Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/742—Spore-forming bacteria, e.g. Bacillus coagulans, Bacillus subtilis, clostridium or Lactobacillus sporogenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/66—Microorganisms or materials therefrom
  • A61K35/74—Bacteria
  • A61K35/741—Probiotics
  • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
  • A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/02—Nutrients, e.g. vitamins, minerals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/04—Preserving or maintaining viable microorganisms
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/20—Bacteria; Culture media therefor
  • C12N1/205—Bacterial isolates
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/07—Bacillus
  • C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
  • C12R2001/00—Microorganisms ; Processes using microorganisms
  • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
  • C12R2001/225—Lactobacillus
  • C12R2001/25—Lactobacillus plantarum

Definitions

• the present invention in some embodiments thereof, relates to methods of generating bacterial compositions, more particularly, but not exclusively, to probiotic compositions, those beneficial to the environment and those used in industry.
• probiotics Living microbial cells which are administered in adequate amounts, confer a beneficial physiological effect on the host, are known as "probiotics". Studies have shown therapeutic effects that probiotic bacteria can provide to the host in maintaining a healthy gut and controlling several types of gastrointestinal infections. Due to their perceived health benefits, probiotic bacteria have been increasingly incorporated into a variety of food and drink products during the last few decades. Some of the most common types of microorganisms used as probiotics are the lactic acid bacteria (LAB), which mainly belong to the genera Lactobacillus and Bifidobacterium. Both these genera are dominant inhabitants in the human intestine and have a long history of safe use and are considered as GRAS (generally recognized as safe).
• LAB lactic acid bacteria
• probiotics are usually available as dry bacterial powders prepared mainly by freeze drying which has been established as a procedure that may cause fatal injury to cells. Therefore, there is a need to develop novel technologies aimed to improve the survival of health-promoting bacteria during food production, as well as through the storage and ingestion processes in order to maintain delivery of probiotics to humans.
• biofilm In most natural ecosystems, bacteria prefer to grow in complex community of multicellular cells called biofilm and not as free-living (planktonic) cells. Biofilm mode of growth is preferable also for bacteria that inhabit the intestinal tract. Cells in a biofilm are bound together by an extracellular matrix that mainly consists of polysaccharides and other macromolecules such as proteins, DNA, lipids and nucleic acids, which are produced by the cells themselves. Interactions between the species embedded in the biofilm and their environment result in the formation of a complex structure, capable of resisting to environmental stress and exposure to antimicrobial agents. Thus, biofilm formation represents a strategy for persistence under unfavorable conditions in diverse environments.
• Bacillus subtilis a spore-forming nonpathogenic bacterium, which is characterized by its ability to produce a robust biofilm.
• Bacillus species principally B. subtilis, have gained recent interest as probiotic microorganism since they were shown to positively effect on host health status mainly by keeping a favorable balance of microflora in the gastrointestinal tract. Since B. subtilis spores are capable of surviving extreme pH conditions and low oxygen, high numbers of dormant but viable microbes may reach the lower intestine which may induce some beneficial effects through secretion of active substances. Furthermore, it was found that B.
• subtilis cells enhance growth and viability of lactobacilli spp., possibly through the production of catalase and subtilisin (Hosoi, Ametani, Kiuchi, & Kaminogawa, 2000). It has also been reported that T- polyglutamic acid produced by B. subtilis as part of an extracellular matrix could be used to improve the survival of probiotic bacteria during freeze drying (A. R. Bhat et al., 2013) and during storage (A. R. Bhat et al., 2015). Likewise, during simulated gastric juice which simulated the acidic conditions of the stomach (A. R. Bhat et al., 2015).
• a method of preparing a bacterial composition comprising:
• a bacterial composition obtainable according to the methods described herein.
• a food/feed product comprising the bacterial composition described herein.
• a method of improving or maintaining the health of a subject comprising administering to the subject a therapeutically effective amount of the probiotic composition described herein, thereby improving or maintaining the health of the subject.
• a method of selecting an agent or culturing condition which is advantageous for preparing a bacterial composition the method comprising co-culturing beneficial bacteria with biofilm-producing bacteria in a growth substrate in the presence of the agent or under the culturing condition, so as to generate a biofilm comprising the beneficial bacteria and the biofilm-producing bacteria, wherein a change in a property of the biofilm is indicative of the agent or culturing condition being advantageous for preparing the bacterial composition.
• the biofilm-producing bacteria are non-pathogenic bacteria.
• the biofilm-producing bacteria are of the bacillus genus.
• the biofilm-producing bacteria are of the B. subtilis species.
• the biofilm-producing bacteria are of the strain 127185/2.
• the growth substrate comprises manganese.
• the growth substrate comprises dextrose.
• the growth substrate comprises manganese.
• the beneficial bacteria are probiotic bacteria.
• the beneficial bacteria are genetically modified to express a therapeutic polypeptide.
• the probiotic bacteria is of the lactobacillales order.
• the biofilm-producing bacteria are of the B. subtilis species.
• the probiotic bacteria are of the L. plantarum species.
• the beneficial bacteria are used in bioremediation.
• the biofilm-producing bacteria express genes of the KinD-SpoOA pathway.
• the growth substrate comprises a growth medium.
• the growth medium is selected from the group consisting of LB, LBGM, milk and MRS.
• the biofilm-producing bacteria are of the bacillus genus and the beneficial bacteria are of the lactobaciUales order, the growth substrate is LBGM, milk or MRS.
• the growth substrate is MRS.
• the conditions comprise a pH of 6.8-
• the growth substrate comprises acetoin.
• the method further comprises dehydrating the biofilm following the isolating.
• the beneficial bacteria comprises no more than 50 bacterial species.
• biofilm-producing bacteria are a single species of biofilm-producing bacteria.
• At least 50 % of the bacteria in the composition are viable.
• the bacterial composition comprises no more than 50 bacterial species of beneficial bacteria.
• the bacterial composition comprises a single species of non-pathogenic bacteria.
• the bacterial composition is edible.
• the bacterial composition is a probiotic bacterial composition.
• the bacterial composition is formulated as a powder, a liquid or a tablet.
• the biofilm-producing bacteria are of the bacillus genus.
• the biofilm-producing bacteria are of the B. subtilis species.
• the beneficial bacteria are probiotic bacteria.
• the probiotic bacteria are of the lactobacillales order.
• the agent alters the pH of a medium of the system.
• FIGs. 1A-B are graphs comparing B. subtilis and L. plantarum growth in co-culture.
• the co-culture generation had no effect on L. plantarum and B. subtilis growth (compared to their growth in pure culture), indicating that there are no antagonistic interactions between these bacteria.
• FIG. 2 are photographs illustrating that modified MRS medium triggers biofilm formation by B. subtilis.
• the effect of the pH modification of MRS on B. subtilis NCIB3610 biofilm formation was analyzed using stereoscopic microscope.
• FIG. 3 are photographs illustrating that the combination of LB with MRS medium triggers biofilm development by B. subtilis.
• FIGs. 4A-B are graphs illustrating that the combination of LB with MRS medium triggers extracellular matrix production by B. subtilis. Increasing MRS concentration induces transcription of tapA-sipW-tasA (A) and epsA-0 (B) operons.
• FIG. 5A are photographs illustrating that the biofilm stimulating effect of MRS is regulated by the matrix synthesis and biofilm forming signaling pathway previously described in B. subtilis.
• Colony development and pellicle formation on MRS (pH 7) by the wild type (WT) and various mutant strains were compared.
• the strains used here were as follows: wild type (NCIB3610), AkinCD (RL4577), AkinAB (RL4573), AspoOA (RL4620), AepsAtasA (RL4566), AabrB (YC668).
• FIG. 5B are photographs illustrating that the effect of MRS in WT cells is comparable to the matrix overproducing mutant cells ⁇ AabrB) in B. subtilis.
• FIG. 6 are photographs illustrating that MRS induces colony biofilm formation in different Bacillus species.
• MRS (pH 7) medium strongly induced colony type biofilm formation of B. paralicheniformis MS303, B. licheniformis MS310, B. licheniformis S 127, B. subtilis
• FIG. 7 are photographs illustrating that MRS induces pellicle formation in different Bacillus species.
• MRS (pH 7) medium strongly induced pellicle formation of B. paralicheniformis MS303, B. licheniformis MS310, B. licheniformis S 127, B. subtilis MS 1577 and fi. cereus 10987.
• FIGs. 8A-B are images illustrating that B. subtilis produces extracellular matrix whilst forming a dual-species biofilm with L. plantarum.
• 8A CLSM images of co-culture biofilm of B. subtilis and L. plantarum in MRS pH 7 at 37 °C and 50 rpm. From left to right: images made using fluorescent light, Nomarski differential interference contrast (DIC) and merged image. Top panel shows the expression of fluorescently tagged B. subtilis cells constitutively express GFP. Bottom panel shows expression of matrix producing B. subtilis cells express CFP under the control of tapA promoter. In all images L. plantarum cells are not stained. 8B. CLSM images of co-culture biofilm of B. subtilis and L. plantarum in LBGM medium.
• DIC Nomarski differential interference contrast
• FIGs. 9A-C are SEM images of (A) B. subtilis cells, (B) L. plantarum cells and (C) dual species biofilm composed of B. subtilis and L. plantarum.
• FIGs. 10A-B are graphs illustrating that dual species biofilm facilitates survival of L. plantarum exposed to unfavorable conditions. Survival of L. plantarum cells in presence or absence (control) of B. subtilis biofilm were determined during (A) heat treatment at 63 °C 1 to 3 min (B) storage at 4 °C for 21 days. The values presented are the average of at least three independent experiments performed in duplicates. *p ⁇ 0.05
• FIGs. 11A-B are graphs illustrating that the extracellular matrix of B. subtilis facilitates increased survival of L. plantarum during heat treatment.
• A The effect of heat treatment at 63 °C for 3 min on WT B. subtilis and its derivatives, a mutant deficient in exopolysaccharide component and protein component of extracellular matrix ⁇ AepsAtasA) and a mutant deficient in a repressor of the matrix genes (AabrB; overproduces biofilm matrix) was tested. The results presented are the average of at least three independent experiments performed in duplicates. *p ⁇ 0.05.
• B The samples were grown in milk for 18 h at 30 °C, 20 rpm. Afterwards they were heat treated at 63 °C for 1 to 3 minutes. Control samples were not heat-treated. The number of viable L. plantarum cells was determined using CFU-method. *p ⁇ 0.05
• FIG. 12 is a graph illustrating that the presence of B. subtilis biofilm increases survival of L. plantarum during gastric and intestinal digestion in vitro (model system). Survival of L. plantarum cells in presence or absence (control) of B. subtilis biofilm were determined during gastro-intestinal digestion in vitro. The results presented are the average of three independent experiments performed in duplicates. *p ⁇ 0.05
• FIG. 13 is a graph of the growth curves of B. subtilis 3610NCIB in MRS (pH 7) and LB.
• FIG. 14 are photographs illustrating the effect of mutations in Histidine kinases on colony surface architecture and pellicle formation in MRS pH 7.
• FIGs. 16A-B are photographs illustrating that acetoin triggers the colony type biofilm formation by Bacillus subtilis
• FIGs. 17A-D are photographs illustrating that the transcription of the tapA operon responsible for the matrix production in B. subtilis is highly upregulated by acetoin.
• FIGs. 18A-B are photographs depicting the biofilm generated from the B. subtilis strains NCIB3610 and 127185/2 respectively.
• FIG. 20 is a graph illustrating the survival of L. plantarum grown in co-culture biofilm with B. subtilis in exposure to high acidity level.
• the sign '+' in the tested cultures indicates a growth with 50 rpm shaking, while the sign '-' indicates a growth without shaking at all.
• the co-cultures of L. plantarum and B. subtilis showed a lower decrease in the survival rates of L. plantarum (compared to the mono- culture of L. plantarum) in transition to an acidic environment as with as well as without shaking.
• FIG. 21 are photographs illustrating that Mn 2+ ions are involved in biofilm formation by
• B. subtilis in modified MRS Effects of exclusion of certain MRS medium components (Mg 2+ , Mn 2+ , sodium acetate, dipotassium phosphate, dextrose, ammonium citrate) on colony development and pellicle formation by the WT B. subtilis cells were observed.
• MRS medium components Mg 2+ , Mn 2+ , sodium acetate, dipotassium phosphate, dextrose, ammonium citrate
• the present invention in some embodiments thereof, relates to methods of generating bacterial compositions, more particularly, but not exclusively, to probiotic compositions, those beneficial to the environment and those used in industry.
• Bacteria are economically important as these microorganisms are used by humans for many purposes.
• the beneficial uses of bacteria include the production of traditional foods such as yoghurt, cheese, and vinegar; biotechnology and genetic engineering, producing substances such as drugs and vitamins; agriculture; fibre retting; production of methane; bioremediation and biological control of pests.
• probiotics are usually available as dry bacterial powders prepared mainly by freeze drying which has been established as a procedure that may cause fatal injury to cells.
• the present inventors co-cultured bacteria of the B. subtilis species together with the probiotic bacteria L. plantarum. They showed that under particular conditions the B. subtilis bacteria generated a biofilm in which the L. plantarum cells were incorporated within the extracellular matrix thereof (Figure 9 A).
• the biofilm-incorporated L. plantarum were shown to be both more heat-resistant and more cold-resistant, and further more acid-resistant than control, non-biofilm incorporated L. plantarum.
• biofilm-producing bacteria can be used to encapsulate a non-biofilm producing bacteria.
• the biofilm-producing bacteria serve as a protective carrier for the beneficial, non-biofilm producing bacteria.
• a method of preparing a bacterial composition comprising:
• bacteria refers to a prokaryotic microorganism, including archaea.
• the bacteria may be gram positive or gram negative.
• the bacteria may also be photosynthetic bacteria (e.g. cyanobacteria).
• waste bacteria refers to any bacteria that bring about a positive effect on human beings.
• the beneficial bacteria do not produce a biofilm when propagated as a monoculture in a growth medium under standard culturing conditions. In another embodiment, the beneficial bacteria do not produce a biofilm when propagated as a monoculture in a growth medium under culturing conditions that are optimal for their propagation.
• the beneficial-bacteria utilize the KinD-SpoOA pathway (for example express the genes histidine kinase kinD, spoOF, spoOB and/or spoOA) - 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.
• KinD-SpoOA pathway for example express the genes histidine kinase kinD, spoOF, spoOB and/or spoOA
• the beneficial bacteria may be one that is typically cultured in Man, Rogosa and Sharpe medium, MRS (solidified using agar or MRS broth).
• the beneficial bacteria should typically not prevent (i.e. antagonize) the biofilm-forming capability of the biofilm-generating bacteria (e.g. B. subtilis). Methods of determining whether bacteria have antagonistic activity towards other bacteria when cultured together are known in the art (see for example Figures 1A-B). In one embodiment, the beneficial bacteria are not soil bacteria.
• any number of strains of beneficial bacteria may be cultured in the co -culture of this aspect of the present invention.
• no more than 500 different strains of beneficial bacteria are cultured in a single culture
• no more than 250 different strains of beneficial bacteria are cultured in a single culture
• no more than 100 different strains of beneficial bacteria are cultured in a single culture
• no more than 90 different strains of beneficial bacteria are cultured in a single culture
• no more than 80 different strains of beneficial bacteria are cultured in a single culture
• no more than 70 different strains of beneficial bacteria are cultured in a single culture
• no more than 60 different strains of beneficial bacteria are cultured in a single culture
• no more than 50 different strains of beneficial bacteria are cultured in a single culture
• no more than 40 different strains of beneficial bacteria are cultured in a single culture
• no more than 30 different strains of beneficial bacteria are cultured in a single culture
• no more than 20 different strains of beneficial bacteria are cultured in a single culture
• the beneficial bacterial strains of a single culture of this aspect of the present invention may belong to a single species or may belong to multiple species.
• the beneficial bacterial strains of a culture may belong to a single species of bacteria.
• multiple species of beneficial bacteria are cultured on a single culture.
• no more than 10 different species of beneficial bacteria are cultured in a single culture, no more than 9 different species of beneficial bacteria are cultured in a single culture, no more than 8 different species of beneficial bacteria are cultured in a single culture, no more than 7 different species of beneficial bacteria are cultured in a single culture, no more than 6 different species of beneficial bacteria are cultured in a single culture, no more than 5 different species of beneficial bacteria are cultured in a single culture, no more than 4 different species of beneficial bacteria are cultured in a single culture, no more than 3 different species of beneficial bacteria are cultured in a single culture, no more than 2 different species of beneficial bacteria are cultured in a single culture only one species of beneficial bacteria is cultured per single culture.
• the beneficial bacteria when ingested promote the health of a human being.
• the beneficial bacteria are used in industry to generate a product that is useful for human beings (e.g. methane, petroleum, insecticide etc.).
• the beneficial bacteria are used in the food industry.
• the beneficial bacteria are used in a silage inoculant.
• the beneficial bacteria are used in agriculture to support the growth of plants.
• the beneficial bacteria are used in bioremediation.
• the beneficial bacteria are probiotic bacteria.
• probiotic bacteria refers to live bacteria which when administered in adequate amounts confer a health benefit on the host (e.g. human).
• enteric pathogens by the production of lactic acid, hydrogen peroxide and bacteriocins; competitive exclusion of enteric pathogens by blocking adhesion sites, competition for nutrients and modulation of the immune system, including inflammation reduction. They also provide benefits to the host, such as lactose intolerance alleviation; cholesterol decrease by assimilation, sustenance of the intestinal normal microbiota and dysbiosis ameliorating suppression of toxin production, degradation of toxin receptors in the intestine, preservation of normal intestinal pH, increase intestinal motility and help to maintain the integrity of the intestine permeability.
• 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 nonsporulating, non-respiring, either rod- or coccus-shaped bacteria that share common metabolic and physiological characteristics. These bacteria produce lactic acid as the major metabolic end product of carbohydrate fermentation.
• LAB lactic acid bacteria
• the beneficial bacteria of the Lactobacillales order are ones which grow (and are typically cultured) in MRS agar (MRS).
• MRS agar MRS agar
• 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.
• the beneficial bacteria of this aspect of the present invention belong to the genus lactobacillus.
• Exemplary species of lactobacillus contemplated by the present invention include but are not limited to L. acetotolerans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. alimentarius, L. amylolyticus, L. amylophilus,
• the species of lactobacillus is L. plantarum.
• the beneficial bacteria of this aspect of the present invention may generate a fermentation product.
• fermentation products include but are not limited to pre-biotics, biofuels, methanol, ethanol, propanol, butanol, alcohol fuels, proteins, recombinant proteins, vitamins, amino acids, organic acids (for 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.
• the beneficial bacteria may be genetically modified to express a beneficial polypeptide.
• the beneficial polypeptides may be intracellular polypeptides (e.g., a cytosolic protein), transmembrane polypeptides, or secreted polypeptides. Heterologous production of proteins is widely employed in research and industrial settings, for example, for production of therapeutics, vaccines, diagnostics, biofuels, and many other applications of interest.
• Exemplary therapeutic proteins that can be produced by employing the subject compositions and methods, include but are not limited to certain native 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 hematostatic proteins (e.g., tissue plasminogen activator and activated protein C), immunological proteins (e.g., interleukin), antibodies and other enzymes (e.g., deoxyribonuclease I).
• 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
• Exemplary vaccines that can be produced by the subject compositions and methods include but are not limited to vaccines against various influenza viruses (e.g., types A, B and C and the various serotypes for each type such as H5N2, H1N1, H3N2 for type A influenza viruses), HIV, hepatitis viruses (e.g., hepatitis A, B, C or D), Lyme disease, and human papillomavirus (HPV).
• examples of heterologously produced protein diagnostics include but are not limited to secretin, thyroid stimulating hormone (TSH), HIV antigens, and hepatitis C antigens.
• Proteins or peptides produced by the heterologous polypeptides can 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.
• enzymes include acetylcholinesterase, lactase, activated protein C, factor VII, collagenase (e.g., marketed by Advance Biofactures Corporation under the name Santyl); agalsidase-beta (e.g., marketed by Genzyme under the name Fabrazyme); dornase-alpha (e.g., marketed by Genentech under the name Pulmozyme);reteplase (e.g., marketed by Genentech under the name Activase); pegylated- asparaginase (e.g., marketed by Enzon under the name Oncaspar); asparaginase (e.g., marketed by Merck under the name Elspar); and imiglucerase (e.g., marketed by Genzyme under the name Ceredase).
• acetylcholinesterase lactase, activated protein C, factor VII, collagenase
• polypeptides or proteins include, but are not limited to 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 (IFNgamma), 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), Leishmnania elongation initiating factor (LEIF), platelet derived growth factor (PDGF), tumor necrosis factor (TNF), growth factors, e.g., epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor, (FGF), nerve growth factor (NGF), brain
• the gpl20 glycoprotein is a human immunodeficiency virus (WIV) envelope protein, and the gpl60 glycoprotein is a known precursor to the gpl20 glycoprotein.
• WIV human immunodeficiency virus
• Other examples include secretin, nesiritide (human B-type natriuretic peptide (hBNP)) and GYP-I.
• Contemplated bacteria for the expression of human interferon beta lb include for example E.coli.
• Contemplated bacteria for the expression of human interferon gamma include for example E.coli.
• Contemplated bacteria for the expression of human growth hormone include for example
• Contemplated bacteria for the expression of human insulin include for example E.coli.
• Contemplated bacteria for the expression of interleukin II include for example E.coli.
• the beneficial polypeptide is an antibody (e.g. Humira, Remicade, Rituxan, Enbrel, Avastin, Herceptin).
• an antibody e.g. Humira, Remicade, Rituxan, Enbrel, Avastin, Herceptin.
• Contemplated bacteria for the expression of antibodies include for example E.coli, Bacillus brevis, Bacillus subtilis and Bacillus megaterium.
• exemplary vaccines contemplated by the present invention include, but are not limited to Vivotif Berna Vaccine (typhoid vaccine, live), Prevnar 13 (pneumococcal 13- valent vaccine), Menactra (meningococcal conjugate vaccine), ActHIB (haemophilus b conjugate (prp-t) vaccine), Bexsero (meningococcal group B vaccine), Biothrax (anthrax vaccine adsorbed), Hiberix (haemophilus b conjugate (prp-t) vaccine), HibTITER (haemophilus b conjugate (hboc) vaccine), Liquid PedvaxHIB (haemophilus b conjugate (prp-omp) vaccine), MenHibrix (haemophilus b conjugate (prp-t) vaccine/meningococcal conjugate vaccine), Menomune A / C / Y
• contemplated beneficial bacteria are those that are useful in bioremediation. Such remediation includes heavy metals, chemical, radiation and hydrocarbon contamination.
• bacteria examples include bacteria that may be used for bioremediation.
• Pseudomonas putida is a gram-negative soil bacterium that is involved in the bioremediation of toluene, a component of paint thinner. It is also capable of degrading naphthalene, a product of petroleum refining, in contaminated soils.
• Dechloromonas aromatica is a rod-shaped bacterium which can oxidize aromatics including benzoate, chlorobenzoate, and toluene, coupling the reaction with the reduction of oxygen, chlorate, or nitrate. It is the only organism able to oxidize benzene anaerobically. Due to the high propensity of benzene contamination, especially in ground and surface water, D. aromatic is especially useful for in situ bioremediation of this substance.
• Nitrifiers and Denitrifiers Industrial bioremediation is used to clean wastewater. Most treatment systems rely on microbial activity to remove unwanted mineral nitrogen compounds (i.e. ammonia, nitrite, nitrate). The removal of nitrogen is a two stage process that involves nitrification and denitrification. During nitrification, ammonium is oxidized to nitrite by organisms like Nitrosomonas europaea. Then, nitrite is further oxidized to nitrate by microbes like Nitrobacter hamburgensis. In anaerobic conditions, nitrate produced during ammonium oxidation is used as a terminal electron acceptor by microbes like Paracoccus denitrificans . The result is N2 gas. Through this process, ammonium and nitrate, two pollutants responsible for eutrophication in natural waters, are remediated.
• nitrogen compounds i.e. ammonia, nitrite, nitrate
• Deinococcus radiodurans is a radiation-resistant extremophile bacterium that is genetically engineered for the bioremediation of solvents and heavy metals.
• An engineered strain of Deinococcus radiodurans has been shown to degrade ionic mercury and toluene in radioactive mixed waste environments.
• nitrate produced during ammonium oxidation is used as a terminal electron acceptor by microbes like Paracoccus denitrificans . The result is dinitrogen gas. Through this process, ammonium and nitrate, two pollutants responsible for eutrophication in natural waters, are remediated.
• Methylibium petroleiphilum (formally known as PM1 strain) is a bacterium capable of methyl tert-butyl ether (MTBE) bioremediation. PM1 degrades MTBE by using the contaminant as the sole carbon and energy source.
• PM1 strain Methylibium petroleiphilum (formally known as PM1 strain) is a bacterium capable of methyl tert-butyl ether (MTBE) bioremediation. PM1 degrades MTBE by using the contaminant as the sole carbon and energy source.
• MTBE methyl tert-butyl ether
• Alcanivorax borkumensis is a marine rod-shaped bacterium which consumes hydrocarbons, such as the ones found in fuel, and produces carbon dioxide. It grows rapidly in environments damaged by oil, and has been used to aid in cleaning the 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 clean up oil include Colwellia and Neptuniibacter.
• the method of this aspect of the present invention contemplates culturing the beneficial bacteria with a biofilm-producing bacteria.
• biofilm refers to a community of bacteria that are comprised
• extracellular polymeric substances typically include exopolysaccharides (such as those synthesized by the products of the epsA-0 operon) and amyloid fibers (such as those encoded by tapA-sipW-tasA operon).
• the matrix typically comprises extracellular DNA and protein, as well as carbohydrates.
• biofilm-producing bacteria may also be beneficial bacteria.
• biofilm-producing bacteria are typically of a different order and/or genus than the beneficial bacteria which are incorporated into the biofilm.
• the biofilm-producing bacteria and the beneficial bacteria may be of distinct strains, species, genus and/or order.
• the biofilm-producing bacteria is non-pathogenic (i.e. do not cause physical harm to, or disease in) a human being.
• any number of strains of biofilm-producing bacteria may be cultured in the co-culture of this aspect of the present invention.
• no more than 500 different strains of biofilm-producing bacteria are cultured in a single culture
• no more than 250 different strains of biofilm-producing bacteria are cultured in a single culture
• no more than 100 different strains of biofilm-producing bacteria are cultured in a single culture
• no more than 90 different strains of biofilm-producing bacteria are cultured in a single culture
• no more than 80 different strains of biofilm- bacteria are cultured in a single culture
• no more than 70 different strains of biofilm- producing bacteria are cultured in a single culture
• no more than 60 different strains of biofilm- producing bacteria are cultured in a single culture
• no more than 50 different strains of biofilm- producing bacteria are cultured in a single culture
• no more than 40 different strains of biofilm- producing bacteria are cultured in a single culture
• no more than 30 different strains of biofilm- producing bacteria are cultured in
• biofilm-producing bacterial strains of a single culture of this aspect of the present invention may belong to a single species or may belong to multiple species.
• the biofilm-producing bacterial strains of a culture belong to a single species of bacteria.
• multiple species of biofilm-producing bacteria are cultured on a single culture.
• no more than 10 different species of biofilm-producing bacteria are cultured in a single culture, no more than 9 different species of biofilm-producing bacteria are cultured in a single culture, no more than 8 different species of biofilm-producing bacteria are cultured in a single culture, no more than 7 different species of biofilm-producing bacteria are cultured in a single culture, no more than 6 different species of biofilm-producing bacteria are cultured in a single culture, no more than 5 different species of biofilm-producing bacteria are cultured in a single culture, no more than 4 different species of biofilm-producing bacteria are cultured in a single culture, no more than 3 different species of biofilm-producing bacteria are cultured in a single culture, no more than 2 different species of biofilm-producing bacteria are cultured in a single culture or only one species of biofilm-producing bacteria is cultured per single culture.
• the biofilm-producing bacteria belong to the genus Bacillus.
• the genus Bacillus includes all members known to those of skill in the art, including but not limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B. stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii, B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, and B. thuringiensis . It is recognized that the genus Bacillus continues to undergo taxonomical reorganization. Thus, it is intended that the genus include species that have been reclassified, including but not limited to such organisms as B.
• stearothermophilus which is now named "Geobacillus stearothermophilus.”
• the production of resistant endospores in the presence of oxygen is considered the defining feature of the genus Bacillus, although this characteristic also applies to the recently named Alicyclobacillus, Amphibacillus, Aneurinibacillus, Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus, Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus, and Virgibacillus .
• the biofilm-producing bacteria are of the species B. subtilis.
• Exemplary strains of B. subtilis contemplated by the present invention include, but are not limited to B. subtilis MS 1577 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 S 127.
• Exemplary strains of B.licheniformis contemplated by the present invention include, but are not limited to B.licheniformis MS310, and B. licheniformis MS307.
• the biofilm-producing bacteria does not comprise the species B. cereus.
• both the beneficial culture and the biofilm- generating culture are cultured separately to generate a starter culture.
• the medium and conditions of the starter culture are typically selected so as to optimize growth of each of the bacteria.
• Contemplated started cultures include a dried starter culture, a dehydrated starter culture, a frozen starter culture, or a concentrated starter culture.
• the starter culture is grown for at least two hours, 4 hours, 8 hours, 12 hours until a sufficient amount of bacteria are propagated.
• the method includes a method of co-culturing, whereby the beneficial bacteria is of the genus lactobacillus (e.g. the species L. plantarum) and the biofilm-producing bacteria is of the genus Bacillus (e.g. of the species B. subtilis).
• the beneficial bacteria is of the genus lactobacillus (e.g. the species L. plantarum) and the biofilm-producing bacteria is of the genus Bacillus (e.g. of the species B. subtilis).
• the method of co-culturing the beneficial bacteria with the biofilm producing bacteria is selected such that it enables the proliferation of both types of microorganisms and incorporation of both microorganisms into the biofilm.
• the co-culturing is carried out in (or on) a growth substrate that is typically used to culture the beneficial bacteria.
• the growth substrate may be a solid or a liquid medium.
• the co-culture is shaken during the culturing.
• growth substrates examples include but are not limited to MRS medium, LB medium, TBS medium, yeast extract, soy peptone, casein peptone and meat peptone.
• Abiotrophia media Recipe for medium appropriate for growth of Abiotrophia genus Acetamide
• Medium - Recipe for Acetamide medium.
• Agrobacterium Agar Recipe - Agar appropriate for growth of Agrobacterium genus
• Ashbya Full Medium Recipe for the production of Ashbya full medium.
• Azotobacter Agar - Agar appropriate for growth of Azobacter genus
• Bennett's Medium - media used for growth of some Actinoplanes species Bennett's Medium - media used for growth of some Actinoplanes species.
• Bacillus agar - Agar used to grow some Bacillus species.
• Bifidobacterium Medium Recipe for Bifidobacterium medium.
• Brain Heart Infusion Glucose Agar Recipe for Brain Heart Infusion Glucose Agar.
• Cantharellus Agar Recipe - Recipe for Cantharellus agar.
• Clostridium thermocellum Medium - Recipe for medium appropriate for growth of Clostridium thermocellum
• Creatinine Medium Recipe for the production of creatinine medium.
• CZA Czapek Agar
• CZA Czapek Agar
• Desulfovibrio Medium Recipe for Desulfovibrio Medium.
• Glucose Yeast Extract Agar - Recipe for Glucose Yeast Extract Agar.
• Halobacterium agar - Recipe for the preparation of Halobacterium Halobacteria Medium - Recipe for Halobacteria Medium.
• Marine agar - Recipe for marine agar Used for the growth of several marine bacteria.
• Marine broth - Recipe for marine broth Used for the growth of several marine bacteria.
• Methylamine Salts Agar - Recipe for methylamine salts
• Methylamine Salts Medium - Recipe for methylamine salts medium
• Modified Chopped Meat Medium Used for the growth of several anaerobic bacteria.
• MY medium - Maltose yeast extract bacterial growth medium Maltose yeast extract bacterial growth medium.
• N4 Mineral Medium - Recipe for the production of N4 mineral medium
• Nitrosomonas europaea medium Recipe for the production of Nitrosomonas europaea medium.
• Nutrient agar - Recipe for nutrient agar suitable for growth of many bacterial species Nutrient broth - Recipe for nutrient broth suitable for growth of many bacterial species.
• MRS media - Recipe for MRS media MRS media has been used for the recovery of lactic acid bacteria (LAB) from various food products.
• LAB lactic acid bacteria
• NZCYM - NZ amine NaCl
• bacto-yeast extract NaCl
• casamino acids and magnesium sulfate.
• NZM - NZ amine, NaCl, and magnesium sulfate NZM - NZ amine, NaCl, and magnesium sulfate.
• NZYM - NZ amine NaCl
• bacto-yeast extract magnesium sulfate
• Oenococcus Medium Recipe for the preparation of Oenococcus medium.
• Osmophilic Medium Recipe for Osmophilic Medium.
• Propionibacterium Agar Recipe - Agar appropriate for the growth of Propionibacterium.
• Propionibacterium Medium Recipe - Medium appropriate for the growth of Propionibacterium.
• PYS agar - agar used to grow some Actinomadura species.
• Starch - Mineral Salt (STMS) Agar - Recipe for starch - mineral salt (STMS) agar.
• Styrene Mineral Salts Medium - Recipe for Styrene Mineral Salts medium.
• Tomato Juice Medium Recipe for the preparation of tomato juice medium.
• Tomato Juice Yeast Extract Agar - Recipe for the preparation of tomato juice yeast extract agar.
• Tomato Juice Yeast Extract Medium Recipe for the preparation of tomato juice yeast extract medium.
• TSY agar - Trypticase soy yeast agar Recipe TSY broth - Trypticase soy yeast broth Recipe.
• TYG Medium Tryptone, yeast, glucose bacterial growth medium.
• TYX Medium Tryptone, yeast, xylose bacterial growth medium.
• YMG agar - Recipe for yeast and malt extract with glucose agar This agar is used for a number of Streptomyces species.
• YMG media - Recipe for yeast and malt extract with glucose media This media is used for a number of Streptomyces species.
• YPD media - Yeast extract/peptone/dextrose bacterial media YPD media - Yeast extract/peptone/dextrose bacterial media.
• the co-culture may be carried out in a growth substrate which comprises LBGM, milk or MRS.
• LBGM genus lactobacillus
• Other media that can be used to generate the co-culture of the present invention include MSgg minimal medium (Shemesh, M., et al (2010). Bacteriol 192, 6352-6356); LB enriched with lactose: Duanis-Assaf D., et al (2016) Front. Microbiol.
• the culturing conditions are selected that encourage incorporation of both the different bacteria into the biofilm.
• the present inventors have uncovered particular components of a growth medium that are important for biofilm generation of bacteria being of the genus Bacillus (e.g. of the species B. subtilis) - see Figure 21.
• the medium used for co- culturing a beneficial bacteria with Bacillus bacteria comprises manganese.
• the medium comprises dextrose.
• the medium used for co-culturing comprises both manganese and dextrose.
• a method of selecting an agent or culturing condition which is advantageous for preparing a bacterial composition comprising co-culturing beneficial bacteria with a biofilm-producing bacteria in a growth substrate in the presence of the agent or under the culturing condition so as to generate a biofilm comprising the beneficial bacteria and the biofilm-producing bacteria, wherein a change in a property of the biofilm is indicative of the agent or culturing condition being advantageous for preparing the bacterial composition.
• Exemplary conditions of the co-culture that may be altered include the properties of the surface on which the culture is carried out (for example the surface chemistry of the solid surface, including but not limited to functional groups, electrostatic charge, coating; surface roughness, surface topography, including but not limited to grooves, cavities, ridges, pores, hexagonally packed (HP) pillars, equilateral triangles surrounded by HP pillars, and the Sharklet topography etc.).
• the solid surface may be of a defined geometry and/or topography such that it promotes encapsulation/incorporation of the beneficial bacteria into the biofilm.
• the solid surface may be of a defined geometry and/or topography such that it promotes generation of a biofilm of a particular thickness.
• Other topographical 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 the culturing can be carried out include a wide range of substrates, ranging from various polymeric materials (silicone, polystyrene, polyurethane, and epoxy resins) to metals and metal oxides (silicon, titanium, aluminum, silica, and gold). Fabrication techniques (soft lithography and double casting molding techniques, microcontact printing, electron beam lithography, nanoimprint lithography, photolithography, electrodeposition methods, etc.) can be carried out on such materials in order to alter the topography of the solid surface.
• conditions of the co-culture that may be altered include, but are not limited to environmental parameters such as pH, nutrient concentration, the ratio between the beneficial bacteria: biofilm producing bacteria and temperature.
• the co-culturing is carried out in a bioreactor.
• bioreactor refers to an apparatus adapted to support the biofilm of the invention.
• the bioreactor will generally comprise one or more supports for the biofilm which may form a film thereover, and wherein the support is adapted to provide a significant surface area to enhance the formation of the biofilm.
• the bioreactor s of the invention may be adapted for continuous throughput.
• the conditions of the co-culture can be altered by altering the microfluidics (e.g. sheer stress) of the system.
• the agents or conditions are selected that bring about an advantageous change in a property of the biofilm.
• the property is an amount of biofilm.
• the property is a thickness of biofilm.
• the property is a density of the biofilm.
• the property is the rate in which the biofilm is formed.
• the property is the amount of beneficial bacteria which is incorporated into the biofilm.
• the property is the resistance to temperature and/or pH.
• the property is the amount of beneficial bacteria released from the biofilm over a period of time. This may be of particular relevance when a controlled release of the beneficial bacteria is required. For example, it may be advantageous to incorporate bacteria which are beneficial for the skin, scalp or dental applications in biofilms of which the rate of release of the beneficial bacteria therefrom is selected for maximum therapeutic effect.
• the present inventors have now found that altering the pH of the growth substrate to higher than 6, encourages bacteria that utilize the KinD-SpoOA pathway (e.g. being of the genus Bacillus, such as of the species B. subtilis) to be incorporated into a biofilm when cultured in MRS.
• the KinD-SpoOA pathway e.g. being of the genus Bacillus, such as of the species B. subtilis
• the co-culturing of the beneficial bacteria being of the genus lactobacillus (e.g. the species L. plantarum) and the biofilm-producing bacteria being of the genus Bacillus (e.g. of the species B. subtilis), carried out in, or on LBGM, milk or MRS (and more specifically MRS) is effected at a pH of between 6.5 and 9; 6.5-and 8; 6.5 and 7.5; 6.8 and 9; 6.8 and 8; 6.8 and 7.5.
• the biofilm producing bacteria is not B. subtilis MS 1577 or 3610.
• the co-culturing of this aspect of the present invention may be carried out in the presence of additional agents that serve to increase propagation of the bacteria and/or enhance biofilm formation.
• agents include for example acetoin.
• the amount of acetoin and the timing of addition may be altered so as to promote optimal biofilm production. 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.
• the present inventors contemplate a culture comprising acetoin, a biofilm comprising a bacillus bacteria and a culture medium.
• the culture medium is one which is mentioned in Table 1 (for example LB).
• 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.
• 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-cultures of this aspect of the present invention are propagated for a length of time sufficient to generate a biofilm which incorporates both the beneficial bacteria and the biofilm generating bacteria.
• the co-cultures are grown to maximal plateau growth phase of the beneficial bacteria, at which time they may be harvested for maximal biofilm production.
• the co-cultures are grown to maximal plateau growth phase of the biofilm-producing bacteria, at which time they may be harvested for maximal biofilm production.
• the bacteria may 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 or 7 days or longer. In one embodiment, the bacteria are not cultured for longer than 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks or 6 weeks.
• the biofilm is harvested (i.e. removed from the growth substrate).
• the biofilm (and/or bacteria incorporated therein) may be subject to drying (i.e. dehydrating), freezing, spray drying, or freeze-drying.
• drying i.e. dehydrating
• freezing i.e. freezing
• spray drying i.e. freezing
• freeze-drying i.e. freezing
• the biofilm is treated in a way that preserves the viability of the bacteria.
• the biofilm-producing bacteria is present in the bacterial composition in an amount of from 10 3 to 1015 colony forming units per gram of the bacterial composition (e.g. probiotic composition).
• the amount (in weight) of non-cellular material (e.g. exopolysaccharides and/or amyloid fibers) in the composition may be higher than the amount (in weight) of cellular material (e.g. bacterial cells).
• the weight of non-cellular material (e.g. exopolysaccharides and/or amyloid fibers) in the composition may be at least 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or 100 % higher than the weight of cellular material (e.g. bacterial cells) in the composition.
• the amount (in weight) of non-cellular material (e.g. exopolysaccharides and/or amyloid fibers) in the composition may be lower than the amount (in weight) of cellular material (e.g. bacterial cells).
• the weight of cellular material (e.g. bacterial cells) in the composition may be at least 5 %, 10 %, 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90 % or 100 % higher than the weight of non-cellular material (e.g. e.. exopolysaccharides and/or amyloid fiber) in the composition.
• the weight ratio of non-cellular material (e.g. exopolysaccharides): bacterial cells in the compositions described herein may be between 99:1 - 1:99. In some embodiments the weight ratio of non-cellular material (e.g. exopolysaccharides): bacterial cells in the compositions described herein may be between 99: 1 - 50:50. In some embodiments the weight ratio of non- cellular material (e.g. exopolysaccharides): bacterial cells in the compositions described herein may be between 99: 1 - 70:30.
• the bacterial composition is a probiotic composition.
• the probiotic composition comprises from about 10 3 to 1015 colony forming units ("CFUs") of the biofilm-producing microorganism per gram of finished product. In some embodiments, the probiotic composition comprises from about 10 4 to about 10 14 CFUs of the biofilm-producing microorganism per gram of finished product. In some embodiments, the probiotic composition comprise from about 10 5 to about 10 15 CFUs of biofilm- producing microorganism per gram of finished product. In some embodiments, the probiotic composition comprises from about 10 6 to 10 11 colony forming units of the biofilm-producing microorganism per gram of finished product. In some embodiments, the probiotic composition comprises from about 10 2 to about 105 colony forming units of the biofilm-producing microorganism per gram of finished product.
• CFUs colony forming units
• At least 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90% or more of the beneficial bacteria of the composition are viable (i.e. propagate). Furthermore, at least 20 %, 30 %, 40 %, 50 %, 60 %, 70 %, 80 %, 90% or more of the biofilm-producing bacteria of the composition are viable (i.e. propagate).
• the bacterial composition is a probiotic composition.
• Exemplary beneficial bacteria that may be present in the probiotic composition are those that belong to the genus lactobacillus (as described herein above).
• the probiotic composition may comprise additional beneficial bacteria such as those belonging to the Bifidobacterium genus.
• Contemplated species of Bifidobacterium that may be present in the probiotic composition of this aspect of the present invention include, but are not limited to Bifidobacterium longum, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium adolecentis, Bifidobacterium lactis, and Bifidobacterium animalis.
• the probiotic composition comprises a species that belongs to the genus lactobacillus e.g.
• the bacterial compositions disclosed herein are in any form suitable for administering the composition to a mammalian subject.
• the composition is in the form of a tablet, a powder or a liquid. If provided as a powder, combining the powder with a suitable liquid (e.g., liquid dairy product, fruit or vegetable juice, blended fruit or vegetable juice product, etc.) is specifically contemplated.
• a suitable liquid e.g., liquid dairy product, fruit or vegetable juice, blended fruit or vegetable juice product, etc.
• the bacterial compositions disclosed herein are administered to a subject prior to, concomitant with or following administration of an antibiotic agent.
• the conditions of the co-culture may be such that the biofilm which is generated releases the beneficial bacteria in the body such that they are not subject to the activity of the antibiotic agent.
• the bacterial compositions described herein are formulated for topical administration - e.g. in a cream, a gel, a lotion, a shampoo, a rinse.
• the bacterial compositions may be administered to the skin or the scalp.
• the bacterial compositions may be useful for dental applications. For such applications they may be administered to the gums.
• compositions described herein are incorporated into a food product.
• food product refers to any substance containing nutrients that can be ingested by an organism to produce energy, promote health and wellness, stimulate growth, and maintain life.
• enriched food product refers to a food product that has been modified to include the composition comprising composition described herein, which provides a benefit such as a health/wellness-promoting and/or disease- preventing/mitigating/treating property beyond the basic function of supplying nutrients.
• the probiotic composition can be incorporated into any food product.
• Exemplary food products include, but are not limited to, protein powder (meal shakes), baked goods (cakes, cookies, crackers, breads, scones and muffins), dairy-type products (including but not limited to cheese, yogurt, custards, rice pudding, mousses, ice cream, frozen yogurt, frozen custard), desserts (including, but not limited to, sherbet, sorbet, water-ices, granitas and frozen fruit purees), spreads/margarines, pasta products and other cereal products, meal replacement products, nutrition bars, trail mix, granola, beverages (including, but not limited to, smoothies, water or dairy beverages and soy-based beverages), and breakfast type cereal products such as oatmeal.
• protein powder meal shakes
• baked goods cakes, cookies, crackers, breads, scones and muffins
• dairy-type products including but not limited to cheese, yogurt, custards, rice pudding, mousses, ice cream, frozen yogurt, frozen
• the probiotic composition described herein may be in solution, suspended, emulsified or present as a solid.
• the enriched food product is a meal replacement product.
• the term "meal replacement product” as used herein refers to an enriched food product that is intended to be eaten in place of a normal meal.
• Nutrition bars and beverages that are intended to constitute a meal replacement are types of meal replacement products.
• the term also includes products which are eaten as part of a meal replacement weight loss or weight control plan, for example snack products which are not intended to replace a whole meal by themselves, but which may be used with other such products to replace a meal or which are otherwise intended to be used in the plan. These latter products typically have a calorie content in the range of from 50-500 kilocalories per serving.
• the food product is a dietary supplement.
• dietary supplement refers to a substance taken by mouth that contains a "dietary ingredient” intended to supplement the diet.
• dietary ingredients includes, but is not limited to, the composition comprising the probiotic composition as described herein as well as vitamins, minerals, herbs or other botanicals, amino acids, and substances such as enzymes, organ tissues, glandulars, and metabolites.
• the food product is a medical food.
• medical food as used herein means a food which is formulated to be consumed or administered entirely under the supervision of a physician and which is intended for the specific dietary management of a disease or condition for which distinctive nutritional requirements, based on recognized scientific principles, are established by medical evaluation.
• probiotic microorganisms can improve animal efficiency and health.
• Specific examples include increased weight gain-to- feed intake ratio (feed efficiency), improved average daily weight gain, improved milk yield, and improved milk composition by dairy cows as described by U.S. Pat. Nos. 5,529,793 and 5,534,271.
• the administration of probiotic organisms can also reduce the incidence of pathogenic organisms in cattle, as reported by U.S. Pat. No. 7,063,836.
• the probiotic composition described herein can be incorporated into an animal feed.
• the probiotic composition is designed for continual or periodic administration to ruminal, cecal or intestinal fermentors throughout the feeding period in order to reduce the incidence and severity of diarrhea and/or overall health.
• the probiotic composition can be introduced into the rumen, cecum and/or intestines of the animal.
• the probiotic composition described herein are incorporated into a pharmaceutical product or composition.
• Pharmaceutical compositions comprise a prophylactically or therapeutically effective amount of the composition described herein and typically one or more pharmaceutically acceptable carriers or excipients (which are discussed below).
• compositions described herein that are, in some embodiments, powdered, tableted, encapsulated or otherwise formulated for oral administration.
• the compositions may be provided as pharmaceutical compositions, nutraceutical compositions (e.g., a dietary supplement), or as a food or beverage additive, as defined by the U.S. Food and Drug Administration.
• nutraceutical compositions e.g., a dietary supplement
• food or beverage additive as defined by the U.S. Food and Drug Administration.
• the dosage form for the above compositions are not particularly restricted. For example, liquid solutions, suspensions, emulsions, tablets, pills, capsules, sustained release formulations, powders, suppositories, liposomes, microparticles, microcapsules, sterile isotonic aqueous buffer solutions, and the like are all contemplated as suitable dosage forms.
• compositions typically include one or more suitable diluents, fillers, salts, disintegrants, binders, lubricants, glidants, wetting agents, controlled release matrices, colorings, flavoring, carriers, excipients, buffers, stabilizers, solubilizers, commercial adjuvants, and/or other additives known in the art.
• any pharmaceutically acceptable (i.e., sterile and acceptably non-toxic as known in the art) liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient, or medium can be used.
• exemplary diluents include, but are not limited to, polyoxyethylene sorbitan monolaurate, magnesium stearate, calcium phosphate, mineral oil, cocoa butter, and oil of theobroma, methyl- and propylhydroxybenzoate, talc, alginates, carbohydrates, especially mannitol, .alpha.-lactose, anhydrous lactose, cellulose, sucrose, dextrose, sorbitol, modified dextrans, gum acacia, and starch.
• Pharmaceutically acceptable fillers can 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, may also be used as fillers in the pharmaceutical compositions.
• Binders may be used to hold the composition together to form a hard tablet.
• exemplary binders include materials from organic products such as acacia, tragacanth, starch and gelatin.
• Other suitable binders include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC).
• an enriched food product further comprises a bioavailability enhancer, which acts to increase the absorption of the sorbable natural product(s) by the body.
• Bioavailability enhancers can be natural or synthetic compounds.
• the enriched food product comprising the composition described herein further comprises one or more bioavailability enhancers in order to enhance the bioavailability of the bioactive natural product(s).
• Natural bioavailability enhancers include ginger, caraway extracts, pepper extracts and chitosan.
• the active compounds in ginger include 6-gingerol and 6-shogoal.
• Caraway oil can also be used as a bioavailability enhancer (U.S. Patent Application 2003/022838).
• Piperine is a compound derived from pepper (Piper nigrum or Piper longum) that acts as a bioavailability enhancer (see U.S. Pat. No. 5,744,161). Piperine is available commercially under the brand name Bioperine R TM (Sabinsa Corp., Piscataway, N.J.).
• the natural bioavailability enhancers is present in an amount of from about 0.02% to about 0.6% by weight based on the total weight of enriched food product.
• suitable synthetic bioavailability enhancers include, but are not limited to surfactants including those composed of PEG-esters such as are commercially available under the tradenames: Gelucire R TM, Labrafil R TM, Labrasol R TM, Lauroglycol R TM, Pleural 01eique R TM (Gattefosse Corp., Paramus, N.J.) and Capmul R TM (Abitec Corp., Columbus, Ohio).
• the amount and administration regimen of the composition is based on various factors relevant to the purpose of administration, for example human or animal age, sex, body weight, hormone levels, or other nutritional need of the human or animal.
• the composition is administered to a mammalian subject in an amount from about 0.001 mg/kg body weight to about 1 g/kg body weight.
• a typical regimen may comprise multiple doses of the composition.
• the composition is administered once per day.
• the composition may be administered to an individual at any time.
• the composition is administered concurrently, or prior to or at the consumption of a meal.
• the bacterial compositions of this aspect of the present invention are formulated for use as an agricultural product.
• the bacterial compositions may be added to an agricultural carrier such as soil or plant growth medium.
• an agricultural carrier such as soil or plant growth medium.
• Other agricultural carriers that may be used include fertilizers, plant-based oils, humectants, or combinations thereof.
• the agricultural carrier may be a solid, such as diatomaceous earth, loam, silica, alginate, clay, bentonite, vermiculite, seed cases, other plant and animal products, or combinations, including granules, pellets, or suspensions.
• Mixtures of any of the aforementioned ingredients are also contemplated as carriers, such as but not limited to, pesta (flour and kaolin clay), agar or flour- based pellets in loam, sand, or clay, etc.
• Formulations may include food sources for the cultured organisms, such as barley, rice, or other biological materials such as seed, leaf, root, plant elements, sugar cane bagasse, hulls or stalks from grain processing, ground plant material or wood from building site refuse, sawdust or small fibers from recycling of paper, fabric, or wood.
• Other suitable formulations will be known to those skilled in the art.
• the agricultural formulation comprises a fertilizer.
• the fertilizer is one that does not reduce the viability of the bacterial composition by more than 20 %, 30 %, 40 %, 50 % or more.
• the agricultural formulation it is advantageous for the agricultural formulation to contain agents such as herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.
• agents such as herbicide, a nematicide, an insecticide, a plant growth regulator, a rodenticide, and a nutrient.
• Such agents are ideally compatible with the plant onto which the formulation is applied (e.g., it should not be deleterious to the growth or health of the plant).
• the agent is ideally one which does not cause safety concerns for human, animal or industrial use (e.g., no safety issues, or the compound is sufficiently labile that the commodity plant product derived from the plant contains negligible amounts of the compound).
• the agricultural formulations comprising the biofilm of the present invention typically contains between about 0.1 to 95% by weight, for example, between about 1% and 90%, between about 3% and 75%, between about 5% and 60%, between about 10% and 50% in wet weight of the biofilm-incorporated beneficial bacterial population of the present invention.
• the formulation contains at least about 10 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, at least about 10 6 CFU or spores per ml of formulation, or at least about 10 CFU or spores per ml of formulation.
• the presently disclosed agricultural composition may be comprised in an article of manufacture which further comprises an agent which promotes the growth of plants.
• the agents may be formulated together with the biofilm in a single composition, or alternatively packaged separately, but in a single container.
• Suitable agents are described herein above.
• Other suitable agents include fertilizers, pesticides (an herbicide, a nematocide, a fungicide and/or an insecticide), a plant growth regulator, a rodenticide, and a nutrient, as further described herein below.
• the agent which promotes the growth of the plant lacks antibacterial activity.
• compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
• a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
• range format is merely for convenience and brevity and should not be construed as 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, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
• method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
• treating includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.
• the probiotic bacterial strain used in this study was Lactobacillus plantarum. This strain routinely is grown in either MRS (Man, Rogosa & Sharpe) broth or MRS broth solidified using 1.5% agar (DifcoTM).
• MRS Man, Rogosa & Sharpe
• MRS broth solidified using 1.5% agar DifcoTM.
• the Bacillus subtilis wild strain NCIB3610 and its derivatives are typically cultured in LB (10 g of tryptone, 5 g of yeast extract, 5 g of NaCl per liter) broth or LB solidified with 1.5% agar. Prior to their use, L. plantarum and B. subtilis were grown on a hard agar plate for 48 h or overnight, respectively, both at 37 Q C. A starter culture of each strain was prepared using a single bacterial colony, L.
• MRS medium at pH 7 was used since it was found to be effective in promoting biofilm formation by B. subtilis and suitable for co-culture cultivation of B. subtilis and probiotic lactic acid bacteria (LAB).
• B. subtilis cells were mixed with an equal amount of L. plantarum cells to a final concentration of 10 cells/mL of each strain, and then diluted 1: 100 into MRS pH 7. The cells in mixed cultures were incubated aerobically at 37°C at 50 rpm for 7-8 h.
• Assay for colony and pellicle biofilm formation For colony architecture analysis, 3 ⁇ )L of starter cultures were spotted onto MRS agar plates or control LB and incubated at 30 °C for 72 h. For pellicle formation analysis, the starter cultures were diluted 1: 100 into 3.5 mL MRS broth or control LB in a 12-well plates and incubated without agitation at 30 °C for 48 h. Images were taken using a Zeiss Stemi 2000-C microscope with an axiocam ERc 5s camera (Zeiss, Germany).
• ⁇ -galactosidase assay Cells were harvested from colonies grown in either LB, LB supplemented with MRS in different ratio (1: 1, 1:5, and 5:1) or MRS with pH adjustment to 7 on solid medium at 30 °C and resuspended in phosphate -buffered saline (PBS) solution. Typical long bundled chains of cells in the biofilm colony were disrupted using mild sonication. The optical density (OD) of the cell samples were normalized to an OD 6 oo of 1.0 in PBS. One milliliter of bacterial cell suspensions were collected and assayed according to standard procedure.
• PBS phosphate -buffered saline
• L. plantarum cells were grown in co-culture as described above with B. subtilis (YC161) aborting GFP or B. subtilis (YC189) aborting CFP in modified MRS broth.
• Cell suspensions of each bacterium grown as monospecies culture served as control samples.
• One milliliter of each culture was collected and centrifuged at 5000 rpm for 2 minutes. After removing supernatant, the cells were washed with 1 mL of PBS buffer and then following centrifugation (at 5000 rpm for 2 minutes) resuspended in 100 ⁇ of the same buffer. 5 ⁇ from each sample were placed on a microscopy glass slide and visualized in a transmitted light microscope using Nomarski differential interference contrast (DIC).
• DIC Nomarski differential interference contrast
• SEM Scanning electron microscopy
• SIF simulated intestinal fluid
• pancreatic enzymes were added to the digestion mixture to achieve following activities in the final mixture: porcine trypsin (SIGMA T0303) (100 U mL “1 ), bovin chymotrypsin (SIGMA C4129) (25 U mL “ l ), porcine pancreatic a amylase (SIGMA A3176) (200 U mL “1 ), porcine pancreatic lipase (SIGAM L3126) (2000 U mL "1 ).
• bile salts SIGMA T4009 were added to give a final concentration of 10 mM in the final mixture and then the samples were incubated again for 2.5 h.
• One milliliter from each sample collected after gastric and intestinal phases and the numbers of viable L. plantarum cells were determined using CFU counting method as described above.
• biofilms have an increased tolerance toward various unfavorable environmental conditions, apparently due to production of extracellular matrix (Friedman, Kolter, & Branda, 2005).
• the present inventors thus hypothesized that extracellular matrix produced by robust biofilm former bacterium B. subtilis may provide increased protection to other species such as probiotic bacteria during their growth in co-culture biofilm system.
• a specialized medium was developed where L. plantarum and B. subtilis are able to grow in co-culture. It was found that by modifying the pH of the MRS to pH 7, it was possible to grow these bacteria in co-culture. As shown in Figure 13, the co-culture cultivation had no effect on L. plantarum and B.
• subtilis growth (compared to their growth in pure culture), indicating that there are no antagonistic interactions between these bacteria at given conditions.
• modification of MRS medium promotes strong biofilm formation by B. subtilis ( Figure 2). Since B. subtilis appears to be sensitive to acidic pH, the pH of MRS medium used for co-culture cultivation was gradually elevated in order to find a pH value suitable for Bacillus growth. The increase of pH from 6 to 8 led to a proportional increase in robustness of biofilm phenotype of both colony and pellicle biofilm ( Figure 2). When the pH was adjusted to 6 weak growth on solid MRS medium was seen and no growth in liquid medium.
• LB medium that is usually used to culture B. subtilis
• MRS MRS 1: 1, 1:5, and 5: 1).
• Figure 3 The effect of increasing MRS concentration on matrix gene expression in B. subtilis using tapA and eps operons was also investigated, since their products are major components of extracellular matrix. It was found that the expression of tap A increased proportionally with the concentration of MRS in LB ( Figures 4A-B). The expression of eps increased proportionally to the concentration of MRS up to 80% MRS, than a decrease of expression for 100% was detected ( Figures 5A-B).
• the present inventors determined whether MRS triggers biofilm formation through the Kin-SpoOA pathway previously described for B. subtilis (Shemesh and Chai, 2013 Journal of Bacteriology, 2013, Vol 195, No.12 pages 2747-2754). They tested different B. subtilis mutants for biofilm formation (AkinA, AkinB, AkinC, AkinD, AkinE, AkinAB, AkinCD, AspoOA, AepsAtasA) or overproducing biofilm (AabrB). Firstly, they determined biofilm phenotype of mutants deficient in histidine kinases responsible for sensing environmental signals that induce biofilm formation.
• the modified MRS medium was used to investigate dual species biofilm by co-culturing fluorescently tagged B. subtilis cells, which constitutively express GFP (YC161), together with L. plantarum cells. Generated biofilm was visualized using CLSM. As can be seen in Figure 8A (top panel), the generated biofilm consisted of both fluorescent and non-fluorescent cells. L. plantarum cells were surrounded by B. subtilis cells which attached to each other to form a biofilm-related structure (bundle). This is further illustrated in Figure 8B which illustrates the co-cultured biofilm of B. subtilis and L. plantarum in LBGM medium.
• biofilm formation in B. subtilis depends on the synthesis of extracellular matrix
• the present inventors sought to determine whether the production of extracellular matrix takes place during dual species biofilm development.
• the level of the matrix gene expression in the formed biofilm was analyzed using transcriptional fusion of the promoter for tapA-sipW-tasA (operon responsible for synthesis of protein components of biofilm matrix in B. subtilis) to the cfp gene encoding cyan fluorescent protein (YC189), as described previously (Shemesh, Kolter, & Losick, 2010, J Bacteriol 192, 6352-6356) (P tap A-cfp).
• subtilis cells grown as monoculture form also biofilm characterized with homogenous structure in which long filaments of the cells are bound together by an extracellular matrix (Figure 9A).
• the L. plantarum cells could not form notable biofilm in monospecies culture. The observations described above indicate that the extracellular matrix produced by B. subtilis cells could be shared with L. plantarum cells and thus provide them with possible protection against environmental stresses.
• the dual species biofilm facilitates survival ofL. plantarum in hostile environments
• L. plantarum cells grown in co-culture biofilm were exposed to heating at 63 °C for 1 and 3 min.
• L. plantarum cells that grew in monospecies culture were used as control. Following 1 and 3 min of heat treatment, L.
• L. plantarum and B. subtilis mutant strains either deficient in biofilm formation (AepsAtasA) or an overproducing biofilm matrix (AabrB)
• the co-cultures were subjected to heat treatment pasteurization.
• L. plantarum cells grown in mono-species culture and in co-culture with wild type B. subtilis were used as control.
• Figure 11 A L. plantarum cells grown with the cells of AepsAtasA double mutant did not show a significant difference in their survival level compare to L. plantarum grown in mono-species culture.
• Acetoin enhances biofilm formation
• Food products are often enriched by different food additives which may improve organoleptic and sensory characteristics of the products.
• additives there are important small molecules such as acetoin which can improve the flavor of different food products.
• Acetoin is a neutral molecule which widely exists in nature. Some microorganisms, higher plants, insects, and higher animals have the ability to synthesize acetoin.
• Those additives can affect the physiology of many bacteria associated with human health, and affect development of multicellular community of bacterial cells known as a biofilm. Biofilm formation depends on the synthesis of an extracellular matrix that holds the constituent cells together.
• the matrix In Bacillus subtilis, a prebiotic bacteria, the matrix has two main components, an exopolysaccharide synthesized by the products of the epsA-0 operon, and amyloid fibers encoded by tapA-sipW-tasA operon.
• acetoin triggers the biofilm bundles formation in Bacillus subtilis. In the absence of acetoin, no biofilm formation is observed when grown in LB medium ( Figure 15A).
• Figures 16A-B illustrate that acetoin triggers a colony type biofilm formation in Bacillus subtilis. Transcription of the tap A operon responsible for the matrix production in B. subtilis was shown to be highly upregulated by acetoin ( Figures 17A-D).
• the cells express high levels of the extracellular matrix components, in response to acetoin, which are crucial for biofilm formation.
• the objective of this experiment was to test the ability of NCIB3610 (isolated from soil) and 127185/2 (isolated from dairy environment) to protect L. plantarum against hostile environmental conditions during growth in co-culture system.
• the growth medium selected for the co-culture system of B. subtilis and L. plantarum was modified (pH adjusted) MRS medium.
• Characterization of biofilm formation was performed using a stereoscopic microscope or confocal laser scanning microscope (for colony or bundles type biofilm, respectively).
• Figures 18A-B are photographs depicting the biofilm generated from the B. subtilis strains NCIB3610 and 127185/2 respectively.

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