydrogel, the hydrogel allowed bacteria to spread to a distance of 8 mm from the sample. Evaluation of all tests was carried out according to the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "]. An in vitro cytotoxicity test of the resulting construct showed that it is not cytotoxic, namely, it has a score of “0” on the cytotoxicity scale [GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effect of medical devices. Part 5. Testing for cytotoxicity: in vitro methods "].

Example 2.

The manufacture of a bioengineered structure is carried out in several sequential stages.

At the first stage, bacterial alginate is also obtained by the method of controlled microbiological biosynthesis. To maintain the culture of bacteria, use Ashby's solid nutrient medium of the following composition (g / l): K ₂ HPO ₄ - 1.05; KH ₂ PO ₄ 0.2; MgSO ₄ 0.2; NaCl 0.2; Na ₂ MoO ₄ - 0.006; CaCO ₃ - 5.0; sucrose - 20, agar - 20. For the biosynthesis of alginate, the producer strain A. chroococcum 7B is cultivated in Burk's liquid medium of the following composition (g / l): KH ₂ PO ₄ - 0.05; K ₂ HPO ₄ - 0.05; MgSO ₄ 0.4; NaCl - 0.1; FeSO ₄ - 0.01; Na ₂ Mo0 ₄ - 0.06; CaCl ₂ - 0.1; sodium citrate - 0.5; sucrose - 20. First, the inoculum is prepared, for which the producer strain A. chroococcum 7B is grown in Burk's liquid medium of the specified composition in 750 ml shaking flasks (from 100 ml of medium) at 250 rpm (initial pH of the medium is 7, 2; cultivation temperature - 28 ° C) for 24 hours. Then biosynthesis of alginate is carried out by the producer strain A. chroococcum 7B, for which it is cultivated in the same medium after adding 4% (vol.) Of inoculum aged 24 hours in shaking flasks of 750 ml (volume of culture medium - 100 ml) on a microbiological shaker Innova 43 (New Brunswick Scientific, USA) at a stirring speed of 200 rpm for 72 hours. All procedures are performed under sterile conditions. To isolate alginate, the biomass of the producer strain is treated with 0.9% NaCl and 50 mM EDTA, after which centrifugation is carried out at 15000 g for 30 minutes. The precipitate is separated and washed with water. Then a 3-fold volume of 96% ethyl alcohol is added to it. The precipitate is again separated and washed with water. After that, the resulting alginate is purified. For this, the alginate is again precipitated with a 3-fold volume of 96% ethanol and dialysis is performed against 0.5% NaCl for 24 h. The precipitate is again separated and dried using a freeze dryer. The resulting powder is stored at 4 ° C for no more than 3 months.

The resulting alginate has the following combination of parameters: molecular weight (determined by viscosimetry) 2.6 × 10 ⁵ g / mol, molar content of guluronic acid residues in the polymer - 46% and acetylation level - 24% (determined by IR spectroscopy).

At the second stage, working solutions of bacterial polymers are prepared. Prepare a solution of poly-3-hydroxybutyrate (PHB) with a molecular weight of 3.2 × 10 ⁵ g / mol, obtained by bacterial biosynthesis, at a total concentration of 112.5 mg / ml (w / v) in a volume of 8 ml in a glass beaker of 50 ml, for which a sample of 90 mg is prepared. Prepare a solution of poly-3-hydroxybutyrate-co-3-hydroxyvalet (PHBV) with a molar content of 3-hydroxyvalerate of 17.6% and a molecular weight of 1.15 × 10 ⁶ g / mol, obtained by bacterial biosynthesis, at a concentration of 24 mg / ml in chloroform in a total concentration of 112.5 mg / ml (w / v) in a volume of 8 ml in a 50 ml glass beaker, for which a 90 mg portion is prepared.

At the third stage, porous substrates of a bioengineered structure are made by a modified leaching method. A solution of PHBV 17.6% 1150 kDa in chloroform at a concentration of 24 mg / ml is mixed with ammonium bicarbonate powder (with a salt particle size in the range of 90-300 microns in a ratio of 10: 1 by weight and thoroughly mixed with a spatula. The mixture is placed in a glass Petri dish with a diameter of 5 cm and left at room temperature until the organic solvent has completely evaporated for 3 hours.Then the mold is placed in hot distilled water (60 ° C) until gassing completely ceases, washed with distilled water 5 times and dried at 37 ° C for during the day.

At the 4th stage, porous microspheres with an average diameter of 160 μm are obtained using the "water phase / oil phase / water phase" (W / O / W) method, followed by washing out the blowing agent. An aqueous solution of ammonium carbonate is also used as a blowing agent. 21 ml of a chloroform solution of PHB 350 kDa at a concentration of 40 mg / ml is homogenized with 6.6 ml of a 5% (w / v) aqueous solution of ammonium carbonate. The resulting colloid is added dropwise to 210 ml of a 1% (w / v) solution of polyvinyl alcohol with constant stirring on an overhead stirrer at a speed of 750 rpm. After complete evaporation of chloroform, the particles are separated from the emulsifier (PVA) by precipitation and washing with distilled water. For a complete exit of the blowing agent, the microspheres are incubated in hot distilled water (70 ° C). Microspheres with an average diameter of 160 ± 30 μm are obtained, having a through porosity with a negligible fraction of closed pores, a total porosity of 94% and an average pore size of 1.8 ± 0.5 μm. Pieces of 2.5 cm in length and 2 in width are cut from the obtained porous film. see The resulting piece is rolled into a tube. The edges of the film are glued with a PHB solution in chloroform and a layer of microspheres is applied to the surface of the tube, for which microspheres are poured onto the surface of the film, without waiting for it to dry, and left to dry for 24 hours. Ready two-layer substrates with a layer of porous microspheres are cut into pieces of the required size and washed three times with sterile buffer solution 4-5 times for at least 2 hours.

At the next stage, the probiotic bacteria Lactobacillus plantarum 8P-A3 (Sputnik-K LLC) are grown. On a semi-liquid medium, the culture of L. plantarum 8P-A3 is grown in 250 ml conical flasks containing 200 ml of MRS broth and 0.3% agar-agar. The culture medium is poured into the flasks, sealed with cotton-gauze stoppers and autoclaved. The finished medium is not stored. Sowing is carried out as soon as the temperature of the medium after autoclaving reaches 40 ° C.

A liquid seed culture is prepared for sowing. For this, 3 ml of MRS broth is added to a test tube with a culture growing on a slant MRS-agar. The cells are removed from the surface of the solid medium by repeated pipetting and the resulting suspension is transferred into a flask containing 150 ml of culture medium. A flask with a liquid seed culture (5 × 10 ⁸ CFU / g) is incubated for 96 hours under aerobic conditions on a microbiological shaker at 37 ° C and a stirring speed of 120 rpm (orbital diameter 1.9 cm). The growth of the culture is monitored by nephelometry, by determining the optical density at a wavelength of 600 nm on a spectrophotometer (Ultrospec 1100 pro, Amersham Biosciences Limited, trademark of Microsoft Corporation, USA.), Morphological homogeneity is checked using light microscopy (microscope Biomed-1, "Biomed" , RF, with a digital camera).

In a 250 ml conical flask, a 1% (wt.) Solution of microbiologically obtained alginate is prepared in 150 ml of MRS broth on a magnetic stirrer with heating at 60 ° C and a stirring speed of 60 rpm. To obtain L. plantarum biomass on a liquid medium under aerobic conditions, 6 ml of a liquid seed culture is introduced into a conical flask containing the resulting 1% alginate solution in 150 ml of MRS broth, and incubated for 96 hours on a microbiological shaker at 37 ° C and a stirring speed of 120 rpm (orbit diameter 1.9 cm). After the end of the incubation, the biomass is precipitated by centrifugation for 15 minutes at 10000g on a Beckman J2-21 centrifuge. The supernatant is discarded. All procedures are performed under sterile conditions.

At the next stage, interleukin 10 (IL-10) is introduced into the biomass of L. plantarum in bacterial alginate. To do this, prepare a solution of IL-10 in water at a concentration of 1 mg / ml and, at the stage of completion of the cultivation of bacteria in bacterial alginate, add a solution of IL-10 in a volume of 1 ml to 99 ml of a polymer solution containing bacteria to achieve the mass fraction of IL-10 in the resulting polymer product 0.0001% (wt.).

At the last stage, the obtained porous structure based on PHB / PHBV is filled with a solution of the previously obtained bacterial capsular sodium alginate containing probiotic bacteria. For this procedure, the PHB / PHBV-based construct is placed in 100 ml of liquid biomass of L. plantarum 8P-A3 and IL-10 bacteria in MRS culture medium containing 1% sodium alginate, and a container with it into a desiccator, where negative pressure is created to displace air from the pores of the microspheres and the substrate and replacing them with a solution of the medium. Outwardly, the process of filling the pores of the microspheres under these conditions looks like the formation of many microbubbles of air around the microspheres. Then the alginate is gelled, for which the structure impregnated with sodium alginate solution is placed in a glass beaker with 100 ml of 50 mM calcium chloride solution for 10 minutes.

Thus, a bioengineered composite polymer structure is obtained in the form of a tube 25 mm long and 6 mm in diameter, containing 1.2 × 10 ⁸ CFU / g of L. plantarum 8P-A3 bacteria. Thus, there was an increase in the content of living bacteria in the structure against the initial (1.9 × 10 ⁷ CFU / g) by 6.3 times as compared to the drop in the content of bacteria in the composition described in the prototype patent, 11 times from 1 × 10 ⁸ CFU / g to 8.8 × 10 ⁶ CFU / g.

The stability test of the structure at a temperature of 37 ° C showed no deformation. In the resistance test in a simulated intestinal environment, the resulting construct ensured the preservation of 1.2 × 10 ⁸ CFU / g of L. plantarum 8P-A3 bacteria, i.e., 99% bacterial survival, assessed by the standard method for determining the CFU number in comparison with the control bacterial preparation. encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using alginate lyase in a simulated intestinal environment, the resulting construct provided a 54% increase in the survival of bacteria, assessed by the standard method for determining the number of CFUs in comparison with a control preparation of bacteria encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using lysozyme in a simulated intestinal environment, the resulting constructs provided a 67% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of enzymatic resistance using lipase in a simulated intestinal environment, the resulting constructs provided a 79% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of resistance in the environment of foreign bacteria, the obtained bioengineering constructs provided no more than 1% of foreign bacteria E. coli BL21 in the hydrogel. In the test for bacteria migration from the hydrogel, the hydrogel allowed bacteria to spread to a distance of 3 mm from the sample. Evaluation of all tests was carried out according to the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "]. An in vitro cytotoxicity test of the resulting construct showed that it is not cytotoxic, namely, it has a score of “0” on the cytotoxicity scale [GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effect of medical devices. Part 5. Testing for cytotoxicity: in vitro methods "].

Example 3.

The manufacture of a bioengineered structure is carried out in several sequential stages.

At the first stage, bacterial alginate is also obtained by the method of controlled microbiological biosynthesis. To maintain the culture of bacteria, use Ashby's solid nutrient medium of the following composition (g / l): K ₂ HPO ₄ - 1.05; KH ₂ PO ₄ 0.2; MgSO ₄ 0.2; NaCl 0.2; Na ₂ MoO ₄ - 0.006; CaCO ₃ - 5.0; sucrose - 20, agar - 20. For the biosynthesis of alginate, the producer strain A. chroococcum 7B is cultivated in Burk's liquid medium of the following composition (g / l): KH ₂ PO ₄ - 0.05; K ₂ HPO ₄ - 0.05; MgSO ₄ 0.4; NaCl - 0.1; FeSO ₄ - 0.01; Na ₂ MoO ₄ - 0.06; CaCl ₂ - 0.1; sodium citrate - 0.5; sucrose - 40. First, the inoculum is prepared, for which the producer strain A. chroococcum 7B is grown in Burk's liquid medium of the specified composition in 750 ml shaking flasks (from 100 ml of medium) at 250 rpm (initial pH of the medium is 7, 2; cultivation temperature - 28 ° C) for 24 hours. Then biosynthesis of alginate is carried out by the producer strain A. chroococcum 7B, for which it is cultivated in the same medium after adding 4% (vol.) Of inoculum aged 24 hours in shaking flasks of 750 ml (volume of culture medium - 100 ml) on a microbiological shaker Innova 43 (New Brunswick Scientific), USA) at a stirring speed of 200 rpm for 72 hours. All procedures are performed under sterile conditions. To isolate alginate, the biomass of the producer strain is treated with 0.9% NaCl and 50 mM EDTA, after which centrifugation is carried out at 15000 g for 30 minutes. The precipitate is separated and washed with water. Then a 3-fold volume of 96% ethyl alcohol is added to it. The precipitate is again separated and washed with water. After that, the resulting alginate is purified. For this, the alginate is again precipitated with a 3-fold volume of 96% ethanol and dialysis is performed against 0.5% NaCl for 24 h. The precipitate is again separated and dried using a freeze dryer. The resulting powder is stored at 4 ° C for no more than 3 months. The resulting alginate has the following combination of parameters: molecular weight (determined by viscosimetry) 7.6 × 10 ⁵ g / mol, molar content of guluronic acid residues in the polymer - 31% and acetylation level - 48% (determined by IR spectroscopy).

At the second stage, working solutions of bacterial polymers are prepared. Prepare a solution of poly-3-hydroxybutyrate (PHB) with a molecular weight of 5.3 × 10 ⁵ g / mol, obtained by bacterial biosynthesis, at a total concentration of 112.5 mg / ml (w / v) in a volume of 8 ml in a glass beaker of 50 ml, for which a sample of 90 mg is prepared. Prepare a PHBV solution with a molar content of 3-hydroxyvalerate of 9.0% and a molecular weight of 8.5 × 10 ⁵ g / mol, obtained by bacterial biosynthesis, at a concentration of 24 mg / ml in chloroform at a total concentration of 112.5 mg / ml (w / vol.) in a volume of 8 ml in a glass beaker of 50 ml, for which a sample of 90 mg is prepared. Prepare a solution of simvastatin in 1 ml of chloroform at a concentration of 9 mg / ml, for which a 9 mg sample is prepared.

At the third stage, porous substrates of a bioengineered structure are made by a modified leaching method and loaded with a medicinal substance, simvastatin. The solutions of PHBV and simvastatin in chloroform are mixed, for which purpose 0.1 ml of a solution of simvastatin in chloroform at a concentration of 9 mg / ml is added to 8 ml of a solution of PHBV in chloroform at a concentration of 24 mg / ml. The resulting joint solution of PHBV and simvastatin in chloroform is mixed with ammonium bicarbonate powder (with a salt particle size in the range of 90-300 microns in a ratio of 10: 1 by weight) and thoroughly mixed with a spatula. The mixture is placed in a glass Petri dish 5 cm in diameter and left at room temperature until the organic solvent has completely evaporated within 3 hours. After that, the mold is placed in hot distilled water (60 ° C) until gassing completely stops, washed with distilled water 5 times and dried at 37 ° C for a day.

At the 4th stage, porous microspheres with an average diameter of 120 µm are obtained using the "water phase / oil phase / water phase" (W / O / W) method, followed by washing out the blowing agent. An aqueous solution of ammonium carbonate is also used as a blowing agent. 21 ml of a chloroform solution of PHB at a concentration of 40 mg / ml is homogenized with 6.6 ml of a 5% (w / v) aqueous solution of ammonium carbonate. The resulting colloid is added dropwise to 210 ml of a 1% (w / v) solution of polyvinyl alcohol with constant stirring on an overhead stirrer at a speed of 750 rpm. After complete evaporation of chloroform, the particles are separated from the emulsifier (PVA) by precipitation and washing with distilled water. For a complete exit of the blowing agent, the microspheres are incubated in hot distilled water (70 ° C). Microspheres with an average diameter of 120 ± 25 μm are obtained, having a through porosity with a negligible fraction of closed pores, a total porosity of 92% and an average pore size of 1.5 ± 0.4 μm.

Strips 15 mm long and 5 mm wide are cut from the obtained porous film and a layer of microspheres is applied to its surface. To do this, microspheres are poured onto the surface of the film, without waiting for it to dry, in a dense layer and left to dry for 24 hours.

On a semi-liquid medium, the culture of Bifidobacterium longum MS-42 (OOO Sputnik-K) is grown in 250 ml conical flasks containing 200 ml of MRS broth and 0.3% agar-agar. The culture medium is poured into the flasks, sealed with cotton-gauze stoppers and autoclaved. The prepared medium is stored for no more than 2 weeks in a household refrigerator at a temperature of 5-8 ° C. Sowing is carried out as soon as the temperature of the medium after autoclaving reaches 40 ° C.

In a 250 ml conical flask, prepare a 1% (wt.) Solution of alginate with a molecular weight of 4.8 × 10 ⁵ g / mol, obtained by microbiology, in 150 ml of MRS broth on a magnetic stirrer with heating at 60 ° C and a stirring speed of 60 vol. / min. Subculture is carried out from the agar-agar medium to the alginate medium. For this, 8 ml is taken from a flask with a 96-hour culture growing in a semi-liquid medium on agar-agar with a glass serological pipette and introduced into a flask with a freshly prepared semi-liquid medium based on bacterial alginate. The contents of the flask are mixed. To obtain a culture of B. longum MC-42 in a semi-liquid medium, 4 ml of a working culture (1 × 10 ⁸ CFU / g) is added to the bottom of the flask. The contents of the flask are not mixed. After inoculation, the flasks are sealed with cotton-gauze stoppers and placed in a microbiological shaker for thermostating. Incubated for 96 hours at 37 ° C without stirring. The growth of the culture is monitored by nephelometry, by determining the optical density at a wavelength of 600 nm on a spectrophotometer (Ultrospec 1100 pro, Amersham Biosciences Limited, trademark of Microsoft Corporation, USA.), Morphological homogeneity is checked using light microscopy (microscope Biomed-1, "Biomed" , RF, with a digital camera).

At the last stage, the obtained porous structure based on PHB / PHBV is filled with a solution of the previously obtained bacterial capsular sodium alginate containing probiotic bacteria. For this procedure, a PHB / PHBV-based construct is placed in a liquid biomass of B. longum MC-42 bacteria in an MRS culture medium containing 1% sodium alginate, and a container with it in a desiccator, where negative pressure is created to displace air from the pores of the microspheres and the substrate. and replacing them with a solution of the medium. Then the alginate is gelled, for which the structure impregnated with sodium alginate solution is placed in a glass beaker with 100 ml of 50 mM calcium chloride solution for 10 minutes.

Thus, a bioengineered composite polymer structure is obtained in the form of a plate 15 mm long and 6 mm wide, containing 1.3 × 10 ⁷ CFU / g of B. longum MC-42 bacteria. Thus, there was an increase in the content of live bacteria in the structure against the initial (2.7 × 10 ⁶ CFU / g) by 4.8 times compared to the drop in the content of bacteria in the composition described in the prototype patent, 11 times from 1 × 10 ⁸ CFU / g to 8.8 × 10 ⁶ CFU / g.

The stability test of the structure at a temperature of 37 ° C showed no deformation. In the resistance test in a simulated intestinal environment, the resulting structure ensured the preservation of 1.3 × 10 ⁷ CFU / g of B. longum MC-42 bacteria, i.e. 96% bacterial survival, assessed by the standard method for determining the CFU number in comparison with the control bacterial preparation. encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using alginate lyase in a simulated intestinal environment, the resulting construct provided an 87% increase in bacterial survival, assessed by the standard method for determining the CFU number in comparison with the control preparation of bacteria encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using lysozyme in a simulated intestinal environment, the resulting constructs provided a 93% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of enzymatic resistance using lipase in a simulated intestinal environment, the resulting constructs provided a 117% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of resistance in the environment of foreign bacteria, the obtained bioengineering constructs ensured the absence of foreign bacteria E. coli BL21. In the test for bacteria migration from the hydrogel, the hydrogel allowed bacteria to spread to a distance of 2 mm from the sample. Evaluation of all tests was carried out according to the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "]. An in vitro cytotoxicity test of the resulting construct showed that it is not cytotoxic, namely, it has a score of “0” on the cytotoxicity scale [GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effect of medical devices. Part 5. Testing for cytotoxicity: in vitro methods "].

Example 4.

The manufacture of a bioengineered structure is carried out in several sequential stages.

At the first stage, bacterial alginate is also obtained by the method of controlled microbiological biosynthesis. To maintain the culture of bacteria, a solid Ashby nutrient medium is used with the following composition (g / l): K ₂ HPO ₄ - 1.05; KH ₂ PO ₄ 0.2; MgSO ₄ 0.2; NaCl 0.2; Na ₂ MoO ₄ - 0.006; CaCO ₃ - 5.0; sucrose - 20, agar - 20. For the biosynthesis of alginate, the producer strain A. chroococcum 7B is cultivated in Burk's liquid medium of the following composition (g / l): KH ₂ PO ₄ - 1.00; K ₂ HPO ₄ 1.00; MgSO ₄ 0.4; NaCl - 0.1; FeSO ₄ - 0.01; Na ₂ MoO ₄ - 0.06; CaCl ₂ - 0.1; sodium citrate - 0.5; sucrose - 30. First, the inoculum is prepared, for which the producer strain A. chroococcum 7B is grown in Burk's liquid medium of the specified composition in 750 ml shaking flasks (from 100 ml of medium) at 250 rpm (initial pH of the medium is 7, 2; cultivation temperature - 28 ° C) for 24 hours. Then biosynthesis of alginate is carried out by the producer strain A. chroococcum 7B, for which it is cultivated in the same medium after adding 4% (vol.) Of inoculum aged 24 hours in shaking flasks of 750 ml (volume of culture medium - 100 ml) on a microbiological shaker Innova 43 (New Brunswick Scientific), USA) at a stirring speed of 200 rpm for 72 hours. All procedures are performed under sterile conditions. To isolate alginate, the biomass of the producer strain is treated with 0.9% NaCl and 50 mM EDTA, after which centrifugation is carried out at 15000 g for 30 minutes. The precipitate is separated and washed with water. Then a 3-fold volume of 96% ethyl alcohol is added to it. The precipitate is again separated and washed with water. After that, the resulting alginate is purified. For this, the alginate is again precipitated with a 3-fold volume of 96% ethanol and dialysis is performed against 0.5% NaCl for 24 h. The precipitate is again separated and dried using a freeze dryer. The resulting powder is stored at 4 ° C for no more than 3 months. The resulting alginate has the following combination of parameters: molecular weight (determined by viscosimetry) 1.0 × 10 ⁶ g / mol, molar content of guluronic acid residues in the polymer - 35% and acetylation level - 10% (determined by IR spectroscopy).

At the second stage, a PHB solution with a molecular weight of 7.6 × 10 ⁵ g / mol obtained by bacterial biosynthesis is prepared at a total concentration of 112.5 mg / ml in a volume of 8 ml in a 50 ml glass beaker, for which a sample of 90 mg is prepared ...

At the third stage, porous microspheres with an average diameter of 560 μm are obtained using the "water phase / oil phase / water phase" (W / 0 / W) method, followed by washing out the pore former. An aqueous solution of ammonium carbonate is used as a pore-forming agent. 21 ml of a chloroform solution of PHB 350 kDa at a concentration of 40 mg / ml is homogenized with 6.6 ml of a 5% (w / v) aqueous solution of ammonium carbonate. The obtained colloid is added dropwise to 210 ml of a 1% (wt.) Solution of polyvinyl alcohol with constant stirring on an overhead stirrer at a speed of 750 rpm. After complete evaporation of chloroform, the particles are separated from the emulsifier (PVA) by precipitation and washing with distilled water. For a complete exit of the blowing agent, the microspheres are incubated in hot distilled water (70 ° C). Microspheres with an average diameter of 560 ± 60 μm are obtained, having through porosity with a negligible fraction of closed pores, a total porosity of 96% and an average pore size of 3.4 ± 0.9 μm

At the 4th stage, genetically modified probiotic bacteria Lactobacillus delbrueckii subsp.bulgaricus NCTC 12712 (Sputnik-K LLC) are grown, containing the pMC1 plasmid with the pC194 chloramphenicol resistance gene, obtained by the standard method [Serror P., Sasaki T., Ehrlich SD, Maguin E. Electrotransformation of Lactobacillus delbrueckii subsp. Bulgaricus and L. delbrueckii subsp. lactis with Various Plasmids. Appl Environ Microbiol. 2002, 68 (1), 46-52]. On a semi-liquid medium, the culture of L. delbrueckii subsp. bulgaricus VI104 are grown in 250 ml conical flasks containing 200 ml of MRS broth and 0.3% agar-agar. The culture medium is poured into the flasks, sealed with cotton-gauze stoppers and autoclaved. The finished medium is not stored. Sowing is carried out as soon as the temperature of the medium after autoclaving reaches 40 ° C.

A liquid seed culture is prepared for sowing. For this, 3 ml of MRS broth is added to a test tube with a culture growing on a slant MRS-agar. The cells are removed from the surface of the solid medium by repeated pipetting and the resulting suspension is transferred into a flask containing 150 ml of culture medium. The flask with a liquid seed culture is incubated for 96 hours under aerobic conditions on a microbiological shaker at 37 ° C and a stirring speed of 120 rpm (orbital diameter 1.9 cm). The growth of the culture is monitored by nephelometry, by determining the optical density at a wavelength of 600 nm on a spectrophotometer (Ultrospec 1100 pro, Amersham Biosciences Limited, trademark of Microsoft Corporation, USA.), Morphological homogeneity is checked using light microscopy (microscope Biomed-1, "Biomed" , RF, with a digital camera).

In a 250 ml conical flask, a 1% (wt.) Solution of microbiologically obtained alginate is prepared in 150 ml of MRS broth on a magnetic stirrer with heating at 60 ° C and a stirring speed of 60 rpm. To obtain biomass L. delbrueckii subsp. bulgaricus NCTC 12712 on a liquid medium under aerobic conditions 6 ml of a liquid seed culture (1 × 10 ⁸ CFU / g) are introduced into a conical flask containing the resulting 1% alginate solution in 150 ml of MRS broth, and incubated for 96 hours on a microbiological shaker at 37 ° C and a stirring speed of 120 rpm (orbital diameter 1.9 cm). After the end of the incubation, the biomass is precipitated by centrifugation for 15 minutes at 10000g in a "Vesktap J2-21" centrifuge. The supernatant is discarded. All procedures are performed under sterile conditions.

At the 5th stage, a culture of Bifidobacterium longum MC-42 is grown on a semi-liquid medium in 250 ml conical flasks containing 200 ml of MRS broth and 0.3% agar-agar. The culture medium is poured into the flasks, sealed with cotton-gauze stoppers and autoclaved. The prepared medium is stored for no more than 2 weeks in a household refrigerator at a temperature of 5-8 ° C. Sowing is carried out as soon as the temperature of the medium after autoclaving reaches 40 ° C.

In a 250 ml conical flask, prepare a 1% (wt.) Solution of alginate with a molecular weight of 4.8 × 10 ⁵ g / mol, obtained by microbiology, in 150 ml of MRS broth on a magnetic stirrer with heating at 60 ° C and a stirring speed of 60 vol. / min. Subculture is carried out from the agar-agar medium to the alginate medium. For this, 8 ml is taken from a flask with a 96-hour culture growing in a semi-liquid medium on agar-agar with a glass serological pipette and introduced into a flask with a freshly prepared semi-liquid medium based on bacterial alginate. The contents of the flask are mixed. To obtain a culture of B. longum MC-42 in a semi-liquid medium, 4 ml of a working culture (1 × 10 ⁸ CFU / g) is added to the bottom of the flask. The contents of the flask are not mixed. After inoculation, the flasks are sealed with cotton-gauze stoppers and placed in a microbiological shaker for thermostating. Incubated for 96 hours at 37 ° C without stirring. The growth of the culture is monitored by nephelometry, by determining the optical density at a wavelength of 600 nm on a spectrophotometer (Ultrospec 1100 pro, Amersham Biosciences Limited, trademark of Microsoft Corporation, USA.), Morphological homogeneity is checked using light microscopy (microscope Biomed-1, "Biomed" , RF, with a digital camera).

At the last stage, the obtained porous structures in the form of microspheres based on PHB / PHBV are filled with a solution of the previously obtained bacterial capsular sodium alginate containing probiotic bacteria. First, a mixture of bacteria in bacterial alginate is prepared. For this, 50 ml of the biomass of the previously grown bacteria B. longum MC-42 in the MRS culture medium containing 1% sodium alginate is mixed with 50 ml of the biomass of the previously grown genetically modified bacteria Lactobacillus delbrueckii subsp. bulgaricus NCTC 12712 in MRS culture medium containing 1% sodium alginate in a 150 ml glass flask and mix thoroughly. Porous microspheres based on PHB / PHBV are placed in a mixture of bacterial biomass in a MRS culture medium containing 1% sodium alginate, and a container with it in a desiccator, where negative pressure is created to displace air from the pores of the microspheres and replace them with a solution of the medium. Then the alginate is gelled, for which the structure impregnated with sodium alginate solution is placed in a glass beaker with 100 ml of 50 mM calcium chloride solution for 10 minutes.

Thus, a bioengineered composite polymer structure is obtained in the form of a tube 25 mm long and 6 mm in diameter, containing 1.2 × 10 ⁷ CFU / g of bacteria L. delbrueckii subsp.bulgaricus NCTC 12712 and 1.0 × 10 ⁷ CFU / g of bacteria B longum MC-42. Thus, there was an increase in the content of live bacteria in the structure against the initial (2.7 × 10 ⁶ CFU / g) by at least 3.7 times compared with the drop in the content of bacteria in the composition described in the prototype patent, 11 times with 1 × 10 ⁸ CFU / g to 8.8 × 10 ⁶ CFU / g.

The stability test of the structure at a temperature of 37 ° C showed no deformation. In a resistance test in a simulated intestinal environment, the resulting construct ensured the preservation of 1.2 × 10 ⁷ CFU / g bacteria L. delbrueckii subsp.bulgaricus NCTC 12712 and 1.0 × 10 ⁷ CFU / g bacteria B. longum MC-42., 98 % bacterial survival, assessed by the standard method for determining the CFU number in comparison with the control preparation of bacteria encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using alginate lyase in a simulated intestinal environment, the resulting construct provided a 61% increase in bacterial survival, assessed by the standard method for determining the CFU number in comparison with a control preparation of bacteria encapsulated in an alginate hydrogel based on algal alginate. In the test of enzymatic resistance using lysozyme in a simulated intestinal environment, the resulting constructs provided a 65% increase in bacterial survival, as measured by the standard method for determining the number of CFU in the construct. In the test of enzymatic resistance using lipase in a simulated intestinal environment, the resulting constructs provided a 77% increase in bacterial survival, assessed by the standard method for determining the number of CFU in the construct. In the test of resistance in the environment of foreign bacteria, the obtained bioengineering constructs ensured the absence of foreign bacteria E. coli BL21. In the test for bacteria migration from the hydrogel, the hydrogel allowed bacteria to spread to a distance of 3 mm from the sample. Evaluation of all tests was carried out according to the standard method for determining the number of CFU in the structure [GOST R 56139-2014 “Functional food products. Methods for the determination and counting of probiotic microorganisms "]. An in vitro cytotoxicity test of the resulting construct showed that it is not cytotoxic, namely, it has a score of “0” on the cytotoxicity scale [GOST ISO 10993-1-2011 “Medical devices. Assessment of the biological effect of medical devices. Part 5. Testing for cytotoxicity: in vitro methods "].

Claims (25)

1. A bioengineered construction for probiotic bacteria of the genera Lactobacillus and Bifidobacterium for the treatment of diseases of the gastrointestinal tract, which is a layer of porous microspheres in a hydrogel located on a porous substrate, while the porous substrate is made of poly-3-hydroxybutyrate and / or its copolymer with 3 -oxyvalerate with a molecular weight from 1 × 10 ⁵ to 2 × 10 ⁶ g / mol and is characterized by the presence of through pores (or interconnected pores), a total porosity of 75 to 99%, an average pore diameter of 0.5 to 20 μm, and porous microspheres are made of poly-3-hydroxybutyrate with a molecular weight from 1 × 10 ⁴ to 1 × 10 ⁶ g / mol and are characterized by the presence of through pores (or interconnected pores), total porosity from 75 to 99%, average pore diameter from 0.5 up to 20 microns; where the pores of the substrate and microspheres are filled with a hydrogel containing probiotic bacteria, while the hydrogel is made of bacterial alginate with a molecular weight of 10 ⁵ to 10 ⁶ g / mol, a molar content of guluronic acid residues in the polymer from 31 mol% to 48 mol% and a level of acetylation - from 10 mol% to 46 mol%. 2. A bioengineering structure according to claim 1, characterized in that the substrate is made with a thickness of 10 μm to 5 cm, and the diameter of the microspheres is from 2 to 2000 μm. 3. A bioengineering structure according to claim 1, characterized in that the microspheres are arranged in several rows - from one to five. 4. Bioengineering structure according to claim 1, characterized in that it is made in the form of a tube obtained by rolling the substrate. 5. A bioengineering structure according to claim 1, characterized in that the bacterial alginate is a biosynthesis product of bacteria of the genus Azotobacter. 6. Bioengineering construction according to claim 1, characterized in that Lactobacillus and / or Bifidobacterium bacteria, or their genetic modifications, are used as probiotic bacteria, in a ratio of CFU from 1: 999 to 999: 1. 7. Bioengineering structure according to claim 1, characterized in that it additionally contains at least one pharmacologically active substance. 8. A bioengineering structure according to claim 7, characterized in that a drug is used as a pharmacologically active substance, which has an anti-inflammatory effect and stimulates the regeneration of tissues of the intestinal wall. 9. A bioengineering structure according to claim 8, characterized in that simvastatin, curcumin, rosveratrol are used as a drug that has an anti-inflammatory effect and stimulates the regeneration of intestinal wall tissues. 10. Bioengineering construction according to claim 7, characterized in that the pharmacologically active substance is directly in a mixture with a copolymer of poly-3-hydroxybutyrate with 3-hydroxyvalerate at a concentration of 0.01% to 50% (wt.) Of the drug in the resulting polymer product. 11. A bioengineering structure according to claim 7, characterized in that the pharmacologically active substance is directly mixed with the bacterial alginate at a concentration of 0.01% to 50% (weight) of the drug in the resulting polymer product. 12. A method of obtaining a bioengineering structure according to claim 1, characterized in that it consists of the following stages: - microbiological biosynthesis of bacterial alginate using a producer strain of the genus Azotobacter, - obtaining a porous support from poly-3-hydroxybutyrate and / or its copolymer with 3-hydroxyvalerate, - obtaining porous microspheres from poly-3-oxybutrate, - attachment of microspheres to the surface of a porous substrate, - growing probiotic bacteria of the genera Lactobacillus and Bifidobacterium in bacterial alginate, - filling the porous substrate with alginate with bacteria, - gelation of bacterial alginate in situ by adding an aqueous solution of calcium chloride. 13. The method according to claim 12, characterized in that the porous substrate is obtained by one of the following methods: lyophilization, drying, leaching, emulsification, electrospinning, rapid prototyping, laser cutting. 14. The method according to claim 12, characterized in that the porous microspheres are obtained by one of the following methods: lyophilization, spray drying, leaching, emulsification, rapid prototyping. 15. The method according to claim. 12, characterized in that the attachment of the porous microspheres to the surface of the porous substrate is carried out by depositing them on a surface wetted with a solvent or by deposition on the surface in solvent vapor. 16. The method according to claim 12, characterized in that before filling the porous substrate and the microspheres located on it with the hydrogel, live probiotic bacteria are grown in the bacterial alginate that forms its basis to a content of not less than 1 × 10 ⁷ CFU / ml. 17. The method according to claim 12, characterized in that at least one pharmacologically active substance is introduced into it at the stage of obtaining the porous substrate. 18. The method according to claim. 12, characterized in that before filling the porous support and located on the microspheres with a hydrogel based on bacterial alginate containing probiotic bacteria, at least one pharmacologically active substance is introduced into it.

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