Inhibition of Bacillus cereus by Garlic Extracts

Abstract

Aqueous extract of garlic (Allium sativum, L.) was prepared from a 1:2 (wt/vol) ratio offresh garlic bulbs to sterilize distilled water. Garlic extracts of 3%, 5 o/o and 10 o/o inhibited the growth of Bacillus cereus on nutrient agar plates 31.3%, 58.2% and 100%, respectively. Extracts from garlic bulbs stored at-18°C are slightly more inhibitory to the growth of B. cereus than extracts from bulbs stored at 15-35°C for 6 months. The greatest extract activity was found when garlic bulbs were extracted and left at 30°C for 4 h before filtration. When the macerate was held at 4 ° C, 6 h of storage were needed for the extract to reach its greatest activity. Gamma irradiation, at the dose of 570 krads, of garlic bulbs with subsequent freezing before extraction decreased the extracts original activity up to 50%. Exposing the extracts to heat treatments of 80-90° C for a total heating time of 5 min completely destroyed the antibacterial activity of the extract. Garlic (Allium sativum, L.) exhibits antibacterial (2,6,8,12,13,15) antifungal (4,9,11), larvicidal (J) and enzyme inhibitory (14) activities. The active inhibitory principal is allicin (diallyl disulfide and diallyl trisulfide) (3-5). Allicin is enzymatically released from a precursor after the garlic bulbs are crushed (5). Al-Delaimy and Ali (2) reported that 4% fresh garlic extract inhibited the gt.·owth of Escherichia coli, Shigella dysenteriae, Salmonella typhi and Staphylococcus aureus. S. aureus is less sensitive to garlic extract than E. coli (12,15). Dewit et al. (6) reported that 1500 1-1g/g in a meat slurry inhibited toxin production by Clostridium botulinum type A but not by types B or E. Fresh garlic ground with meat prolongs the shelf-life offresh meat (1). The inhibition of Bacillus cereus by garlic extracts has not been reported. The purpose of this study was to examine the effect of the concentration of garlic extract, the storage of garlic bulbs, the extraction and filtration technique, irradiation and heat treatment on the inhibition of Bacillus cereus by garlic extract. MATERIALS AND METHODS ., Preparation of garlic extract Garlic bulbs were purchased from a Baghdad, Iraq, market. The extract was prepared by a modification of the procedure of Al-Delaimy and Ali (2). Hulled garlic segments (50 g) were blended with 100 ml of sterile distilled water in a sterilized Waring Blendor glass jar at medium speed for 3 min. The macerate was vacuum filtered through a sterilize Buchner funnel and Whatman No. 2 filter paper. The filtrates were refiltered through a Seitz filter (0.45-~Jm). The filtered extract was used for inhibition studies within 8 h of extract preparation. Storage of garlic bulbs Freshly harvested garlic bulbs were stored at room temperature (15 to 35°C) in a cloth bag for 6 months. Garlic bulbs from the same lot were also placed in polyethylene bags and stored at-18°C, Extracts were prepared from garlic bulbs from each storage treatment and compared for inhibitory activity against Bacillus cereus. All other studies utilized garlic bulbs that had been stored at-18 ° C. Time between extraction and filtration Extracts of garlic bulbs were prepared from frozen samples as described above. Separate extracts were stored at 5 and 30°C. After 0,2,4,6,12 and 24 h of storage, the extracts were filtered as previously described and assayed for inhibition of B. cereus growth. Irradiation Fresh garlic bulbs (0.5-kg lots) were irradiated with 6 °Co at the following doses : 0,10, 100,200, 300 and 570 krad. All samples were then stored at-18 ° C for 3 months before extraction and assessment of antibacterial activity. Heat treatment Seitz-filtered garlic extract was prepared from frozen bulbs and 5-ml portions added to sterilized test tubes. The extracts were then heated at SO, 60, 70, 80 and 90°C for 5 min. An unheated sample served as a control. The inhibitory activity of the heated extracts were examined. Bacillus cereus inhibition The test organism, B. cereus was obtained from the Bacteriology Laboratory, College of Agriculture, University of Baghdad. A fresh culture was prepared by inoculating 10 ml of nutrient broth with a loopful of the stock culture and incubating the inoculated tube at 37°C for 18-20 h. Pour plates containing 0.1 ml of B. cereus suspension (320-420 viable cells), garlic extract and 15 ml of nutrient agar (Oxoid) were prepared. Extracts were added to the plates at a final concentration of 3, 5 or 10o/o (vollvol). The inoculated plates were incubated at 35°C for 48 h. The o/o inhibition was calculated from the number of colonies on duplicate plates with and without garlic extract.