JP7228533B2 - Method for Producing Bacterial Compositions Comprising Biofilms with Beneficial Bacteria - Google Patents

Description

The present invention, in some embodiments thereof, relates to methods of producing bacterial compositions, and more particularly, but not limited to, probiotic compositions, environmentally beneficial probiotic compositions and industrial It relates to a probiotic composition for use in

Live microbial cells that confer beneficial physiological effects on the host when administered in appropriate amounts are known as "probiotics." Studies have shown that probiotic bacteria can provide therapeutic benefits that contribute to maintaining a healthy gut in the host and controlling some types of gastrointestinal infections. Due to their recognized health benefits, probiotic bacteria have been increasingly included in a variety of food and beverage products over the last few decades. Some of the most common types of microorganisms used as probiotics are lactic acid bacteria (LAB), which mainly belong to the genera Lactobacillus and Bifidobacterium. Both of these genera are predominant commensals in the human gut, have a long history of safe use and are generally recognized as safe (GRAS). To ensure their beneficial effects in the body, these organisms must survive during food processing, storage and transit through the upper gastrointestinal tract (GIT) and reach their site of action alive. must. However, previous studies have shown that the survival levels of probiotic bacteria in finished foods are low, and the highly acidic conditions of the stomach and high bile concentrations in the small intestine significantly reduce their survival. Additionally, probiotics are usually provided as dried bacterial powders, which are primarily prepared by freeze-drying, an established procedure that can cause lethal damage to cells. Therefore, to sustain delivery of probiotics to humans, there is a need to develop new techniques to improve the survival of health-promoting bacteria during food manufacturing and throughout the storage and consumption process.

In most natural ecosystems, bacteria prefer to grow within multicellular, complex cell populations called biofilms, rather than as free-living (planktonic) cells. Biofilm mode of growth is also favorable for bacteria that live in the intestinal tract. Cells in biofilms are connected by an extracellular matrix that consists primarily of polysaccharides produced by the cells themselves and other macromolecules such as proteins, DNA, lipids and nucleic acids. The interaction of species embedded in biofilms with their environment creates complex structures that can withstand environmental stress and antimicrobial exposure. Biofilm formation is therefore a strategy for survival under unfavorable conditions in diverse environments.

One of the most studied biofilm formers is Bacillus subtilis, a spore-forming, non-pathogenic bacterium characterized by its ability to generate robust biofilms. Bacillus species, primarily B. subtilis, have recently gained interest as probiotic microorganisms, primarily due to their positive effect on host health by maintaining a favorable balance of the microflora of the gastrointestinal tract. Since B. subtilis spores can survive extreme pH conditions and hypoxia, large numbers of dormant but viable microorganisms reach the lower intestine and secrete active substances to produce several beneficial effects. can induce In addition, B. subtilis cells were found to enhance the growth and viability of Lactobacillus species, possibly by production of catalase and subtilisin (Hosoi, Ametani, Kiuchi, & Kaminogawa, 2000). γ-Polyglutamic acid produced by B. subtilis as part of the extracellular matrix has been shown to increase probiotic bacteria during lyophilization (A. R. Bhat et al., 2013) and storage (A. R. Bhat et al., 2015). It was also reported that it could be used to improve survival. The same was seen in simulated gastric fluid that mimics the acidic conditions of the stomach (A. R. Bhat et al., 2015).

Other background art includes US Patent Application Publication No. 20100203581 and Salas Jara et al., Microorganisms 2016, 4, 35; doi:10.3390.

According to one aspect of the invention, a method for preparing a bacterial composition comprising:
(a) co-cultivating beneficial bacteria and biofilm-forming bacteria in a growth substrate in vitro under conditions that produce a biofilm comprising beneficial bacteria and non-pathogenic bacteria;
(b) isolating the biofilm from the growth substrate to prepare said bacterial composition is provided.

According to one aspect of the invention there is provided a bacterial composition obtainable by the method described herein.

According to one aspect of the invention there is provided a food or feed comprising a bacterial composition as described herein.

According to one aspect of the invention, a method of improving or maintaining the health of a subject comprises administering to the subject a therapeutically effective amount of a probiotic composition described herein, thereby improving the health of the subject. or maintaining.

According to one aspect of the invention, there is provided a method of selecting agents or culture conditions favorable for the preparation of a bacterial composition, wherein beneficial bacteria and biofilm-forming bacteria are grown in a growth substrate, in the presence of an agent, or under culture conditions. co-cultivating to produce a biofilm comprising beneficial bacteria and biofilm-producing bacteria, wherein a change in the properties of the biofilm is determined by an agent or culture condition that favors the preparation of the bacterial composition. A method is provided to indicate that.

According to embodiments of the invention, the biofilm-forming bacteria are non-pathogenic.

According to an embodiment of the invention, the biofilm-producing bacterium is of the genus bacillus.

According to an embodiment of the invention, the biofilm-producing bacteria are B. subtilis species.

According to an embodiment of the invention, the biofilm-producing bacterium is strain 127185/2.

According to embodiments of the invention, the growth substrate comprises manganese.

According to embodiments of the invention, the growth substrate comprises dextrose.

According to an embodiment of the invention, when the biofilm-forming bacterium is of the genus bacillus, the growth substrate comprises manganese.

According to an embodiment of the invention, beneficial bacteria are probiotic bacteria.

According to embodiments of the invention, the beneficial bacterium is genetically modified to express a therapeutic polypeptide.

According to an embodiment of the invention, the probiotic bacterium is of the order Lactobacillales.

According to an embodiment of the invention, the biofilm-producing bacteria are B. subtilis species.

According to an embodiment of the invention, the probiotic bacterium is L. plantarum species.

According to embodiments of the invention, the beneficial bacteria are for bioremediation.

According to embodiments of the present invention, the biofilm-producing bacteria express genes of the KinD-SpoOA pathway.

According to embodiments of the invention, the growth substrate comprises growth medium.

According to an embodiment of the invention, the growth medium is selected from the group consisting of LB medium, LBGM medium, milk medium and MRS medium.

According to an embodiment of the invention, the biofilm-producing bacterium is of the genus bacillus, the beneficial bacterium is of the order Lactobacillales, and the growth substrate is LBGM medium, milk medium or MRS medium.

According to an embodiment of the invention, the growth substrate is MRS medium.

According to embodiments of the present invention, the conditions include a pH of about 6.5-8.

According to an embodiment of the invention the conditions comprise pH 6.8-7.5.

According to embodiments of the invention, the growth substrate comprises acetoin.

According to embodiments of the invention, the method further comprises dehydrating the biofilm after isolation.

According to embodiments of the present invention, beneficial bacteria include 50 or fewer bacterial species.

According to embodiments of the invention, the biofilm-forming bacterium is a single species of biofilm-forming bacterium.

According to an embodiment of the invention, at least 50% of the bacteria in the composition are viable cells.

According to an embodiment of the invention, the bacterial composition comprises 50 or fewer bacterial species as beneficial bacteria.

According to embodiments of the invention, the bacterial composition comprises a single species of non-pathogenic bacteria.

According to embodiments of the present invention, the bacterial composition is edible.

According to an embodiment of the invention the bacterial composition is a probiotic bacterial composition.

According to embodiments of the invention, the bacterial composition is formulated as a powder, liquid or tablet.

According to an embodiment of the invention, the biofilm-producing bacterium is of the genus bacillus.

According to an embodiment of the invention, the biofilm-producing bacteria are B. subtilis species.

According to an embodiment of the invention, beneficial bacteria are probiotic bacteria.

According to an embodiment of the invention, the probiotic bacterium is of the order Lactobacillales.

According to embodiments of the invention, the agent alters the pH of the medium within the system.

Unless defined otherwise, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not necessarily intended to be limiting.

Several embodiments of the present invention are described herein, by way of illustration only, with reference to the accompanying drawings. It is emphasized that the details, hereinafter with particular reference to the drawings, are for the purpose of illustration and detailed description of the embodiments of the invention. Likewise, it will become apparent to those skilled in the art how the embodiments of the invention can be practiced, when viewed in conjunction with the drawings.

Figures 1A-B are graphs comparing the growth of B. subtilis and L. plantarum in co-culture. Co-culture production had no effect on the growth of L. plantarum and B. subtilis (compared to their growth in pure culture), indicating no antagonistic interactions between these bacteria. there is Figure 2 is a photograph showing that modified MRS medium induces biofilm formation by B. subtilis. The effect of modifying the pH of MRS medium on biofilm formation of B. subtilis NCIB3610 was analyzed by stereomicroscopy. Figure 3 is a photograph showing that the combination of LB medium and MRS medium induces biofilm formation by B. subtilis. Effect of LB medium enriched with different concentrations of MRS medium (pH 7) on biofilm formation by colonies (upper row) and pellicles (lower row). Figures 4A-B are graphs showing that the combination of LB medium and MRS medium induces extracellular matrix production by B. subtilis. Increasing MRS concentration induces transcription of the tapA-sipW-tasA (A) and epsA-O (B) operons. FIG. 5A is a photograph showing that the biofilm-stimulating effect of MRS medium is regulated by previously reported substrate synthesis and biofilm formation signaling pathways in B. subtilis. Colony and pellicle formation on MRS medium (pH 7) by wild-type (WT) and various mutant strains were compared. The strains used here were: wild-type (NCIB3610), ΔkinCD (RL4577), ΔkinAB (RL4573), Δspo0A (RL4620), ΔepsΔtasA (RL4566), ΔabrB (YC668). FIG. 5B is a photograph showing that the effect of MRS medium on WT cells is comparable to that of substrate overproducing mutant cells (ΔabrB) on B. subtilis. Figure 6 is a photograph showing that MRS medium induces colony-type biofilm formation in various Bacillus species. MRS medium (pH 7) strongly induced colony-type biofilm formation of B. paralicheniformis MS303, B. licheniformis MS310, B. licheniformis S127, B. subtilis MS1577 and B. cereus 10987. FIG. 7 is a photograph showing that MRS medium induces pellicle formation in various Bacillus species. MRS medium (pH 7) strongly induced pellicle formation of B. paralicheniformis MS303, B. licheniformis MS310, B. licheniformis S127, B. subtilis MS1577 and B. cereus 10987. FIG. 8A is an image showing that B. subtilis produces extracellular matrix and forms a dual-species biofilm with L. plantarum. CLSM image of a co-cultivated biofilm of B. subtilis and L. plantarum at MRS medium pH 7, 37° C. and 50 rpm. From left to right: fluorescence image, image generated using Nomarski differential interference contrast (DIC) and overlay image. Top, expression of fluorescently labeled B. subtilis cells constitutively express GFP. Bottom, expression of substrate-producing B. subtilis cells expressing CFP under the control of the tapA promoter. L. plantarum cells are unstained in all images. Figure 8B is an image showing that B. subtilis produces extracellular matrix and forms a dual-species biofilm with L. plantarum. CLSM images of co-cultured biofilms of B. subtilis and L. plantarum in LBGM medium. From left to right: fluorescence image, image generated using Nomarski differential interference contrast (DIC) and overlay image. Top, expression of fluorescently labeled B. subtilis cells constitutively express GFP. Bottom, expression of substrate-producing B. subtilis cells expressing CFP under the control of the tapA promoter. L. plantarum cells are unstained in all images. Figures 9A-C are SEM images of (A) B. subtilis cells, (B) L. plantarum cells and (C) dual-species biofilms composed of B. subtilis and L. plantarum. Figures 10A-B are graphs showing that dual-species biofilms promote survival of L. plantarum exposed to unfavorable conditions. Survival of L. plantarum cells in the presence or absence (control) of B. subtilis biofilms was measured during (A) heat treatment at 63°C for 1-3 minutes and (B) at 4°C for 21 days. Measured during storage. Values shown are the average of at least three independent experiments performed in duplicate. *p<0.05 FIG. 11A is a graph showing that the extracellular matrix of B. subtilis promotes increased viability of L. plantarum during heat treatment. WT B. subtilis and its derivatives, a mutant lacking the exopolysaccharide and protein components of the extracellular matrix (ΔepsΔtasA), and a mutant lacking the repressor of the substrate gene (ΔabrB; ) was tested for the effect of heat treatment at 63° C. for 3 minutes. Results shown are the average of at least three independent experiments performed in duplicate. *p<0.05 FIG. 11B is a graph showing that the extracellular matrix of B. subtilis promotes increased viability of L. plantarum during heat treatment. Samples were grown in milk medium for 18 hours at 30°C and 20 rpm. They were then subjected to heat treatment at 63° C. for 1-3 minutes. No heat treatment was performed on the control sample. The number of viable L. plantarum cells was determined by the CFU method. *p<0.05 Figure 12 is a graph showing that the presence of B. subtilis biofilm increases the survival of L. plantarum upon in vitro gastric and intestinal digestion (model system). Viability of L. plantarum cells in the presence or absence of B. subtilis biofilm (control) was measured during in vitro gastrointestinal digestion. Results shown are the average of at least three independent experiments performed in duplicate. *p<0.05 Figure 13 is a graph of growth curves of B. subtilis 3610NCIB in MRS medium (pH 7) and LB medium. FIG. 14 is a photograph showing the effect of histidine kinase mutations on colony surface structure and pellicle formation in MRS medium pH7. FIG. 15A-C are CLSM images of fluorescently labeled B. subtilis cells (Pspank-gfp) after 24 hours of incubation in LB medium in the presence and absence of acetoin. Figure 16A-B are photographs showing that acetoin induces colony-type biofilm formation by Bacillus subtilis. Figures 17A-D are photographs showing that transcription of the tapA operon responsible for substrate production in B. subtilis is highly upregulated by acetoin. CLSM images of B. subtilis cells with PtapA-cfp transcriptional fusions after 24 hours of incubation in LB medium, which does not promote biofilm formation. Figures 18A-B are photographs showing biofilms produced by B. subtilis strains NCIB3610 and 127185/2, respectively. Figure 19 is a graph showing survival of L. plantarum growing in co-culture biofilms with B. subtilis during migration in an in vitro model of the digestive system. The t=0 culture was the experimental control. Co-cultures of L. plantarum + B. subtilis 127185/2 showed the highest survival rates after incubation in stomach-like fluid. L. plantarum + B. subtilis NCIB3610 co-cultures showed slightly higher survival than L. plantarum alone. After further incubation, there was a significant decrease in the intestinal-like fluid when the viability trends in the various cultures were maintained. Figure 20 is a graph showing survival of L. plantarum growing in co-culture biofilms with B. subtilis upon exposure to high acidity levels. The sign "+" in the test cultures indicates growth with shaking at 50 rpm, the sign "-" indicates growth without any shaking. In general, the survival of L. plantarum decreased dramatically when transitioning from pH 7 to pH 3 growth media. Co-cultures of L. plantarum and B. subtilis showed less loss of viability of L. plantarum (compared to monocultures of L. plantarum) upon transition to an acidic environment, with shaking as well as without shaking. . FIG. 21 is a photograph showing that Mn ²⁺ ions are involved in biofilm formation by B. subtilis in modified MRS medium. We observed the effect of exclusion of certain MRS media components (Mg ²⁺ , Mn ²⁺ , sodium acetate, dipotassium phosphate, dextrose, ammonium citrate) on colony and pellicle formation by WT B. subtilis cells.

The present invention, in some embodiments thereof, relates to methods of producing bacterial compositions, more particularly, but not limited to, probiotic compositions, environmentally beneficial probiotic compositions and industrial of probiotic compositions.

Before describing at least one embodiment of the present invention in detail, the present invention must be described in detail of construction and elements whose application is set forth in the following description and/or illustrated in the drawings and/or examples. It should be understood that the arrangement and/or methods are not limited. The invention is capable of other embodiments and of being practiced or carried out by various means.

Bacteria are of economic importance. This is because these microorganisms are used by humans for multiple purposes. Beneficial uses of bacteria include the production of traditional foods such as yogurt, cheese and vinegar, biotechnology and genetic engineering to produce substances such as drugs and vitamins, agriculture, textile softening, methane production, bioremediation and harmful effects. Biological control of organisms.

To serve these purposes, bacteria are often exposed to harsh conditions that reduce their viability and thus their effectiveness.

For example, to ensure the beneficial effects of probiotics in the body, these organisms must survive during food processing, storage and passage through the upper gastrointestinal tract (GIT) and live at their site of action. must be reached by However, previous studies have shown that the survival levels of probiotic bacteria in finished foods are low, significantly reducing their viability to the highly acidic conditions of the stomach and high bile concentrations in the small intestine. In addition, probiotics are usually available as dried bacterial powders prepared primarily by freeze-drying, an established procedure that can be fatally damaging to cells.

While conducting studies on bacterial biofilms, we found that, under appropriate conditions, biofilm-forming bacteria incorporate non-biofilm-forming bacteria into their biofilms and expose them to extreme temperatures (cold, heat, respectively). 10A-B and 11A-B) can be made more tolerant.

Specifically, the inventors co-cultivated bacteria of the B. subtilis species together with the probiotic bacterium L. plantarum. They showed that under certain conditions, B. subtilis bacteria generate biofilms and L. plantarum cells are incorporated into their extracellular matrix (Fig. 9A). L. plantarum incorporated into biofilms was shown to be more heat and cold tolerant and more acid tolerant than the control L. plantarum not incorporated into biofilms.

Taken together, we propose to use biofilm forming bacteria to encapsulate non-biofilm forming bacteria. Biofilm-forming bacteria therefore serve as protective carriers for beneficial non-biofilm-forming bacteria.

Thus, according to a first aspect of the present invention, a method for preparing a bacterial composition comprising:
(a) co-cultivating beneficial bacteria and biofilm-forming bacteria in a growth substrate in vitro under conditions that produce a biofilm comprising beneficial bacteria and non-pathogenic bacteria;
(b) isolating the biofilm from the growth substrate to prepare a bacterial composition is provided.

As used herein, the term "bacteria" refers to prokaryotic microorganisms, including archaea. Bacteria can be Gram-positive or Gram-negative. Bacteria may also be photosynthetic bacteria (eg, cyanobacteria).

As used herein, the term "beneficial bacterium" refers to any bacterium that has a positive effect on humans.

In one embodiment, the beneficial bacterium does not form a biofilm when grown as a monoculture under standard culture conditions in a growth medium.

In another embodiment, the beneficial bacteria do not form biofilms when grown as monocultures in a growth medium under optimal culture conditions for their growth.

In yet another embodiment, the beneficial bacterium utilizes the KinD-Spo0A pathway (eg, expresses genes for the histidine kinases kinD, spo0F, spo0B and/or spo0A). See, for example, Shemesh and Chai, 2013 Journal of Bacteriology, 2013, Vol 195, No.12 pages 2747-2754. The contents of which are incorporated herein by reference.

Beneficial bacteria can generally be cultured on Man, Rogosa and Sharpe (MRS) medium (solidified with agar or MRS broth).

Beneficial bacteria generally should not inhibit (ie, antagonize) the ability of biofilm-forming bacteria (eg, B. subtilis) to form biofilms. Methods for determining whether a bacterium has antagonistic activity when co-cultivated against another bacterium are known in the art (see, eg, FIGS. 1A-B). In one embodiment, beneficial bacteria are not soil bacteria.

Any number of beneficial bacterial strains can be cultured in the co-cultivation of this aspect of the invention. In one embodiment, no more than 500 different beneficial bacterial strains are cultured as single cultures, no more than 250 different beneficial bacterial strains are cultured as single cultures, and no more than 100 different beneficial bacterial strains are cultured as single cultures. cultivated as one culture, cultivated up to 90 different beneficial bacterial strains as a single culture, cultivated up to 80 different beneficial bacterial strains as a single culture, cultivated up to 70 different beneficial bacterial strains as a single culture; Cultivated as a monoculture, culturing up to 60 different beneficial bacterial strains as a monoculture, culturing up to 50 different beneficial bacterial strains as a monoculture, culturing up to 40 different beneficial bacterial strains as a monoculture as a monoculture, no more than 30 different beneficial bacterial strains are cultivated as monocultures, no more than 20 different beneficial bacterial strains are cultivated as monocultures, no more than 10 different beneficial bacteria are cultivated as monocultures Strains are cultivated as monocultures, up to 9 different beneficial bacterial strains are cultivated as monocultures, up to 8 different beneficial bacterial strains are cultivated as monocultures, up to 7 different beneficial bacterial strains are cultivated as monocultures, culturing bacterial strains as monocultures, culturing up to 6 different beneficial bacterial strains as monocultures, culturing up to 5 different beneficial bacterial strains as monocultures, culturing up to 4 different beneficial bacterial strains as monocultures different bacterial strains as monocultures, no more than 3 different beneficial bacterial strains are cultivated as monocultures, no more than 2 different beneficial bacterial strains are cultivated as monocultures, each single In culture, only one beneficial strain of bacteria is cultivated.

The monoculture beneficial bacterial strains of this aspect of the invention may belong to a single species or to multiple species. Preferably, the cultivated beneficial bacterial strain belongs to one bacterial species. In another embodiment, multiple species of beneficial bacteria are grown as a single culture. Preferably, no more than 10 different species of beneficial bacteria are grown as a single culture, no more than 9 different species of beneficial bacteria are grown as a single culture, no more than 8 different species of beneficial bacteria are grown as a single culture. cultured as one culture, culturing up to 7 different species of beneficial bacteria as a single culture, culturing up to 6 different species of beneficial bacteria as a single culture, culturing up to 5 different species of beneficial bacteria no more than 4 different species of beneficial bacteria as monocultures, no more than 3 different species of beneficial bacteria as monocultures, no more than 2 different species of beneficial bacteria Different species of beneficial bacteria are cultivated as single cultures, and in each single culture only one beneficial bacterial strain is cultivated.

In one embodiment, beneficial bacteria improve human health when ingested. In another embodiment, beneficial bacteria are used in industry to produce products that are useful to humans (eg, methane, petroleum, pesticides, etc.). In another embodiment, beneficial bacteria are used in the food industry. In another embodiment, beneficial bacteria are used for silage inoculum. In yet another embodiment, the beneficial bacteria are used in agriculture to help grow plants. In yet another embodiment, beneficial bacteria are used for bioremediation.

In one embodiment, beneficial bacteria are probiotic bacteria.

As used herein, the term "probiotic bacteria" refers to live bacteria that confer a health benefit to the host (eg, human) when administered in appropriate amounts.

The major mechanisms of probiotic action include suppression of intestinal pathogens through the production of lactic acid, hydrogen peroxide and bacteriocins, adhesion site sealing, nutrient competition and regulation of the immune system, including inflammation suppression. Competitive exclusion of pathogens can be found. These include alleviation of lactose intolerance, anabolic cholesterol reduction, maintenance of normal intestinal flora and suppression of dysbiotic toxin production, degradation of toxin receptors in the intestine, maintenance of normal intestinal pH, and improvement of intestinal motility. It also provides benefits to the host, such as increasing and helping to maintain the integrity of intestinal permeability.

In one embodiment, the beneficial bacteria belong to the order Lactobacillales (commonly known as lactic acid bacteria (LAB)). These bacteria are Gram-positive, low GC, acid-tolerant, generally non-spore-forming, non-respiring, rod-shaped or cocciform bacteria that share metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end product of carbohydrate fermentation.

Preferably, the beneficial bacteria of the order Lactobacillales grow (and are generally cultured) in MRS agar (MRS).

Exemplary contemplated genera of the order Lactobacillales include, but are not limited to, Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Aerococcus, Carnobacterium, Enterococcus, Oenococcus, SporoLactobacillus, Tetragenococcus, Vagococcus, and Weissella.

According to one preferred embodiment, the beneficial bacterium of this aspect of the invention belongs to the genus Lactobacillus. Exemplary species of Lactobacillus contemplated by the present invention include L. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius, L. amylolyticus, L. amylophilus , L. amylotrophicus, L. amylovorus, L. animalis, L. antri, L. apodemi, L. aviarius, L. bifermentans, L. brevis, L. buchneri, L. camelliae, L. casei, L. catenaformis, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. coryniformis, L. crispatus, L. crustorum, L. curvatus, L. delbrueckii subsp. bulgaricus, L. delbrueckii subsp. lactis, L. dextrinicus, L. diolivorans, L. equi, L. equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fornicalis, L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. ghanensis, L. hilgardii, L. homohiochii, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. johnsonii, L. kalixensis, L. kefiranofaciens, L. kefiri, L. kimchii, L. kitasatonis, L. kunkeei, L. leichmannii, L. lindneri, L. malefermentans, L. mali, L. manihotivorans, L. mindensis, L. mucosae , L. murinus, L. nagelii, L. namurensis, L. nantensis, L. oligofermentans, L. oris, L. panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracasei, L. paracollinoides, L. parafarraginis, L. parakefiri, L. paralimentarius, L. paraplantarum, L. pentosus, L. perolens, L. plantarum, L. pontis, L. protectus, L. psittaci, L. rennini, L. reuteri, L. rhamnosus , L. rimae, L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L. salivarius, L. sanfranciscensis, L. satsumensis, L. secaliphilus, L. sharpeae, L. siliginis, L. spicheri, L. suebicus, L. thailandensis, L. ultunensis, L. vaccinostercus, L. vaginalis, L. versmoldensis, L. vini, L. vitulinus, L. zeae and L. zymae. not.

In one particular embodiment, the Lactobacillus species is L. plantarum.

Beneficial bacteria of this aspect of the invention are capable of producing fermentation products. Examples of fermentation products include prebiotics, biofuels, methanol, ethanol, propanol, butanol, alcohol fuels, proteins, recombinant proteins, vitamins, amino acids, organic acids (e.g. lactic acid, propionic acid, acetic acid, succinic acid , malic acid, glutamic acid, aspartic acid and 3-hydroxypropionic acid), enzymes, antigens, antibiotics, organic chemicals, bioremediation treatments, preservatives and metabolites.

Thus, beneficial bacteria can be genetically modified to express beneficial polypeptides.

Beneficial polypeptides can be intracellular (eg, cytoplasmic), transmembrane or secreted polypeptides. Heterologous production of proteins is widely employed in research and industrial settings, for example, in the production of therapeutics, vaccines, diagnostics, biofuels, and numerous other applications of interest. Exemplary therapeutic proteins that can be produced using the compositions and methods of the invention include certain natural and recombinant human hormones (e.g., insulin, growth hormone, insulin-like growth factor 1, follicle-stimulating hormone). and chorionic gonadotropin), hematopoietic proteins (e.g. erythropoietin, C-CSF, GM-CSF and IL-11), thrombotic and hemostatic proteins (e.g. tissue plasminogen activator and activated protein C), Non-limiting examples include immunological proteins (eg interleukins), antibodies and other enzymes (eg deoxyribonuclease I). Exemplary vaccines that can be produced by the compositions and methods of the present invention include a variety of influenza viruses (e.g., types A, B and C, and in the case of influenza virus type A, H5N2, H1N1, H3N2, etc.). types of various serotypes), HIV, hepatitis viruses (eg, hepatitis A, B, C or D), Lyme disease and human papillomavirus (HPV). Examples of heterologous protein diagnostic agents include, but are not limited to, secretin, thyroid stimulating hormone (TSH), HIV antigen and hepatitis C antigen.

Proteins or peptides produced by heterologous polypeptides include, but are not limited to, cytokines, chemokines, lymphokines, ligands, receptors, hormones, enzymes, antibodies and antibody fragments, and growth factors. Non-limiting examples of receptors include TNF type I receptor, IL-1 receptor type II, IL-1 receptor antagonist, IL-4 receptor and any chemically or genetically modified Soluble receptors are included. Examples of enzymes include acetylcholinesterase, lactase, activated protein C, factor VII, collagenase (for example sold by Advance Biofactories Corporation under the name Santyl); (for example sold by Genzyme under the name Fabrazyme). agalsidase-beta; dornase-alpha (eg sold by Genentech under the name Pulmozyme); alteplase (eg sold by Genentech under the name Activase); PEG (eg sold by Enzon under the name Oncaspar) asparaginase (eg sold by Merck under the name Elspar); and imiglucerase (eg sold by Genzyme under the name Ceredase). Examples of specific polypeptides or proteins include granulocyte macrophage colony stimulating factor (GM-CSF), granulocyte colony stimulating factor (G-CSF), macrophage colony stimulating factor (M-CSF), colony stimulating factor (CSF) , interferon beta (IFN-beta), interferon gamma (IFN gamma), interferon gamma-inducing factor I (IGIF), transforming growth factor beta (IGF-beta), RANTES (regulated upon activation, normal T-cell expressed and presumably secreted ), macrophage inflammatory proteins (e.g. MIP-1-alpha and MIP-1-beta), Leishmania elongation initiation factor (LEIF), platelet-derived growth factor (PDGF), tumor necrosis factor (TNF), growth factors such as , epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-2 (NT- 2), neurotrophin-3 (NT-3), neurotrophin-4 (NT-4), neurotrophin-5 (NT-5), glial cell-derived neurotrophic factor (GDNF), ciliary nerve Non-limiting examples include trophic factor (CNTF), TNF alpha type II receptor, erythropoietin (EPO), insulin and soluble glycoproteins such as gp120 and gp160 glycoproteins. The gp120 glycoprotein is the human immunodeficiency virus (WIV) envelope protein and the gp160 glycoprotein is the known precursor of the gp120 glycoprotein. Other examples include secretin, nesiritide (human B-type natriuretic peptide (hBNP)) and GYP-I.

Contemplated bacteria for expression of human interferon beta 1b include, for example, E. coli.

Contemplated bacteria for expression of human interferon gamma include, for example, E. coli.

Contemplated bacteria for expression of human growth hormone include, for example, E. coli.

Contemplated bacteria for expression of human insulin include, for example, E. coli.

Contemplated bacteria for expression of interleukin II include, for example, E. coli.

According to one particular embodiment, the beneficial polypeptide is an antibody (eg Humira, Remicade, Rixan, Enbrel, Avastin, Herceptin).

Bacteria contemplated for expression of antibodies include, for example, E. coli, Bacillus brevis, Bacillus subtilis and Bacillus megaterium.

Other beneficial bacteria contemplated by the present invention include those used as bacterial vaccines. Exemplary vaccines contemplated by the present invention include Vivotiv Veruna vaccine (typhoid vaccine, live), Prevnar13 (pneumococcal 13-valent vaccine), Menactra (meningococcal conjugate vaccine), ActHIB (haemophilus b conjugate vaccine). (prp-t) vaccine), Bexsero (meningococcal group B vaccine), Biothrax (adsorbed anthrax vaccine), Hiberix (haemophilus b conjugate (prp-t) vaccine), HibTITER (haemophilus b conjugate (hboc) vaccine) ), liquid Pedvax HIB (haemophilus b conjugate (prp-omp) vaccine), MenHibrix (haemophilus b conjugate (prp-t) vaccine/meningococcal conjugate vaccine), Menomune A/C/Y/W-135 ( meningococcal polysaccharide vaccine), Menveo (meningococcal conjugate vaccine), Pneumovax23 (pneumococcal 23-valent vaccine), Prevnar (pneumococcal 7-valent vaccine), Te Anatoxal Berna (tetanus toxoid), Adsorbed tetanus toxoid (tetanus toxoids), TheraCys (bcg), Tice BCG (bcg), Trumenba (meningococcal group B vaccine), Typhim Vi (typhoid vaccine, inactivated), Vaxchora, cholera vaccine, live and Vivotiv Veruna (typhoid vaccine, live) include, but are not limited to.

Other contemplated beneficial bacteria are those useful in bioremediation. Such remediations include heavy metals, chemicals, radiation and hydrocarbon pollution.

Examples of bacteria that can be used for bioremediation are listed below.

Pseudomonas putida: Pseudomonas putida is a Gram-negative soil bacterium involved in the bioremediation of toluene, a component of paint diluents. It can also decompose petroleum refinery product naphthalene in contaminated soil.

Dechloromonas aromatica: Dechloromonas aromatica are rods capable of oxidizing aromatic compounds including benzoic acid, chlorobenzoic acid and toluene, coupling the reaction with the reduction of oxygen, chlorate or nitrate. It is also the only organism capable of anaerobically oxidizing benzene. D. aromatic is particularly useful for the in situ bioremediation of this material due to its high propensity for benzene contamination, especially in ground and surface waters.

Nitrifying and denitrifying bacteria: Industrial bioremediation is used for wastewater purification. Most treatment systems rely on the activity of microorganisms to remove unwanted inorganic nitrogen compounds (ie ammonia, nitrites, nitrates). Nitrogen removal is a two-step process involving nitrification and denitrification. During nitrification, ammonium is oxidized to nitrites by organisms such as Nitrosomonas europaea. Nitrites are then further oxidized to nitrates by microorganisms such as Nitrobacter hamburgensis. Under anaerobic conditions, the nitrates produced during ammonium oxidation are used as final electron acceptors by microorganisms such as Paracoccus denitrificans. The result is _N2 gas. Through this process, ammonium and nitrates, two contaminants responsible for eutrophication of natural waters, are treated.

Deinococcus radiodurans: Deinococcus radiodurans is a radiation-tolerant extremophile bacterium genetically engineered for the bioremediation of solvents and heavy metals. Engineered strains of Deinococcus radiodurans have been shown to degrade ionic mercury and toluene in mixed radioactive waste environments.

Under anaerobic conditions, the nitrates produced during ammonium oxidation are used as final electron acceptors by microorganisms such as Paracoccus denitrificans. The result is dinitrogen gas. Through this process, ammonium and nitrates, two contaminants responsible for eutrophication of natural waters, are treated.

Methylibium petroleiphilum: Methylibium petroleiphilum (officially known as PM1 strain) is a bacterium capable of bioremediation of methyl tert-butyl ether (MTBE). PM1 degrades MTBE by using pollutants as the sole carbon and energy source.

Alcanivorax borkumensis: Alcanivorax borkumensis is a marine bacillus that consumes hydrocarbons, such as those present in fuel, and produces carbon dioxide. It grows rapidly in oil-contaminated environments and was used to help remove more than 830,000 gallons of oil from the Deepwater Horizon oil spill in the Gulf of Mexico. Other contemplated bacteria that can be used to remove oil include Colwellia and Neptuniibacter.

As noted above, the method of this aspect of the invention contemplates culturing beneficial bacteria together with biofilm-forming bacteria.

As used herein, the term "biofilm" refers to a population of bacteria contained (eg, embedded or encapsulated) in a matrix of extracellular polymeric material produced by the population of bacteria. In general, bacteria exhibit different phenotypes in terms of growth rate and gene transcription when present in biofilms compared to freely migrating planktonic bacteria. Examples of extracellular polymeric substances that may be present in biofilms include exopolysaccharides (such as those synthesized by products of the epsA-O operon) and amyloid fibrils (such as those encoded by the tapA-sipW-tasA operon). ). Substrates therefore generally include extracellular DNA and proteins as well as carbohydrates.

It is understood that biofilm-producing bacteria can also be beneficial bacteria.

Biofilm-producing bacteria are generally of a different order and/or genus than the beneficial bacteria that are incorporated into biofilms. Thus, biofilm-producing bacteria and beneficial bacteria can be different strains, species, genera and/or orders.

Preferably, the biofilm-forming bacteria are non-pathogenic to humans (ie, do not cause physical harm or disease).

Any number of strains of biofilm-producing bacteria can be cultured in the co-cultivation of this aspect of the invention. In one embodiment, no more than 500 different strains of biofilm-forming bacteria are cultured as a single culture, no more than 250 different strains of biofilm-forming bacteria are cultured as a single culture, and no more than 100 different strains of biofilm-forming bacteria are cultured as a single culture. culturing biofilm-forming bacteria as a monoculture, culturing 80 or less different strains of biofilm-forming bacteria as a monoculture, culturing 70 or less different strains of biofilm-forming bacteria as a monoculture. , culturing up to 60 different strains of biofilm-producing bacteria as a monoculture, culturing up to 50 different strains of biofilm-producing bacteria as a monoculture, and culturing up to 40 different strains of biofilm-producing bacteria as a monoculture. Cultivated as a monoculture, no more than 30 different strains of biofilm-producing bacteria were cultivated as a monoculture, no more than 20 different strains of biofilm-producing bacteria were cultivated as a monoculture, no more than 10 different strains of biofilm-forming bacteria as a monoculture, no more than 9 different strains of biofilm-forming bacteria as a monoculture, no more than 8 different strains of biofilm-forming bacteria as a monoculture and culturing no more than 7 different strains of biofilm-producing bacteria as a monoculture, culturing no more than 6 different strains of biofilm-producing bacteria as a monoculture, and no more than 5 different strains of biofilm-producing bacteria. culturing bacteria as monocultures, culturing no more than 4 different strains of biofilm-forming bacteria as monocultures, culturing no more than 3 different strains of biofilm-forming bacteria as monocultures, culturing no more than 2 different strains of biofilm-forming bacteria as single cultures, or only one strain of biofilm-forming bacteria in each single culture.

A single culture of biofilm-forming bacterial strain of this aspect of the invention may belong to one species or may belong to multiple species. Preferably, the biofilm-forming bacterial strains of the culture belong to a single bacterial species. In another embodiment, multiple species of biofilm-forming bacteria are cultured as a single culture. Preferably, no more than 10 different species of biofilm-producing bacteria are cultivated as a single culture, no more than 9 different species of biofilm-producing bacteria are cultivated as a mono-culture, and no more than 8 different species of biofilm-producing bacteria are cultivated as a monoculture. culturing the bacteria as a monoculture, culturing no more than 7 different species of biofilm-forming bacteria as a monoculture, culturing no more than 6 different species of biofilm-forming bacteria as a monoculture, and no more than 5 different species of biofilm-forming bacteria as a monoculture, no more than 4 different species of biofilm-forming bacteria as a monoculture, no more than 3 different species of biofilm-forming bacteria as a monoculture two or fewer different species of biofilm-forming bacteria as single cultures, or only one biofilm-forming bacterium in each single culture.

In one embodiment, the biofilm-producing bacterium belongs to the genus Bacillus.

As used herein, "Bacillus genus" includes B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, All members known to those skilled in the art are included, including but not limited to B. coagulans, B. circulans, B. lautus and B. thuringiensis. It should be recognized that the genus Bacillus continues to undergo taxonomic reorganization. Thus, this genus is intended to include reclassified species including, but not limited to, organisms such as B. stearothermophilus, now referred to as "Geobacillus stearothermophilus." The production of resistant endospores in the presence of oxygen is considered a defining feature of the genus Bacillus, a feature that has recently been named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus and Paenibacillus. , Salibacillus, Thermobacillus, Ureibacillus and Virgibacillus.

In one embodiment, the biofilm-forming bacterium is a B. subtilis species.

Exemplary strains of B. subtilis contemplated by the present invention include, but are not limited to, B. subtilis MS1577 and 127185/2 (MS302; dairy isolate) and NCIB3610.

Exemplary strains of B paralicheniformis contemplated by the present invention include, but are not limited to, B. paralicheniformis MS303 and B. paralicheniformis S127.

Exemplary strains of B. licheniformis contemplated by the present invention include, but are not limited to, B. licheniformis MS310 and B. licheniformis MS307.

According to one particular embodiment, the biofilm-forming bacteria do not comprise B. cereus species.

To generate co-cultures, generally both the beneficial culture and the biofilm-producing culture are cultured separately to generate a starter culture. Starter culture media and conditions are generally chosen to optimize the growth of each of the bacteria.

Contemplated starter cultures include dried starter cultures, dehydrated starter cultures, frozen starter cultures or concentrated starter cultures.

The starter culture is grown for at least 2, 4, 8, 12 hours until a sufficient amount of bacteria has grown.

According to one particular embodiment, the method comprises a method of co-cultivation, wherein the beneficial bacterium is of the genus Lactobacillus (e.g., L. plantarum species) and the biofilm-forming bacterium is of the genus Bacillus (e.g., B. subtilis species).

Methods for co-cultivating beneficial bacteria with biofilm-forming bacteria are chosen to allow growth of both types of microorganisms and incorporation of both microorganisms into biofilms.

In one embodiment, the co-cultivation takes place in (or on) a growth substrate commonly used for culturing beneficial bacteria. Growth substrates may be solid or liquid media. Preferably, the co-culture is shaken during culturing.

Examples of growth substrates that can be used to culture bacteria include, but are not limited to, MRS medium, LB medium, TBS medium, yeast extract, soy peptone, casein peptone and meat peptone.

Further examples of media are listed in Table 1 below.

Thus, for example, if the beneficial bacterium is of the genus Lactobacillus (e.g., L. plantarum sp.) and the biofilm-forming bacterium is of the genus Bacillus (e.g., B. subtilis sp.), the co-culture may be LBGM medium, milk medium or MRS. It can be done in a growth substrate containing medium. Alternative media that can be used to generate co-cultures of the invention include MSgg minimal medium (Shemesh, M., et al (2010). J Bacteriol 192, 6352-6356); lactose-enriched LB medium : Duanis-Assaf D., et al (2016) Front. Microbiol. 6:1517; Butyrate-added LB medium: Pasvolsky R., et al., Int. J. Food Microbiol.181C: 19-27. In general, culture conditions are chosen that promote the incorporation of both different bacteria into the biofilm.

The inventors have identified specific components of growth media that are important for biofilm formation of bacteria of the genus Bacillus (eg, B. subtilis species). See FIG. Thus, we propose that the medium used to co-cultivate beneficial bacteria with Bacillus bacteria contains manganese. In another embodiment, the medium contains dextrose. In yet another embodiment, the medium used for co-cultivation contains both manganese and dextrose.

Thus, according to another aspect of the invention, there is provided a method of selecting an agent or culture conditions favorable for the preparation of a bacterial composition, comprising: co-cultivating under or under culture conditions to produce a biofilm comprising beneficial bacteria and biofilm-forming bacteria, wherein the agent or culture conditions alter the properties of the biofilm to the preparation of the bacterial composition. A method is provided that is indicative of an advantage to

Exemplary modifiable conditions for co-cultivation include the nature of the surface on which the culture is performed (e.g. surface chemistry of the solid surface, including but not limited to functional groups, electrostatic charges, coatings; grooves, Surface roughness, surface topography, including but not limited to cavities, ridges, holes, hexagonal packing (HP) columns, equilateral triangles enclosed by HP columns, and Sharklet topography). The solid surface can be of defined shape and/or topography to facilitate encapsulation/uptake of beneficial bacteria into the biofilm. Additionally, the solid surface can have a distinct shape and/or topography to facilitate the formation of biofilms of a particular thickness. Other morphological patterns contemplated by the present invention are described in Graham and Cady, Coatings, 2014, 4, pages 37-59, the contents of which are incorporated herein by reference.

Exemplary solid surfaces on which culture can be performed include substrates ranging from various polymeric materials (silicone, polystyrene, polyurethane and epoxy resins) to metals and metal oxides (silicon, titanium, aluminum, silica and gold). is mentioned. Fabrication techniques (soft lithography and double casting molding techniques, microcontact printing, e-beam lithography, nanoimprint lithography, photolithography, electrodeposition, etc.) can be performed on these materials to alter the topography of solid surfaces.

Other conditions of co-cultivation that can be altered include, but are not limited to, environmental parameters such as pH, nutrient concentration, ratio of beneficial bacteria to biofilm-forming bacteria, and temperature.

In one embodiment, co-cultivation is performed in a bioreactor.

As used herein, the term "bioreactor" refers to a device adapted to support the biofilms of the invention.

A bioreactor generally includes one or more biofilm supports for biofilm formation thereon, the supports adapted to provide a significant surface area to promote biofilm formation. It is what was done. A bioreactor in the present invention can be adapted for continuous processing.

It should be appreciated that when biofilms are generated in a bioreactor system, co-cultivation conditions can be altered by altering the microfluidics (eg, shear stress) of the system.

As noted above, agents or conditions are selected that produce beneficial changes in biofilm properties. In one embodiment, the property is the amount of biofilm. In one embodiment, the property is biofilm thickness. In another embodiment, the property is biofilm density. In yet another embodiment, the property is the rate at which biofilms form. In yet another embodiment, the property is the amount of beneficial bacteria incorporated into the biofilm. In yet another embodiment, the property is temperature and/or pH resistance.

In yet another embodiment, the property is the amount of beneficial bacteria released from the biofilm over time. This may be of particular relevance when controlled release of beneficial bacteria is required. For example, it may be advantageous to load the biofilm with bacteria that are beneficial for skin, scalp or tooth applications and to select the rate of release of beneficial bacteria from the biofilm such that maximum therapeutic effect is obtained.

The present inventors have now found that when the pH of the growth substrate is increased above 6, bacteria that utilize the KinD-SpoOA pathway (e.g., Bacillus genus such as B. subtilis species), when cultured in MRS medium, produce biofilms. I found that it becomes easier to be taken into.

In one embodiment, a beneficial bacterium of the genus Lactobacillus (e.g., L. plantarum spp.) and the genus Bacillus (e.g., Lactobacillus sp.) performed in or on LBGM medium, milk medium or MRS medium (especially MRS medium). For example, co-cultivation with biofilm-forming bacteria (B. subtilis spp.) yields pH 6.5-9, 6.5-8, 6.5-7.5, 6.8-9, 6.8-8 , 6.8-7.5.

In one particular embodiment, the biofilm-forming bacterium is neither B. subtilis MS1577 nor 3610 when the co-cultivation is performed in milk medium.

The co-cultivation of this aspect of the invention can be performed in the presence of additional agents that help increase bacterial growth and/or promote biofilm formation. Such agents include, for example, acetoin.

The amount of acetoin and timing of addition can be varied to promote optimal biofilm formation. In one embodiment, about 0.01-5% acetoin is used. In another embodiment, about 0.01-4% acetoin is used. In another embodiment, about 0.01-3% acetoin is used. In another embodiment, about 0.01-2% acetoin is used. In another embodiment, about 0.01-1% acetoin is used. In another embodiment, about 0.01-0.5% acetoin is used.

We therefore contemplate cultures, biofilms and media containing Bacillus bacteria that contain acetoin. In one embodiment, the medium is one shown in Table 1 (eg, LB medium).

In one embodiment, about 0.05-5% acetoin is used. In another embodiment, about 0.05-4% acetoin is used. In another embodiment, about 0.05-3% acetoin is used. In another embodiment, about 0.05-2% acetoin is used. In another embodiment, about 0.05-1% acetoin is used. In another embodiment, about 0.05-0.5% acetoin is used.

In one embodiment, about 0.1-5% acetoin is used. In another embodiment, about 0.1-4% acetoin is used. In another embodiment, about 0.1-3% acetoin is used. In another embodiment, about 0.1-2% acetoin is used. In another embodiment, about 0.1-1% acetoin is used. In another embodiment, about 0.1-0.5% acetoin is used.

The co-culture of this aspect of the invention is grown for a period of time sufficient to produce a biofilm that incorporates both beneficial and biofilm-forming bacteria.

According to one embodiment, the co-culture is grown to the maximum stationary growth phase of beneficial bacteria, at which point it can be harvested for maximum biofilm production.

According to another embodiment, the co-culture can be grown to the maximum stationary growth phase of the biofilm-forming bacteria, at which point it can be harvested for maximal biofilm production.

Accordingly, the bacteria can be cultured for at least 3 hours, at least 6 hours, at least 12 hours, at least 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days or more. In one embodiment, the bacteria are not cultured for longer than 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.

Once a sufficient amount of beneficial bacteria have grown (and become encapsulated in the biofilm), the biofilm is harvested (ie, removed from the growth substrate).

After isolation from the growth substrate, the biofilm (and/or bacteria incorporated therein) can be dried (ie, dehydrated), frozen, spray-dried, or freeze-dried. Preferably, the biofilm is treated to maintain bacterial viability.

Therefore, according to another aspect of the invention, there is provided a bacterial composition obtainable by the method described herein.

Biofilm forming bacteria are present in the bacterial composition at 10 ³ to 10 ¹⁵ colony forming units per gram of bacterial composition (eg, probiotic composition).

The amount (by weight) of non-cellular material (eg, exopolysaccharide and/or amyloid fibrils) in the composition can be greater than the amount (by weight) of cellular material (eg, bacterial cells). For example, the weight of non-cellular material (e.g., exopolysaccharide and/or amyloid fibrils) in the composition is at least 5%, 10%, 20% greater than the weight of cellular material (e.g., bacterial cells) in the composition. , 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more.

The amount (weight) of non-cellular material (eg, exopolysaccharide and/or amyloid fibrils) in the composition can be less than the amount (weight) of cellular material (eg, bacterial cells). For example, the weight of cellular material (e.g., bacterial cells) in the composition is at least 5%, 10%, 20% greater than the weight of non-cellular material (e.g., exopolysaccharide and/or amyloid fibrils) in the composition. , 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more.

Thus, the weight ratio of non-cellular material (eg, exopolysaccharide):bacterial cells in the compositions described herein can be from 99:1 to 1:99. In some embodiments, the weight ratio of non-cellular material (eg, exopolysaccharide):bacterial cells in the compositions described herein can be from 99:1 to 50:50. In some embodiments, the weight ratio of non-cellular material (eg, exopolysaccharide):bacterial cells in the compositions described herein can be from 99:1 to 70:30.

In one embodiment the bacterial composition is a probiotic composition.

In some embodiments, the probiotic composition comprises about 10 ³ -10 ¹⁵ colony forming units (“CFU”) of biofilm-forming microorganisms per gram of final product. In some embodiments, the probiotic composition comprises from about 10 ⁴ to about 10 ¹⁴ CFU of biofilm-forming microorganisms per gram of final product. In some embodiments, the probiotic composition comprises from about 10 ⁵ to about 10 ¹⁵ CFU of biofilm-forming microorganisms per gram of final product. In some embodiments, the probiotic composition comprises about 10 ⁶ to 10 ¹¹ colony-forming units of biofilm-forming microorganisms per gram of final product. In some embodiments, the probiotic composition comprises about 10 ² to about 10 ⁵ colony forming units of biofilm-forming microorganisms per gram of final product.

Understand that at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the beneficial bacteria in the composition are viable (i.e. grow) want to be Further, at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the biofilm-forming bacteria in the composition are viable (i.e., grow) .

According to one particular embodiment, the bacterial composition is a probiotic composition.

Exemplary beneficial bacteria that may be present in the probiotic composition belong to the genus Lactobacillus (as described above).

A probiotic composition can include additional beneficial bacteria, such as Bifidobacterium spp. Contemplated Bifidobacterium species that may be present in the probiotic compositions of this aspect of the invention include Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolecentis, Bifidobacterium lactis and Bifidobacterium animalis, It is not limited to these. In some embodiments, the probiotic composition is from Lactobacillus species (e.g., Lactobacillus plantarum) and the following Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolecentis, Bifidobacterium lactis, and Bifidobacterium animalis. and at least two selected microorganisms.

In one embodiment, the bacterial compositions disclosed herein are in any form suitable for administering the composition to a mammalian subject. In some embodiments, the composition is in tablet, powder or liquid form. If provided as a powder, it is specifically contemplated to mix the powder with a suitable liquid (eg, liquid dairy products, fruit or vegetable juices, mixed fruit or vegetable juice products, etc.).

In some embodiments, the bacterial compositions disclosed herein are administered to the subject prior to, concurrently with, or after antibiotic administration. The co-cultivation conditions can be such that the biofilm that forms releases the beneficial bacteria within the body so that the beneficial bacteria are not exposed to the activity of the antibiotic.

In some embodiments, the bacterial compositions described herein are formulated for topical administration, eg, creams, gels, lotions, shampoos, rinses. The bacterial composition can be applied to the skin or scalp. Bacterial compositions can be useful in dental applications. In such applications, they can be administered gingivally.

In some embodiments, the compositions described herein are added to food. As used herein, the term "food" means any substance that contains nutrients that can be taken up by an organism, the uptake of which by the organism produces energy, promotes health and comfort, stimulates growth, and promotes life. It refers to something to maintain. As used herein, the term "fortified food" refers to food products that have been modified to contain compositions, including the compositions described herein, to provide health benefits beyond their basic function of supplying nutrients. / Refers to foods that provide benefits such as increased comfort and/or prevention/mitigation/treatment of disease.

A probiotic composition can be added to any food product. Exemplary foods include protein powders (shake meals), bakery items (cakes, cookies, crackers, bread, scones and muffins), dairy products (cheese, yogurt, custard, rice pudding, mousse, ice cream, frozen yogurt, desserts (including but not limited to sorbets, sorbets, ice creams, granitas and frozen fruit purees), spreads/margarines, pasta products and other grain products, meal replacements foods, nutrition bars, trail mixes, granola, beverages (including but not limited to smoothies, water or dairy drinks and soy-based beverages), and breakfast grain products such as oatmeal. . For beverages, the probiotic compositions described herein can be in solution and can be present as suspensions, emulsions or solids.

In one embodiment, the fortified food is a meal replacement. As used herein, the term "meal replacement" refers to fortified foods intended to be eaten in place of regular meals. Nutrition bars and beverages intended to be meal replacements are a type of meal replacement. The term refers to meal replacement products that are eaten as part of a weight loss or weight management regimen, e.g., are not intended to replace an entire meal by themselves, but may be combined with other such products to replace a meal. , or snacks intended for use in planning. These latter products generally have a caloric content of 50-500 kcal per serving.

In another embodiment the food product is a dietary supplement. As used herein, the term "dietary supplement" refers to substances taken by mouth, including "dietary ingredients" intended to supplement the diet. The term "dietary ingredient" includes any composition, including the probiotic composition described herein, as well as vitamins, minerals, herbal or other botanicals, amino acids, and enzymes, organ tissues, glandulars. , metabolites, and other substances.

In yet another embodiment the food product is a medical food product. As used herein, the term "medical food" is any food formulated to be consumed or administered under the full supervision of a physician and whose specific nutritional requirements are based on accepted scientific principles. means a food intended for use in a specific diet for a disease or condition established by

It has also been established that the addition of probiotic microorganisms to animal feed can improve animal efficiency and health. Examples include increased weight gain/feed intake ratio (feed efficiency), improved average daily weight gain, as described in U.S. Pat. Nos. 5,529,793 and 5,534,271. , improved milk yield, and improved milk composition by dairy cows. As reported in US Pat. No. 7,063,836, administration of probiotic organisms can also reduce pathogen incidence in cattle.

Therefore, according to another embodiment, the probiotic compositions described herein can be added to animal feed.

In one embodiment, the probiotic composition is designed to reduce the incidence and severity of diarrhea and/or reduce the overall health of the ruminal, cecal or gut bacteria (fermentors) throughout the feeding period. designed to be administered continuously or periodically to In this embodiment, the probiotic composition can be introduced into the animal's rumen, cecum and/or intestine.

In yet another embodiment, the probiotic composition described herein is added to a medicament or composition. Pharmaceutical compositions comprise a prophylactically or therapeutically effective amount of a composition described herein, and generally one or more pharmaceutically acceptable carriers or excipients (discussed below). .

The present disclosure contemplates formulation of the bacterial compositions described herein, which in some embodiments are powdered, tableted, encapsulated, or formulated for oral administration. . The composition can be provided as a pharmaceutical composition, a functional food composition (eg, a dietary supplement), or an additive for food or beverages as defined by the US Food and Drug Administration. The dosage form of the composition is not particularly limited. For example, solutions, suspensions, emulsions, tablets, pills, capsules, sustained-release preparations, powders, suppositories, liposomes, microparticles, microcapsules, sterile isotonic aqueous buffer solutions, etc. All are contemplated as suitable dosage forms.

Compositions generally comprise one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorants, flavoring agents, carriers, excipients. agents, buffers, stabilizers, solubilizers, commercially available adjuvants, and/or other additives known in the art.

Any pharmaceutically acceptable (i.e. acceptable to be sterile and non-toxic known in the art) liquid, semi-solid or Solid diluents can be used. Exemplary diluents include polyoxyethylene sorbitan monolaurate, magnesium stearate, calcium phosphate, mineral oil, cocoa butter and cocoa butter, methyl and propyl hydroxybenzoates, talc, alginates, carbohydrates, especially mannitol, alpha lactose, Non-limiting examples include anhydrous lactose, cellulose, sucrose, dextrose, sorbitol, modified dextran, gum arabic, and starch.

Pharmaceutically acceptable fillers include, for example, lactose, microcrystalline cellulose, dicalcium phosphate, tricalcium phosphate, calcium sulfate, dextrose, mannitol and/or sucrose. Salts including calcium triphosphate, magnesium carbonate and sodium chloride can also be used as fillers in pharmaceutical compositions.

Binders can be used to hold the composition together to form a hard tablet. Exemplary binders include materials of organic origin, such as gum arabic, tragacanth, starch, gelatin, and the like. Other suitable binders include methylcellulose (MC), ethylcellulose (EC) and carboxymethylcellulose (CMC).

In some embodiments, the fortified food product further comprises a bioavailability enhancer that acts to increase absorption of one or more adsorbable natural products by the body. Bioavailability-enhancing agents can be natural or synthetic compounds. In one embodiment, the fortified food product comprising the compositions described herein comprises one or more bioavailable bioactive natural products to enhance the bioavailability of one or more bioactive natural products. Further comprising an enhancer.

Natural bioavailability enhancers include ginger, caraway extract, pepper extract and chitosan. Active compounds in ginger include 6-gingerol and 6-shogaol. Caraway oil can also be used as a bioavailability enhancer (US Patent Application Publication No. 2003/022838). Piperine is a pepper-derived compound (Piper nigrum or Piper longum) that acts as a bioavailability enhancer (see US Pat. No. 5,744,161). Piperine is commercially available under the tradename Bioperine® (Sabinsa Corp., Piscataway, NJ). In some embodiments, the natural bioavailability enhancer is present in an amount from about 0.02% to about 0.6% by weight based on the total weight of the fortified food.

Examples of suitable synthetic bioavailability enhancers include, but are not limited to, surfactants composed of PEG esters. Such surfactants include those available under the trade names Gelucire®, Labrafil®, Labrasol®, Lauroglycol®, Pleurol Oleique® (Gattefosse Corp., Paramus, NJ). (manufactured by Abitec Corp., Columbus, Ohio) and Capmul® (manufactured by Abitec Corp., Columbus, Ohio).

The amount and dosing regimen of the composition is based on a variety of factors relevant to the purpose of administration, such as the age, sex, weight, hormone levels of the human or animal, or nutritional requirements of the human or animal. In some embodiments, the composition is administered to a mammalian subject in an amount of about 0.001 mg/kg body weight to about 1 g/kg body weight.

A typical dosing regimen may comprise multiple doses of the composition. In one embodiment, the composition is administered once daily. The composition can be administered to the individual at any time. In some embodiments, the composition is administered concurrently with, prior to, or at the time of ingestion of a meal.

In some embodiments, the bacterial composition of this aspect of the invention is formulated for agricultural use. Bacterial compositions can be added to agricultural carriers such as soil and plant growth media. Other agricultural carriers that can be used include fertilizers, vegetable oils, water retention agents, or combinations thereof. Alternatively, the agricultural carrier is solid such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed case, other plant and animal products, or combinations including granules, pellets or suspensions. can be Mixtures of any of the above ingredients are also contemplated as carriers such as, but not limited to, pesta (wheat flour and kaolin clay) in loam, sand or clay, agar or flour-based pellets. Preparations may be barley, rice or seeds, leaves, roots, plant elements, sugar cane bagasse, hulls or stems from grain processing, crushed plant material from construction site waste or from wood, paper, textiles or wood recycling. Food sources for cultured organisms can be included such as sawdust or other biological material such as fibrils. Other suitable formulations will be known to those skilled in the art.

In one embodiment, the agricultural formula includes fertilizer. Preferably, the fertilizer does not reduce the viability of the bacterial composition by more than 20%, 30%, 40%, 50%.

In some cases, agricultural formulations advantageously contain agents such as herbicides, nematicides, insecticides, plant growth regulators, rodenticides, nutrients and the like. Such agents are ideally compatible with the plants to which they are applied (eg, should not be detrimental to plant growth or health). Furthermore, the drug should ideally not raise safety concerns for human, animal or industrial use (e.g. no safety issues or the compound is sufficiently unstable that it is The amount of the compound contained in the derived plant product is negligible).

Agricultural formulations comprising the biofilms of the present invention generally contain a beneficial population of bacteria incorporated into the biofilms of the present invention from about 0.1 to 95% by weight, based on the wet weight of the biofilm, for example about 1%-90%, about 3%-75%, about 5%-60%, about 10%-50%. The formulation contains at least about 10 ² CFU or spores per ml of formulation; at least about 10 ³ CFU or spores per ml of formulation; at least about 10 ⁴ CFU or spores per ml of formulation; at least about 10 ⁵ CFU or spores per ml of formulation; It preferably contains at least about 10 ⁶ CFU or spores, or at least about 10 ⁷ CFU or spores per ml of formulation.

The inventors also contemplate that the agricultural compositions of this disclosure may be included in products that further include agents that promote plant growth.

The agent can be formulated together with the biofilm into a single composition. Alternatively, they can be packaged in separate but single containers.

Suitable agents are described above. Other suitable agents include fertilizers, pesticides (herbicides, nematicides, fungicides and/or insecticides), plant growth regulators, rodenticides and nutrients, as further described below.

In one embodiment, the plant growth promoting agent has no antimicrobial activity.

As used herein, "about" refers to ±10%.

The terms "comprises," "comprising," "includes," "including," "having," and conjugations thereof include, but are not limited to, means including but not limited to.

The term "consisting of" means "including and limited to."

The term "consisting essentially of" means that the composition, method or structure may contain additional components, steps and/or moieties. This is so long as the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed compositions, methods or structures.

As used herein, the singular forms "a," "an," and "the" refer to the plural unless the context clearly dictates otherwise. For example, reference to "a compound" or "at least one compound" includes multiple compounds and can include mixtures thereof.

Throughout this application, various embodiments of this invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and is not an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, the description of a range such as 1 to 6 includes not only partial ranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, but also individual numerical values within that range, For example, 1, 2, 3, 4, 5 and 6 are also specifically disclosed. This applies regardless of the size of the range.

Whenever a numerical range is indicated herein, it is intended to include any cited number (fractional or integral) within the indicated range. The phrases "range between" a first indicated number and a second indicated number and "range to" a first indicated number "to" a second indicated number are interchangeable herein. is intended to include the first index number and the second index number and all fractions and integers therebetween.

As used herein, the term "method" means a modality, means, technique or procedure for accomplishing a given task and can be used by practitioners in the fields of chemistry, pharmacology, biology, biochemistry and medicine. or can be readily developed by the practitioner from known modalities, means, techniques and procedures.

The term "treating" as used herein in connection with an abnormal activity, disease or condition includes arresting, substantially inhibiting, slowing or reversing the progression of the condition, reducing clinical or aesthetic symptoms of the condition. It includes substantial remission or substantial prevention of worsening of the clinical or aesthetic symptoms of the condition.

It should be understood that certain features of the invention that are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, several features of the invention, which are, for brevity, described in the context of a single embodiment, may also be combined separately or in any suitable subcombination or other suitable described embodiment. can also be provided for Certain features described in association with various embodiments should not be construed as essential requirements of that embodiment, unless that particular embodiment is inoperable without that element.

As noted above, the various embodiments and aspects of the invention described and claimed herein find experimental support in the following examples.

Reference is now made to the following examples which, together with the above description, illustrate but do not limit the invention.

In general, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are explained fully in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books , New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); 4,683,202, 4,801,531, 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds) , "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980). Available immunoassays are extensively described in the patent and scientific literature, for example, US Pat. , 850,578, 3,853,987, 3,867,517, 3,879,262, 3,901,654, 3,935,074, 3,984 , 533, 3,996,345, 4,034,074, 4,098,876, 4,879,219, 5,011,771 and 5,281,521 vol., "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual," CSHL Press (1996). All of which are incorporated herein by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All information contained therein is incorporated herein by reference.

Biofilm Formation Materials and Methods Strains and Growth Conditions: The probiotic strain used in this study was Lactobacillus plantarum. The strain is routinely grown in MRS (Man, Rogosa & Sharpe) broth or MRS broth solidified with 1.5% agar (Difco™). Bacillus subtilis wild strain NCIB3610 and its derivatives are generally grown in LB medium (10 g tryptone, 5 g yeast extract, 5 g NaCl per liter) in broth or 1.5% agar-solidified LB medium. culture. Prior to their use, L. plantarum and B. subtilis were grown on hard agar plates for 48 hours or overnight, respectively, both at 37°C. A starter culture of each strain was prepared using a single bacterial colony, L. plantarum was inoculated into 5 mL MRS broth and incubated for 8 hours without agitation, B. subtilis was inoculated into LB medium and incubated for 5 hours at 37 C. and 150 rpm until _OD600 reached approximately 1.5. For co-culture experiments, pH 7 MRS medium was used. This is because it was found to be effective in promoting biofilm formation by B. subtilis and suitable for co-cultivation of B. subtilis and probiotic lactic acid bacteria (LAB). B. subtilis cells were mixed with an equal volume of L. plantarum cells to a final concentration of 10 ⁸ cells/mL for each strain and then diluted 1:100 in MRS medium pH 7. Cells in mixed culture were incubated aerobically at 37° C., 50 rpm for 7-8 hours.

Single-species biofilms of B. subtilis were generated at 30° C. using MRS (Hy-lab) medium or MRS medium supplemented with LB medium at various ratios (1:5, 1:2, 5:1). bottom. To determine the optimal conditions for Bacillus strains to form biofilms, the pH of MRS medium was gradually increased from 6 to 8 using 1M NaOH. All strains used in this study are shown in Table 2. These are isogenic unless otherwise stated.

Analysis of Colony and Pellicle Biofilm Formation: For analysis of colony structure, 3 μL of starter culture was spotted onto MRS agar plates or control LB medium and incubated at 30° C. for 72 hours. For analysis of pellicle formation, starter cultures were diluted 1:100 in 3.5 mL of MRS broth or control LB medium in 12-well plates and incubated at 30° C. for 48 hours without agitation. Images were taken with a Zeiss Stemi2000-C microscope (Zeiss, Germany) equipped with an axiocam ERc5s camera.

β-galactosidase assay: Cells were plated on solid media, either LB medium, LB medium supplemented with MRS medium at various ratios (1:1, 1:5 and 5:1), or MRS medium adjusted to pH 7. were harvested from colonies grown at 30° C. and resuspended in phosphate-buffered saline (PBS) solution. Typical bundled long cell chains in biofilm colonies were disrupted by light sonication. Optical densities (OD) of cell samples were normalized to PBS as 1.0 at _OD600 . One milliliter of bacterial cell suspension was collected and analyzed according to standard procedures.

Growth curve analysis of L. plantarum growing in co-culture: Overnight cultures of B. subtilis and L. plantarum were grown to stationary phase in LB medium or MRS medium, respectively, and modified MRS broth with high pH (up to 7). Diluted 1:100 with 25 mL. Co-culture samples generated as described above were grown under aerobic conditions at 37° C. and 150 rpm for 8 hours. Monocultures of B. subtilis and L. plantarum were also prepared and used as control samples. Each hour, 1 mL was harvested from each culture for microbial count by colony forming unit (CFU) counting method. CFU counting was performed by making appropriate dilutions with PBS buffer and culturing these on MRS agar. Plates were incubated aerobically at 37° C. for 48 hours.

Visualization of biofilm-forming cells by confocal laser scanning microscopy (CLSM): MRS modified L. plantarum cells together with B. subtilis (YC161) that suppresses GFP or B. subtilis (YC189) that suppresses CFP. Grow by co-culture as described above in broth. A cell suspension of each bacterium grown as a monoculture was used as a control sample. One milliliter of each culture was harvested and centrifuged at 5000 rpm for 2 minutes. After removing the supernatant, the cells were washed with 1 mL of PBS buffer, then centrifuged (5000 rpm for 2 minutes) and resuspended in 100 μl of the same buffer. 5 μl of each sample was placed on a glass microscope slide and visualized using Nomarski differential interference contrast (DIC) in a transmitted light microscope.

Analysis by Scanning Electron Microscopy (SEM): Co-cultured cells grown as described above were plated on polylysine-coated glass slides overnight. The slides were then washed twice with DDW to remove unbound cells and media residues. Slides were exposed to 40 μl of 4% formaldehyde and incubated for 15 minutes at room temperature. The slides were washed again with DDW and analyzed by SEM.

Analysis of Viability After Cold Heat Treatment: Co-culture samples generated as described above were grown aerobically at 37° C. and 50 rpm for 7-8 hours. L. plantarum cells grown as monocultures were used as controls. The samples were subjected to load tests such as cold heat treatment. Samples were taken before and after treatment, sonicated to disrupt biofilm bundles (Time: 20 sec, Pulse: 10 sec, Pause: 5 sec, Amp: 30%) and CFU counts on MRS agar plates. did

Analysis of L. plantarum viability during migration in the in vitro digestive system: L. plantarum monocultures and co-cultures with B. subtilis cells to examine the viability of L. plantarum during migration in the gastrointestinal tract. A sample of the product was monitored for 4 hours using an in vitro digestion model (Minekus et al., 2014). To simulate the gastric phase of digestion, a 5 mL aliquot of each sample suspension was mixed 1:1 with simulated gastric fluid (SGF) to a final volume of 10 mL. Porcine pepsin (SIGMA P9700) was added to 2000 UmL ⁻¹ in the final digestion mixture, followed by CaCl ₂ to 0.075 mM in the final digestion mixture. The pH was lowered to 3.0 with 1M HCl and the sample was placed in a water bath with a magnetic stirrer for 2 hours at 37°C. Each sample was divided into two tubes of 5 mL each. 50 μl of PMSF (phenylmethylsulfonyl fluoride, SIGMA P7626) was added to one tube to stop the reaction and then examined for viability of L. plantarum. The other tube was used as the next digestive phase, the intestine. To simulate the intestinal phase of digestion, 2.5 mL of gastric chyme was mixed 1:1 with simulated intestinal fluid (SIF) to a final volume of 5 mL or less. 1M NaOH was added to neutralize the mixture to pH 7.0 and pancreatic enzymes were added to the digestion mixture such that the final mixture exhibited the following activities: porcine trypsin (SIGMA T0303) (100 UmL ⁻¹ ), bovine chymotrypsin. (SIGMA C4129) (25 UmL ⁻¹ ), porcine pancreatic α-amylase (SIGMA A3176) (200 UmL ⁻¹ ), porcine pancreatic lipase (SIGAM L3126) (2000 UmL ⁻¹ ). Additionally, bile salts (SIGMA T4009) were added to a final concentration of 10 mM in the final mixture, then the samples were incubated again for 2.5 hours. One milliliter of each sample was collected after gastric and intestinal phases and the number of viable L. plantarum cells was determined by CFU counting as described above.

Results Development of a system for interproliferation of B. subtilis and L. plantarum in co-culture Biofilms have previously been shown to be highly resistant to a variety of unfavorable environmental conditions through the production of extracellular matrix. (Friedman, Kolter, & Branda, 2005). Thus, we believe that the extracellular matrix produced by B. subtilis, a robust biofilm-forming bacterium, may be useful against other species, such as probiotic bacteria, in co-culture biofilm systems. hypothesized that it could provide enhanced protection. To this end, we developed a specialized medium that allows L. plantarum and B. subtilis to grow in co-culture. We found that these bacteria could be grown in co-culture by changing the pH of the MRS medium to pH 7. As shown in Figure 13, co-cultivation did not affect the growth of L. plantarum and B. subtilis (compared to their growth in pure cultures) and there was an antagonistic interaction between these bacteria in the given conditions. indicates no effect. Surprisingly, we found that modification of MRS medium promoted strong biofilm formation by B. subtilis (Fig. 2). Since B. subtilis appears to be sensitive to acidic pH, the pH of the MRS medium used for co-cultivation was gradually increased in order to find a suitable pH value for Bacillus growth. Increasing the pH from 6 to 8 proportionally increased the robustness of the biofilm phenotype of both colony and pellicle biofilms (Fig. 2). When the pH was adjusted to 6, weak growth was seen on solid MRS medium and no growth in liquid medium. When the pH was adjusted to 6.5, not only was bacterial growth observed on both solid and liquid MRS media, but surprisingly, initiation of biofilm formation on solid media was also observed. As the pH was increased to 7 and 8, a very robust biofilm phenotype was observed in both growth configurations. Next, the growth rate of B. subtilis was compared between pH 7 MRS medium and LB medium. As seen in FIG. 13, a smaller delay in the beginning of microbial growth was observed in MRS medium than in LB medium. However, a higher rate of growth was subsequently observed for B. subtilis cells in MRS medium than in LB medium.

Modified MRS medium promotes biofilm formation and substrate gene expression via the KinD-Spo0A pathway. LB medium (usually used) was supplemented with various amounts of MRS medium (1:1, 1:5 and 5:1). A direct proportional correlation between biofilm phenotype and increasing MRS medium concentration was demonstrated (Fig. 3). The effect of increasing MRS medium concentration on the expression of substrate genes by B. subtilis using the tapA and eps operons was also examined. This is because the products of these operons are the major components of the extracellular matrix. We found that tapA expression increased in proportion to MRS medium concentration in LB medium (FIGS. 4A-B). Expression of eps increased proportionally with the concentration of MRS medium up to 80% MRS medium, and a decrease in expression was detected at 100% (FIGS. 5A-B).

Next, we determined whether MRS medium induces biofilm formation via the Kin-SpoOA pathway previously reported for B. subtilis (Shemesh and Chai, 2013 Journal of Bacteriology, 2013, Vol 195 , No. 12 pages 2747-2754). They tested biofilm formation (ΔkinA, ΔkinB, ΔkinC, ΔkinD, ΔkinE, ΔkinAB, ΔkinCD, ΔspoOA, ΔepsΔtasA) or biofilm overproduction (ΔabrB) by different B. subtilis mutants. First, we determined the biofilm phenotype of mutants lacking histidine kinase, which is responsible for sensing environmental signals that induce biofilm formation. Although single mutants of either kinase showed no significant defect in the biofilm phenotype, we found that the ΔkinC and ΔkinD mutants showed a slight reduction in biofilm formation compared to controls (Figure 14). . However, the ΔkinCD double mutant completely abolished the biofilm phenotype (Fig. 5A). On the other hand, double mutation of ΔkinAB did not prevent biofilm formation, but some changes in biofilm phenotype were observed (for colony-type biofilms). Mutation of the key transcription factor spo0A and double mutation of eps and tasA fully abolished biofilm formation (Fig. 5A). Mutation of the transcriptional repressor ΔabrB did not result in a further increase in biofilm formation compared to control WT cells (Fig. 5B). This result again emphasizes the dramatic increase in substrate production during growth of B. subtilis WT cells in modified MRS medium.

To determine whether the biofilm-promoting effect of MRS medium is conserved among Bacillus species, another B. subtilis strain and another Bacillus species were tested. A biofilm promoting effect was seen as judged by wrinkled colonies (Fig. 6) and rigid floating pellicles (Fig. 7).

Co-culture growth of B. subtilis and L. plantarum results in dual-species biofilm development Using modified MRS medium, fluorescently labeled B. subtilis cells (YC161) constitutively expressing GFP were grown to L. Dual-species biofilms were examined by co-culturing with plantarum cells. The generated biofilms were visualized using CLSM. As seen in FIG. 8A (top), the biofilms that formed consisted of both fluorescent and non-fluorescent cells. L. plantarum cells were surrounded by B. subtilis cells that bound together to form biofilm-associated structures (bundles). This is further illustrated in FIG. 8B. Figure 8B shows a co-culture biofilm of B. subtilis and L. plantarum in LBGM medium.

Since biofilm formation in B. subtilis is dependent on extracellular matrix synthesis, we sought to determine whether extracellular matrix production occurs during dual-species biofilm development. Matrix gene expression levels in formed biofilms were measured using tapA-sipW-tasA (biofilm matrix protein in B. subtilis Analysis was performed using a transcriptional fusion (P _tapA -cfp) of the cfp gene encoding the cyan fluorescent protein (YC189) with the promoter of the operon responsible for the synthesis of the components. Notable CFP expression was observed, indicating that the tapA-sipW-tasA operon was activated and thus substrate production was induced in dual-species biofilms (FIGS. 8A-B, bottom panel). To determine whether L. plantarum cells could be surrounded by extracellular polymeric material derived from B. subtilis biofilm formation, dual-species biofilms were analyzed by SEM (FIGS. 9A-C). The resulting image (Fig. 9C) showed the formation of a three-dimensional heterogeneous structure of biofilm, in which L. plantarum cells appeared to be incorporated into the extracellular matrix produced by B. subtilis. Importantly, B. subtilis cells grown as a monoculture form also form biofilms characterized by homogeneous structures, with long filaments of cells connected by an extracellular matrix (Fig. 9A). In contrast, L. plantarum cells failed to form noticeable biofilms in monoculture. According to the above observations, the extracellular matrix produced by B. subtilis cells can be shared with L. plantarum cells, thus protecting them against environmental stress.

Dual-species biofilms facilitate survival of L. plantarum in hostile environments Determining whether substrates produced by B. subtilis in co-culture biofilms can protect L. plantarum against unfavorable environmental conditions To do so, we tested the survival of L. plantarum cells during heat treatment (conditions simulating industrial processes such as pasteurization) and cooling (conditions simulating storage conditions). For heat treatment pasteurization, L. plantarum cells grown in co-culture biofilms were subjected to heating at 63° C. for 1 and 3 minutes. For chilling treatments, L. plantarum cells grown in co-culture biofilms were stored at 4° C. for up to 21 days. L. plantarum cells grown in monoculture were used as controls. After heat treatment for 1 and 3 minutes, L. plantarum cells grown in co-culture biofilms showed approximately 1.25 Log CFU/mL and 1.06 Log CFU/mL increases in viable L. plantarum cell numbers, respectively, compared to controls. (FIGS. 10A-B). Moreover, the results of the cold treatment experiments showed that L. plantarum cells grown in co-culture biofilms were much better protected through storage conditions, with their viability ranging from about 0.44 to 0.89 Log CFU/mL. increased (FIGS. 10A-B).

Extracellular matrix produced during dual-species biofilm formation facilitates survival of L. plantarum during heat treatment Increased resistance of L. plantarum to unfavorable environmental conditions is facilitated by extracellular matrix To further demonstrate, co-cultures of L. plantarum and B. subtilis mutants, deficient in biofilm formation (ΔepsΔtasA) or overproducing biofilm substrate (ΔabrB), were generated. The co-culture was subjected to heat pasteurization. L. plantarum cells grown in monoculture and co-culture with wild-type B. subtilis were used as controls. As shown in FIG. 11A, L. plantarum cells grown together with cells of the ΔepsΔtasA double mutant had no significant difference in survival levels compared to L. plantarum grown in monoculture. However, there was a significant increase in viability of L. plantarum cells grown in co-culture with wild-type B. subtilis. Interestingly, there was an approximately 1.78 Log CFU/mL increase in viability of L. plantarum cells grown in the presence of the ΔabrB mutant cells compared to that of L. plantarum grown in monoculture. Admitted.

In another experiment, samples were grown in milk medium for 18 hours at 30°C and 20 rpm. They were then heat treated at 63° C. for 1-3 minutes. Control samples were not heat treated. The number of viable cells of L. plantarum was determined by the CFU method. *p<0.05. As shown in FIG. 11B, B. subtilis biofilms facilitate survival of L. plantarum upon heating in milk medium.

Extracellular matrix produced during dual-species biofilm formation facilitates survival of L. plantarum under conditions resembling the human digestive system To investigate the viability of L. plantarum during migration through the gastrointestinal tract Finally, the viability of L. plantarum cells was examined using an in vitro digestion model (Fig. 12). After 2 hours of incubation in simulated gastric conditions, an increase in viable cell concentration of approximately 0.86 Log CFU/mL over monocultured L. plantarum cells grew in co-cultured biofilms with B. subtilis. observed in L. plantarum cells. Cells were then incubated for 2 h under simulated intestinal conditions and an increase in viable cell concentration of approximately 0.9 Log CFU/mL compared to free L. plantarum viable cells was observed in biofilm-protected L. plantarum. found in cells.

Acetoin Promotes Biofilm Formation Foods are often rich in various food additives that can improve the organoleptic properties and organoleptic properties of the product. These additives include important small molecules such as acetoin that can improve the flavor of various foods. Acetoin is a neutral molecule that occurs widely in nature. Some microorganisms, higher plants, insects and higher animals have the ability to synthesize acetoin. These additives affect the physiology of many bacteria relevant to human health and can influence the development of multicellular populations of bacterial cells known as biofilms. Biofilm formation depends on the synthesis of an extracellular matrix that holds constituent cells together. In the prebiotic bacterium Bacillus subtilis, substrates are composed of two major components: exopolysaccharides synthesized by the products of the epsA-O operon and amyloid fibrils encoded by the tapA-sipW-tasA operon. have

Results As shown in Figures 15A-C, acetoin induces biofilm bundle formation in Bacillus subtilis. In the absence of acetoin, no biofilm formation is observed when grown in LB medium (Fig. 15A). Figures 16A-B show that acetoin induces colony-type biofilm formation in Bacillus subtilis. Transcription of the tapA operon responsible for substrate production in B. subtilis was shown to be highly upregulated by acetoin (FIGS. 17A-D).

These results indicate that B. subtilis cells develop into complex bundles during growth in the presence of acetoin. Cells express high levels of extracellular matrix components in response to acetoin that are important for biofilm formation.

The purpose of this experiment was to demonstrate the ability of NCIB3610 (isolated from soil) and 127185/2 (isolated from a dairy environment) to protect L. plantarum under harsh conditions during growth in a co-culture system. is to test

Materials and methods
The growth medium of choice for the B. subtilis and L. plantarum co-culture system was modified (pH adjusted) MRS medium.

Characterization of biofilm formation was performed using a stereomicroscope (for colonies) or a confocal laser scanning microscope (for bundled biofilms).

Experiments examining the viability of L. plantarum grown in co-culture biofilms with B. subtilis and exposed to low pH in an in vitro model of gastrointestinal migration were performed using the CFU method, as described above. .

Results Figures 18A-B are photographs showing biofilms produced from B. subtilis strains NCIB3610 and 127185/2, respectively.

Counts of L. plantarum surviving co-culture with NCIB3610 were higher than L. plantarum grown in mono-culture. This effect was enhanced when the cultures were shaken (Figure 19). Furthermore, the number of L. plantarum that survived co-cultures with NCIB3610 or 127185/2 under acidic conditions was 30-fold higher than monocultures grown under the same conditions (Figure 20).

To elucidate the components of MRS medium that are important for the induction of biofilm development, the contributions of the following components involved in colony-type biofilm formation by B. subtilis were analyzed: Mg ²⁺ , Mn ²⁺ , sodium acetate, phosphate. Dipotassium, dextrose, ammonium citrate. Interestingly, the most incomplete biofilm phenotype was observed in the absence of Mn2 ⁺ . B. subtilis was unable to form a grown pellicle and colony-type biofilm on MRS medium to which Mn ²⁺ was not added (Fig. 21). Notably, biofilms generated in the absence of dextrose showed some inhibition and had a wrinkled phenotype, whereas those generated in the absence of Mn2 ⁺ were completely flat. There was (Fig. 21). From these results, we concluded that the presence of ^Mn2+ in the MRS medium is most important for biofilm development by B. subtilis.

Although the invention has been described in relation to specific embodiments thereof, many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, all such alternatives, modifications and variations are intended to be included within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent and patent application were incorporated herein by specific and individual reference. is incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. Also, to the extent section headings are used, they should not necessarily be construed as limiting.

Claims (8)

A method for preparing a bacterial composition, comprising:
(a) a probiotic bacterium of the order Lactobacillales and a biofilm-forming bacterium of the genus Bacillus in a growth medium containing manganese under conditions to form a biofilm comprising said probiotic bacterium and said biofilm-forming bacterium; , co-cultured in vitro,
(b) preparing said bacterial composition by isolating said biofilm from said growth medium. 2. The method of claim 1 , wherein said biofilm-forming bacterium is a B. subtilis species. 2. The method of claim 1 , wherein said probiotic bacteria are L. plantarum species. The method of any one of claims 1-3 , wherein the growth medium comprises dextrose. A method according to any one of claims 1 to 3 , wherein said growth medium is selected from the group consisting of LB medium, LBGM medium, milk medium and MRS medium. 2. The method of claim 1, wherein said growth medium is LBGM medium, milk medium or MRS medium. 7. The method of claim 6 , wherein said conditions comprise pH 6.5-8. The method of any one of claims 1-7 , wherein the growth medium comprises an amount of acetoin that promotes biofilm formation .

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