Antibacterial effect of hydrosoluble extracts of garlic (Allium sativum) against Bifidobacterium spp. and Lactobacillus acidophilus

The antimicrobial effects of garlic (Allium sativum) against pathogenic microorganisms have been well documented. It is generally stated that garlic exhibits differential inhibition between pathogenic and beneficial bacteria. Though there is substantial evidence to support the claim for pathogens, there is limited literature on its effects on beneficial bacteria, specifically probiotic bifidobacteria. This study aimed to investigate the antimicrobial effects of different garlic preparations on five strains of bifidobacteria. The disk diffusion assay revealed antibacterial activity of different garlic preparations characterised by zones of inhibition ranging from 13.0 ± 1.7 to 36.7 ± 1.2 mm. Minimum inhibitory concentration (MIC) values for garlic clove extract ranged from 75.9 to 303.5 mg/ml (estimated 24.84 to 99.37 μg/ml allicin). Bifidobacterium lactis Bi-07 300B was on average the most resistant to garlic, followed by B. lactis Bb12, B. longum LMG 13197, B. longum Bb356 and B. bifidum 11041, being most sensitive. This study reveals for the first time, susceptibility of bifidobacteria to antibacterial activity of garlic. Caution is therefore advised when using probiotic bifidobacteria and garlic simultaneously.The National Research Foundation (NRF) and the University of Pretoria.http://www.academicjournals.org/AJMRam201

Fourier transform infra-red spectroscopy and flow cytometric assessment of the antibacterial mechanism of action of aqueous extract of garlic (Allium sativum) against selected probiotic Bifidobacterium strains

BACKGROUND: It is generally reported that garlic (Allium sativum) harms pathogenic but not beneficial bacteria. Although numerous studies supporting the alleged garlic effects on pathogens are available, there are limited studies to prove this claim for beneficial bacteria. We have recently shown that garlic exhibits antibacterial activity against probiotic bifidobacteria. The aim of the current study was to elucidate the mechanism of action of garlic clove extract (GCE) on Bifidobacterium bifidum LMG 11041, B. longum LMG 13197 and B. lactis Bb12 using Fourier transform infrared (FT-IR) spectroscopy and flow cytometry. METHODS: Cultures (1 × 108 CFU ml-1) were individually incubated for 6 h at 37°C in garlic clove extract containing allicin at a corresponding predetermined minimum bactericidal concentration for each strain. For FTIR, an aliquot of each culture was deposited on CaF2 slide and vacuum dried. The slides were immediately viewed using a Bruker Vertex 70 V FT-IR spectrometer equipped with a Hyperion microscope and data analyzed using OPUS software (version 6, Bruker). Spectra were smoothed with a Savitsky-Goly function algorithim, base-line corrected and normalized. Samples for flow cytometry were stained using the Live/Dead BacLight bacterial viability kit L7012. Data compensation and analysis was performed using a BD FACSAria and FlowJo (version 7.6.1). RESULTS: Fourier transform infrared spectroscopy showed changes in spectral features of lipids and fatty acids in cell membranes, proteins, polysaccharides and nucleic acids. Spectral data as per principle component analysis (PCA) revealed segregation of control and GCE-treated cells for all the tested bifidobacteria. Flow cytometry not only showed increase in numbers of membrane damaged and possibly lysed cells after GCE treatment, but also displayed diffuse light scatter patterns for GCE treated cells, which is evidence for changes to the size, granularity and molecular content of the cells. CONCLUSION: Garlic has multiple target sites in bifidobacteria, penetrating the cell membrane and entering the cytoplasm, where it causes changes to carbohydrates, fatty acids, proteins and nucleic acids. These changes, for example, modification of membrane properties, may prevent exposed bifidobacteria from colonizing the intestinal mucosa. Loss of colonization potential would render them less efficient as probiotics.The National Research Foundation and University of Pretoriahttp://www.biomedcentral.com/bmccomplementalternmedam201

In vitro antibacterial mechanism of action of crude garlic (Allium sativum) clove extract on selected probiotic Bifidobacterium species as revealed by SEM, TEM, and SDS-PAGE analysis

There has been much research on the effects of garlic (Allium sativum) on numerous pathogens, but very few, if any, studies on its effect on beneficial, probiotic bifidobacteria. We have recently shown that garlic exhibits antibacterial activity against bifidobacteria. The mechanism by which garlic kills bifidobacteria is yet to be elucidated. This study sought to determine the mechanism of action of garlic clove extract on selected Bifidobacterium species using scanning and transmission electron microscopy and SDS-PAGE analysis. SEM micrographs revealed unusual morphological changes such as cell elongation, cocci-shaped cells with cross-walls, and distorted cells with bulbous ends. With TEM, observed changes included among others, condensation of cytoplasmic material, disintegration of membranes, and loss of structural integrity. SDS-PAGE analysis did not reveal any differences in whole-cell protein profiles of untreated and garlic clove extract-treated cells. The current study is the first to reveal the mechanism of action of garlic clove extract on probiotic Bifidobacterium species. The results indicate that garlic affects these beneficial bacteria in a manner similar to that exhibited in pathogens. These results therefore further highlight that caution should be taken especially when using raw garlic and probiotic bifidobacteria simultaneously as viability of these bacteria could be reduced by allicin released upon crushing of garlic cloves, thereby limiting the health benefits that the consumer anticipate to gain from probiotics.National Research Foundation (NRF) and the University of Pretoria.http://link.springer.com/journal/12602hb201

Antibacterial activity of garlic (Allium sativum) against probiotic Bifidobacterium species

During the past decade there has been an explosion in the probiotic industry due to an increase in concern for health. It is well known that these probiotic products offer consumers numerous health benefits and that viability of cultures in these products need to be maintained at high levels. It is therefore important to test for antimicrobial compounds or substances that may come into contact with probiotics and thereby negatively affect and decrease their viability. Garlic (Allium sativum) has been used as a natural medicinal remedy for thousands of years and research has shown that it has antimicrobial activity against a wide variety of microorganisms. Although it has been tested against numerous pathogenic microorganisms, there have been few studies on its effect on beneficial bacteria, specifically probiotic Bifidobacterium species. A great amount of work and money is put into preparing probiotic products with sufficient numbers of viable bacterial cells. All these are devoted to ensure that the consumers seize the optimal purported health benefits from probiotic cultures incorporated within the different products. Hence it is necessary to recognize any compound or substance that poses a threat to viability of these probiotic cells, thereby rendering them ineffective. Therefore, the current study aimed at determining whether garlic had any antibacterial activity towards selected Bifidobacterium spp. In vitro studies revealed that garlic has an inhibitory effect on these specific probiotic bacteria. The disk diffusion assay revealed antibacterial activity of garlic preparations characterized by inhibition zones ranging from 13.0 ± 1.7 to 36.7 ± 1.2 mm. Minimum inhibitory concentration (MIC) values for garlic clove extract ranged from 75.9 to 303.5 mg/ml (estimated to contain 24.84 to 99.37 μg/ml allicin) while the minimum bactericidal concentration (MBC) ranged from 10.24 to 198.74 μg/ml xix allicin. Susceptibility of the tested Bifidobacterium species to garlic varied between species as well as between strains even within a small numbers of the tested bifidobacteria. Among the tested Bifidobacterium spp., B. bifidum LMG 11041 was most susceptible to garlic, whereas B. lactis Bi-07 300B was the most resistant. These results were contrary to what has been generally published in literature, that garlic selectively kills pathogens without negatively affecting beneficial bacteria. Garlic clove, garlic powder, garlic paste and garlic spice showed varying degrees of potency, with fresh garlic clove extract and garlic paste extract having the highest and lowest antibifidobacterial activity, respectively. It became necessary to investigate the actual antibacterial mechanism of action of garlic on Bifidobacterium spp., upon realization that its extracts inhibits growth of or kills some of these bacteria, whose contribution to health and well being of consumers is to a large extent dependent on their viability. This was determined by using scanning electron microscopy (SEM) and Fourier-transform infrared (FT-IR) spectroscopy. Scanning electron microscopy was used to investigate the effect of garlic on the morphology and cell surface properties of the tested strains while FT-IR spectroscopy was used to determine any biochemical changes taking place in garlic-treated bifidobacteria. Scanning electron microscopy showed various morphological changes such as cell elongation, distorted cells with bulbous ends and cocci-shaped cells. Behavioural changes were also observed such as swarming of cells was also observed. FT-IR spectra confirmed that garlic damaged Bifidobacterium cells by inducing biochemical changes within the cells. It identified some of the main targets sites of garlic on bifidobacteria, mainly, the nucleic acids and fatty acids (lipids) in the cell membrane. Flow cytometry analysis was used to determine the level at which the garlic decreased the viability of Bifidobacterium cells as well as the extent of damage induced by the garlic. Results revealed a drop in viability with associated decrease in stainability of some the cells, for all strains upon treatment with garlic clove extract. The inability of cells to be stained by nucleic acid stains, hence presence of cells referred to as ‘ghost cells’, has been associated with extensive damage and lysis of cellular membranes resulting in loss nucleic acids. Interestingly, re-inoculation of the cells analysed by flow cytometry into a fresh growth medium and their subsequent reanalysis using the same technique showed an increase in percentage of viable cells and a decrease in percentages of damaged, unstained and dead cells. This suggested that injured cells were able to recover and regress to their active state. Therefore, Bifidobacterium cells exposed to sub lethal amounts of garlic can repair any damage and regrow. However, it was not determined how long active compounds of garlic remain stable within the gastrointestinal tract. This study is the first, according to our knowledge, to show that garlic exhibits antibacterial activity against beneficial bacteria specifically, probiotic bifidobacteria. Furthermore, the results revealed that the mechanism of action of garlic towards bifidobacteria is similar to that which was reported for pathogenic bacteria. Bacterial death and growth inhibition occurs due to damage to the fatty acids/lipids in the cell membrane, modification of the nucleic acids (DNA and RNA). This study is of significant importance to consumers, medical practitioners as well as to the probiotic industry. It suggests that if garlic comes into contact with probiotic bifidobacteria, they die and thus become unable to deliver the promised health benefits to the consumers. Therefore, consumers should be advised against ingestion of probiotic products and garlic simultaneously, as this study reveals that garlic does indeed inhibit some probiotic Bifidobacterium spp. The probiotic industry should also consider including this information on their product labels to make consumers aware of this fact. Failure to include this information may lead to market deterioration due to loss of interest in the products as soon as consumers realize they do not get their money’s worth from the products. Lastly, medical practitioners should also be made aware of this as they also prescribe probiotics to patients for various health reasons. The effect of food matrices on the antibacterial effects, as well as determination of how long the active compounds of garlic remain within the gastrointestinal tract, in relation to levels of garlic ingested will confirm whether indeed there is concern. But for now, in light of results of the current study, caution needs to be taken in simultaneous use of probiotics and garlic, until further testing indicates otherwise.Dissertation (MSc)--University of Pretoria, 2013.gm2014Microbiology and Plant Pathologyunrestricte