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LACTOBACILLUS

Lactobacillus derive their name from the production of lactic acid. Lactic acid production is a common denominator for all Lactobacillus but it varies in relation to species (Lactobacillus Acidophilus, Lactobacillus Paracasei…) and the strains utilized.

Every species of Lactobacillus, depending on its metabolic activity, produces in addition to lactic acid, other volatile fatty acids that have lower acidifying activity. The higher the pH of the medium, the lesser the lactic acid releasing capacity of the Lactobacillus that naturally develop in the medium. The COBIOTEX bacterial strains have the advantage to allow the implementation and development of Lactobacillus strains with high lactic acid releasing capacity in alkaline media and to eliminate by competition the Lactobacillus that are adapted to alkaline media.

The disappearance of alkaline media friendly Lactobacillus that develops in high pH media contribute to the reduction of nauseating smell from animal bedding and slurry that are treated with COBIOTEX bacterial strains due to the reduced production of volatile fatty acids other than lactic acid.

Among the different families of Lactobacillus there are important differences in the production capability of lactic acid based on strain. This requires a fine selection among existing strains collected on the same natural biotop to retain Lactobacillus with a high lactic acid production capacity. The same variability is observed among Lactobacillus strains for the utilization of ammoniacal nitrogen as nutrient that decreases the release of ammonia from litter in poultry houses.

In addition to the production variability of lactic acid by different Lactobacillus strains, it is also essential to take into consideration that each strain produces specific quantities of two types of lactic acid: d. and l. lactic acid that differ in their properties..

These two forms of lactic acid are called optic isomers. Their molecules are symmetric in relation to a plane. (Item and its reflection in a mirror)

The d. lactic acid does not have direct metabolic activity. Its principal action is to influence the pH of the medium where it is released. Its acidifying power is important compared to other volatile fatty acids. As it is not directly metabolized, d. lactic acid persists and accumulates in the medium, thus maintaining a prolonged acidifying activity.

The l. lactic acid has, as its optic isomer, also an activity on the pH of the medium. This activity is complemented by important metabolic properties. The l. lactic acid is captured at the level of the cell membranes by specific receptors that facilitate its penetration into the cells where it is directly incorporated in energy metabolism. In this case, the acid is metabolized and loses its acidifying power. As cell membrane receptors have a limited capacity for activity, the surplus l. lactic acid accumulates in contact with cell membranes and will participate in the activity against these cells in the same manner as the d. lactic acid.

Due to its accumulation in the intracellular space, the acid molecule due to its gradient of concentration, will penetrate non-dissociated into the cell. Once inside the cell, the acid molecule will release its proton H+ and acidify the intracellular media. This action will modify the potential of cellular membranes causing a disorganization of transport mechanisms across membranes. Depending on the surface area of the membrane affected by these disturbances, there is either, a reduction of the number of cells capable of multiplying with an extension of the time needed between two cellular divisions, resulting in bacteriostatic activity or, a total stop of the capacity of the cell to divide followed by cellular death resulting in bactericidal activity. (Refer to Table I)

Some bacteria, in particular the Lactobacillus can collect certain DNA fragments from other bacterial cells. The DNA fragment can integrate itself or not, onto the DNA of the receptor bacteria. In case of non-integration, the DNA fragment remains isolated inside the cytoplasm and forms a plasmid. At the time of cellular division, the plasmid will split into two as the DNA cell. The split cell receives the parental DNA plus the DNA from the plasmid which remains non integrated in the cytoplasm but will express its potential to synthesize specific molecules that take on a significant importance due to important properties that strengthen their position within bacterial ecosystems. Thus, many plasmids are described to code the production of certain molecules with antibiotic activity such as nysine, lacticidine etc.

The molecules thus produced are all named becteriocines or “bacteriocine like”, depending on whether their activity is on cells belonging to the same family as the parent cell or to different families.

When placed under non strict anaerobic conditions, in order to eliminate excess oxygen, anaerobic bacteria excrete hydrogen peroxide (H2O2). This metabolite is often deadly to strict anaerobic microorganisms. In contrast, there are other cells that are either less sensitive or not sensitive at all to this metabolite. It is therefore important that the COBIOTEX Lactobacillus strains are either not sensitive or to have very low sensitivity to hydrogen peroxide because the media where these bacteria must function are often poor in oxygen but not totally deprived. The method of control that is applied to COBIOTEX Lactobacillus strains, utilizes a medium of lowered O2 but not a strict anaerobic condition.

The release of oxygen peroxide by COBIOTEX Lactobacillus strains allows the elimination of competitive strains that are sensitive to hydrogen peroxide.

BACILLUS

Bacilli derives their name from a rod like appearance. They have a multiplication cycle that goes through a sporulation phase when exposed to unfavorable conditions. The cell will produce a spore for protection. The sporulation phase takes place along with physiological modifications such as cessation of energy metabolism and other metabolic processes that favor the secretion of lipopeptides, such as surfactine and others.

The selection of COBIOTEX Bacillus strains depends on two criteria: ·

Enzymatic activity ·

Lipopeptitdes secretion activity

Currently 7 types of lipopeptides exist with known structure and with different properties (Refer to Table II).

The number of Bacillus strains that release lipopeptides is limited. There are approximately twenty surfactine producing strains of Bacillus subtilis. Bacillus licheniformis and Bacillus pumilus secrete lichenysine and pumilacidine that are variants of surfactine.

Laboratory research has demonstrated that many of the strains in the COBIOTEX Bacillus collection release inhibitory substances with biosurfactant properties. The specific properties of biosurfactant molecules synthesized by different Bacillus strains, in particular of the B. Subtilis in the COBIOTEX collection, indicate that these molecules belong to a particular group of lipopeptides that combine the properties of surfactines and iturines.

Research on the secretion kinetic of lipoproteic molecules has established at least two different molecules for the same cell. These molecules are synthesized with a time lag of several hours and the cells sensitive to these different molecules are different from each other, which allows a broader spectrum of activity.

Lipopeptide have multiple and diverse properties: ·

-Adhesion and penetration into cell membranes.

Lipopeptides play an important role of adhesion that allow bacteria to adhere to each other. They improve the adhesion mechanisms of bacteria as well as the capability of bacteria to aggregate. The targeted cells are therefore agglutinated around the Bacillus thus allowing the Bacillus to act with improved efficiency. Lipopeptides can also insert themselves between the different layers of cell membranes of bacteria and modify the properties of these membranes. ·

-Excretion of certain proteins

Some lipopeptides intervene in the excretion mechanism of proteins in particular of certain enzymes such as alpha amylase. ·

-Inhibitory Activity

Lipopeptides have also inhibitory activity on certain gram positive and gram negative bacteria and on certain fungi. Iturine is an antifungal, it inhibits Aspergillus Fumigatus at very low concentration, by modification of the cell membranes thus causing cellular death. Sufactine forms a complex with Ca++ ions which simultaneously facilitate the interaction of iturine with phospholipides in cell membranes, altering their integrity thus causing cellular death. Sufactine has also an activity against mycoplasma acting on cell membranes. This mode of action is similar for all lipopeptides: interaction with cell membranes, disorganization of this membrane and cellular death. ·

-Biosurfactant activity

The majority of lipopeptides have a biosurfactant activity. Their presence in a medium decreases the tension on the surface of aqueous solutions. They modify the molecular distribution in contact with cell membranes. The surfactine produced by Bacillus Subtilis strain is one the most effective biosurfactant.

I Organic acid that do not dissociate and penetrate into cells by diffusion. (passive transport)

II Release of protons (H+) in the Cytoplasm.

III The influx of protons inducing acidification of the cytoplasm.

IV Decreasing the movement potential of proton through cell membranes.(D pH)

V Inhibition of transport mechanisms, energy dependent mechanisms and synthesis of macromolecules.

References.

CRAMER AND PRESTEGARD – 1997 Biochemical and Biophysical Research Communication – 75. 295-301

DIEZ GONZALES AND RUSSEL – 1997 Microbiology 143. 1175-1180

Jean Penaud

January 29, 2002