FIELD AND BACKGROUND OF THE INVENTION

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.

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 generaLactobacillusandBifidobacterium. 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). To assure their beneficial effects in the body, these organisms must survive during food processing, storage and the passage through the upper gastrointestinal tract (GIT) and arrive alive to their site of action. However, previous studies have shown low survival level of probiotic bacteria in the final food product and a considerable loss in their viability to high acidic conditions of the stomach and high bile concentration in the small intestine. In addition, 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.

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.

One of the mostly studied biofilm formers isBacillus subtilis, a spore-forming non-pathogenic bacterium, which is characterized by its ability to produce a robust biofilm.Bacillusspecies, principallyB. 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. SinceB. subtilisspores 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 thatB. subtiliscells 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 γ-polyglutamic acid produced byB. subtilisas 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).

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

SUMMARY OF THE INVENTION

According to an aspect of the present invention there is provided a method of preparing a bacterial composition comprising:

(a) in vitro co-culturing beneficial bacteria with biofilm-producing bacteria in a growth substrate under conditions that generate a biofilm which comprises the beneficial bacteria and the non-pathogenic bacteria; and

(b) isolating the biofilm from the growth substrate, thereby preparing the bacterial composition.

According to an aspect of the present invention there is provided a bacterial composition obtainable according to the methods described herein.

According to an aspect of the present invention there is provided a food/feed product comprising the bacterial composition described herein.

According to an aspect of the present invention there is provided 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.

According to an aspect of the present invention there is provided 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.

According to embodiments of the present invention the biofilm-producing bacteria are non-pathogenic bacteria.

According to embodiments of the present invention the biofilm-producing bacteria are of thebacillusgenus.

According to embodiments of the present invention the biofilm-producing bacteria are of theB. subtilisspecies.

According to embodiments of the present invention the beneficial bacteria are probiotic bacteria.

According to embodiments of the present invention the beneficial bacteria are genetically modified to express a therapeutic polypeptide.

According to embodiments of the present invention the probiotic bacteria is of the lactobacillales order.

According to embodiments of the present invention the biofilm-producing bacteria are of theB. subtilisspecies.

According to embodiments of the present invention the probiotic bacteria are of theL. plantarumspecies.

According to embodiments of the present invention the beneficial bacteria are used in bioremediation.

According to embodiments of the present invention the biofilm-producing bacteria express genes of the KinD-Spo0A pathway.

According to embodiments of the present invention the growth substrate comprises a growth medium.

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

According to embodiments of the present invention the biofilm-producing bacteria are of thebacillusgenus and the beneficial bacteria are of the lactobacillales order, the growth substrate is LBGM, milk or MRS.

According to embodiments of the present invention the growth substrate is MRS.

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

According to embodiments of the present invention the conditions comprise a pH of 6.8-7.5.

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

According to embodiments of the present invention the method further comprises dehydrating the biofilm following the isolating.

According to embodiments of the present invention the beneficial bacteria comprises no more than 50 bacterial species.

According to embodiments of the present invention the biofilm-producing bacteria are a single species of biofilm-producing bacteria.

According to embodiments of the present invention, at least 50% of the bacteria in the composition are viable.

According to embodiments of the present invention the bacterial composition comprises no more than 50 bacterial species of beneficial bacteria.

According to embodiments of the present 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 embodiments of the present invention the bacterial composition is a probiotic bacterial composition.

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

According to embodiments of the present invention the biofilm-producing bacteria are of thebacillusgenus.

According to embodiments of the present invention the biofilm-producing bacteria are of theB. subtilisspecies.

According to embodiments of the present invention the beneficial bacteria are probiotic bacteria.

According to embodiments of the present invention the probiotic bacteria are of the lactobacillales order.

According to embodiments of the present invention the agent alters the pH of a medium of the system.

Unless otherwise defined, 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 the invention pertains. 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 are not intended to be necessarily limiting.

