كلية الصيدلة

Faculty of Pharmacy

Submitted in partial fulfillment for Master degree in Pharmaceutical Sciences (Microbiology & Immunology)

Includes Bibliographic Referances, p121:157.

1. Introduction
Lactic acid bacteria (LAB) constitute a group of gram-positive bacteria united by a constellation of morphological, metabolic, and physiological characteristics, The boundaries of the group have been subject to some controversy, but historically the genera Lactobacillus, Leuconostoc, Pediococcus, and Streptococcus form the core of the group. However, from a practical, food-technology point of view, the following genera are considered the principal LAB: Aerococcus, Carnobacterium, Enterococcus, Lactobacillus, Lactococcus, Leuconostoc,Oenococcus, Pediococcus, Streptococcus, Tetragenococcus, Vagococcus, and Weissella and The genus Bifidobacterium, often considered in the same context as the genuine lactic acid bacteria (Axelsson, 2004).
LAB display a wide range of antimicrobial activities. Amongst these activities, the production of lactic acid and acetic acid is obviously the most important. However, certain strains of LAB are further known to produce bioactive molecules such as ethanol, formic acid, fatty acids, hydrogen peroxide, diacetyl, reuterin, and reutericyclin (De-Vuyst and Leroy, 2007).
Lactic acid bacteria produce the antimicrobial substances lactic acid, hydrogen peroxide, diacetyl, acetoin as well as small heat-stable inhibitory peptides. LAB that produce antimicrobial substances have considerable advantage in the competition with other micro-organisms, including pathogens and other harmful bacteria (Simova et al., 2009).
Another beneficial substance reported to be produced by several LAB is the bacteriocin or bacteriocin-like substance. Bacteriocins can be regarded as antibiotics, but
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they differ from antibiotics in that they are ribosomally synthesized, host cells are immune to them, have different mode of action than antibiotics and have a narrow killing spectrum and thus they are generally able to kill only bacteria closely related to the producing strain (Nes et al., 2007a).
These bacteriocins have been grouped into 4 different classes. Class I consists of the small, post-translationally modified peptides, often referred to as lantibiotics due to the presence of the modified residues lanthionine and methyl-lanthionine. Class II consists of peptide bacteriocins without modified residues. Both Class I and Class II bacteriocins are relatively small heat stable proteins. The third class, Class III, consists of large, heat labile antimicrobial proteins. Class IV consists of a complex bacteriocins where protein is with lipids and/or carbohydrates (Nes et al., 2007b).
In light of searching for new, cheap, alternative, and implementable strategies to combat H. pylori infections on a large scale; bacteriocin could be considered to fulfill such strategy. This substance has the advantage of possibility to be used as probiotics, which can be delivered simply and economically by the oral route; thus constituting a new development in the treatment and eradication of infection by H. pylori (Hamilton-Miller, 2003; Lesbros-Pantoflickova et al., 2007). Summary:
In the present study, a total of 138 lactic acid bacteria (LAB) isolates were recovered from different dairy and non-dairy products as well as infant stool samples. Of these isolates, cell-free supernatant (CFS) of 73 ones were active against enteropathogenic indicator organisms (Escherichia coli, Salmonella enteritidis and Helicobacter pylori). Twenty five of these active isolates were found to grow under strict aerobic condition, while the remaining 48 isolates were found to be facultative anaerobes. Out of these isolates, 45 were considered to be promising isolates and were selected for further investigations as they have shown zones of inhibition equal to or greater than 15 mm in diameter.
The next step was to determine the reason for the anti-enterics activity exerted by the LAB isolates; whether it is due to acid release, hydrogen peroxide production or due to the production of other inhibiting compound(s). This was achieved by ruling out the inhibiting activity both of the organic acid, mainly lactic acid, and of the hydrogen peroxide produced by the LAB. As for the effect of the organic acids, it was eliminated by neutralizing the pH of the CFS with few drops of NaOH. This led to loss of activity in about 31% of the promising isolates. However, only one isolate have shown activity greater than that of the untreated CFS. Concerning the hydrogen peroxide as inhibitory agent against the tested enteric bacteria, it was cleaved by the addition of catalase enzyme. This action resulted in losing the inhibitory action of hydrogen peroxide in 38% of the isolates. Upon subjecting the CFS to the combined treatment with NaOH and
catalase enzyme, only one aerobic and 4 facultative anaerobic LAB remained active and exerted a moderate activity against the three standard enteropathogenics.
Identification of the five selected (active) LAB isolates was performed using 16S Ribosomal DNA probing; revealing that the isolate LAB 1(aerobic) was identified as Lactobacillus johnsonii, while the other four (facultative anaerobic) LAB 26, LAB 27, LAB 29 and LAB 30 were identified as Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus paracasei and Lactobacillus delbrueckii subsp. bulgaricus respectively.
The fact that, the cell-free supernatants (neutralized and treated with catalase) inhibited the growth of the indicator strains gives evidence (as reported in literature) that the antimicrobial activity is due to the production of bacteriocins.
The selected active Lactobacillus plantarum (the most active isolate) was subjected to extraction of bacteriocin with ammonium sulphate precipitation followed by purification using ion exchange chromatography. Finally FPLC reverse chromatography was used and revealed a fraction with anti-helicobacter activity.
The purified bacteriocin was considered to be heat stable and active at pH ranged from 3.0 to 8.0. The bacteriocins preserved maximum activity during 90 days of storage at 20°C. The inhibitory activity of bacteriocin produced by Lactobacillus plantarum was the most stable at 4° and 20°C for90 days. SDS PAGE electrophoresis revealed that the purified bacteriocin was about 3.5 kDa.
Concerning probiotic properties; bile and acid tolerance was done for some selected active lactobacilli (focusing on the most active Lactobacillus plantarum).The data showed that isolates Lactobacillus johnsonii (LAB1), Lactobacillus plantarum (LAB27) and Lactobacillus paracasei (LAB29) were able to survive in acidic pH 3.0. The strains, resistant to low pH, were screened for their ability to tolerate 0.3% bile salt for 4 h. Although the three selected active isolates showed resistance to the 0.3% bile salt, LAB27 and LAB1 were indicated to be more tolerant to bile than LAB29.
In conclusion, it was found that the majority of LAB isolates recovered in this work showed inhibition of the enteropathogenic indicators tested (Escherichia coli, Salmonella enteritidis and Helicobacter pylori). This activity was indicated neither to be due to the organic acid produced, nor to be due to the hydrogen peroxide obtained during the metabolic activity of LAB. Activity was revealed to be related to bacteriocin or bacteriocin-like compound. Of special interest was the strain Lactobacillus plantarum, whose bacteriocin showed high activity against Helicobacter pylori. In addition, being resistant to acid and bile; this strain Lactobacillus plantarum might be useful as probiotic to be included in food e.g. yoghurt in prophylaxis or as co-therapy for H. pylori infection.

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Includes Abstract in Arabic