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Non-Commercial Use Only Microbiology Research 2018; volume 9:7465 Inactivation of food borne activities like, antibacterial, antifungal, pathogens by lipid fractions of anticancer, antimutagenic, antidiabetic, Correspondence: Ayeza Naeem, Department antiviral, anti-inflammatory, and antiproto- of Microbiology, University of Karachi, culinary condiments and their zoal properties are assigned to them.3 The 75270 Karachi, Pakistan. nutraceutical properties antimicrobial activity of lipid fractions is E-mail: [email protected] allotted to many terpenoid and phenolic Acknowledgments: the authors would like to 1 1 compounds, which also in crude form have Ayeza Naeem, Tanveer Abbas, thank Miss Tahira Mohsin Ali for basically 2 2 been shown to possess antibacterial or anti- Tahira Mohsin Ali, Abid Hasnain 4 perusing the manuscript and worthwhile dis- 1 fungal activity. The antibacterial properties cussions. Department of Microbiology; of these compounds are in part related to 2Department of Food Science and their lipophilic attribute, leading to accumu- Key words: Black seed; Fennel seed; Technology, University of Karachi, lation in membranes and to subsequent Coriander seeds; Bay leaf; lipid fractions. Karachi, Pakistan membrane-associated events such as energy depletion.5 However, there are often large Contributions: the authors contributed variances in the stated antibacterial activity coequally. of oils from the same source. The justifica- tion for this diversity can be due to the geo- Conflict of interest: the authors declare no Abstract potential conflict of interest. graphical sources, the harvesting seasons, Lipid fraction from four different culi- the genotype, the climate, the drying and nary condiments namely black seed Funding: none. the distilled part of the plant which are sig- (Nigella sativa), fennel seeds (Foeniculum nificant factors influencing the chemical vulgare), bay leaf (Laurus nobilis) and Received for publication: 24 October 2017. composition and relative magnitudes of the coriander seeds (Coriandrum sativum) were Accepted for publication: 1 December 2017. individual elements in the lipid fractions of investigated for total phenolic content, the plant. Also, several lipid fraction com- This work is licensed under a Creative antioxidant activity, total flavonoid content, ponents show noteworthy antimicrobial Commons Attribution NonCommercial 4.0 total flavonol content and antibacterial only characteristics when tested discretely.6 License (CC BY-NC 4.0). attributes. Antimicrobial properties were However, there is confirmation that lipid determined against food-borne bacteria ©Copyright A. Naeem et al., 2018 fractions are more intensely antimicrobial through agar well diffusion, drop agar diffu- Licensee PAGEPress, Italy than their major antimicrobial constituents.7 sion, macrobroth dilution with simultane- use Microbiology Research 2018; 9:7465 The present study deals with the isola- doi:10.4081/mr.2018.7465 ous determination of their minimum tion of lipid fractions of Nigella sativa linn. inhibitory concentrations and changes in (Family: Ranunculaceae), Coriandrum cellular morphology was analyzed through sativum L. (Family: Apiaceae Scanning electron microscopy. Generally, Umbelliferae), Foeniculum vulgare Miller ethanolic lipid fractions were more effec- Extraction of lipid fractions (Family: Apiaceae) and Laurus nobilis L. tive bioactively as compared to methanolic Lipid fractions were extracted by the LFs. Parallel results were obtained for anti- (Family: Lauraceae) and to compare these solvent extraction method as proposed by bacterial activities with the highest antibac- lipid fractions in terms of their antioxidant, Cheikh-Rouhou et al. 20078 using methanol terial activities exhibited by ethanolic LFs. total phenolic, total flavonoid and total and ethanol as an extractant. Dried spice The results positively support the use of flavonol contents. Moreover, the antimicro- powder (50g) was extracted separately with these lipid fractions in generating new sys- bial activities of these fractions were also 250ml of each solvent. After mixing in a tems to inhibit bacterial growth, extend the investigated using different assays against shaking water bath for four hours at 40˚C, shelf life and enhance the safety of the five food borne pathogens. the mixture was centrifuged for 15 minutes packaged food product. The examined oils at 1000g. The supernatant was filtered can also be used for therapeutic purposes. through a Whatman® No. 2 filter paper. Non-commercialMaterials and Methods The extraction procedure was repeated Chemicals twice and the solvent was removed using a Introduction All chemicals used in this research were rotary evaporator (Rotavapor R-210, Buchi Food poisoning is still a major appre- of analytical grade and were obtained from laboratories, Switzerland) at 40˚C. The con- hension simultaneously for consumers and Sigma Aldrich (Sigma Aldrich GmbH, centrated lipid fraction was pooled in an the food industry regardless of the use of Sternheim, Germany). Mueller Hinton agar amber colored bottle and tightly sealed and numerous conservation procedures. Due to and broth were purchased from Thermo stored at freezing temperature until ana- the resistance that pathogens build to count- Scientific™ Oxoid™. lyzed. Extraction yield of each ethanolic er antibiotics, there is an increasing aware- and methanolic lipid fraction was calculated ness to make use of natural antibacterial Seed material in terms of percent extraction yield and tab- derivatives for food preservation and safety, Four different dried spices i.e. black ulated by the formula: like extracts of culinary herbs and condi- seeds, fennel seeds, coriander seeds and bay ments.1 leaf were purchased from a local market Extraction yield of lipid fraction (%) = Lipid fractions have since quite a while during the month of February 2015. The ago served as enhancing flavors in food and spices were ground to fine powder using a drinks, and because of their versatile com- Waring® Spice Grinder WSG60K and pre- position of antimicrobial complexes, they served in zip-lock® bags and stored at Analytical methods have potential for food preservation.2 freezing temperature until analyzed. Antioxidant activity Various pharmaceutical and biological The free radical scavenging ability of [Microbiology Research 2018; 9:7465] [page 33] Article the lipid fractions was determined using the flavonols were communicated as mg of Determination of MIC assay was accom- method as described by Han, Weng, & Bi, quercetin counterparts per gram of dry plished as described by Weerakkody et al. 2008.9 Two hundred microlitres of different weight (mg QE/ml of lipid fraction) utiliz- 2010.14 concentrations (10µg/ml, 100µg/ml and ing the calibration curve with quercetin. 250µg/ml) of lipid fractions was mixed with Analysis of disruption in cellular morphol- 2.7ml of 0.06mM methanolic solution of Determination of antibacterial activity ogy using Scanning electron microscopy DPPH (2,2- diphenyl - 1-picryl - hydrazyl). Bacterial cultures Selected bacteria were standardized to a The absorbance of the resulting mixture Five food borne pathogens (Escherichia 0.5 Mcfarland scale and incubated for 18h was measured after 15 minutes at 517 nm coli ATCC 8739, Vibrio parahaemolyticus at 35˚C. Same strains were exposed to lipid using UV-Vis spectrophotometer (JascoV- ATCC 17802, Listeria monocytogenes fractions at MIC overnight at 35˚C. After 670 UV-VIS-NIR Spectrophotometer ATCC 13932, Bacillus cereus ATCC 11778 18h of incubation, 10 µl of crystal violet Tokyo, Japan). and Vibrio alginolyticus ATCC 17749) was added to Eppendorf tubes and left for were selected as test microorganisms. The 1min. Subsequently, the content of the tubes Determination of total phenolic content cultures were grown overnight on nutrient was washed with 70%, 80% and 90% The concentration of total phenols in agar plates for 16 hours and next day a loop- ethanol and centrifuged at 13000 rpm for lipid fractions was analysed using Folin- ful of each test bacteria were inoculated in 10min each. The bacterial cells were coated Ciocalteu Micro method; Waterhouse 3ml of Mueller Hinton broth and were incu- up to 300°A with gold and viewed under 10 2002; and calibrating with standard curve bated at 37˚C until turbidity of 0.5 (1.5×108 SEM (JSM-6380A) at Centralized Science of gallic acid. Briefly, 20 µL of lipid frac- CFU/ml) Mcfarland index was achieved. Laboratories, University of Karachi. tion was mixed with 1.58ml of distilled water and 100 µL of Folin-Ciocalteu Lipid fraction dilutions Statistical analysis reagent. The mixture was homogenized Lipid fractions of black seeds, fennel, Analysis of variance was employed to completely and incubated for 8min. bay leaf and coriander seeds were diluted in compute significant differences between the Subsequently, 300 µL of aqueous 15% sodi- 40% DMSO according to the method of means, and Duncan’s test at P<0.05 was um bicarbonate was added, and the mixture 12 Martins et al. 2013. The concentrations of usedonly to separate means using SPSS soft- was allowed to stand for 120 minutes with the lipid fractions used were 1000µg/ml, ware (version 24, SPSS Inc., USA). Adobe intermittent shaking. The absorbance was 500µg/ml, 250µg/ml,125µg/ml and photoshop was used to create canvas of measured at 765 nm using a UV-Vis spec- 62.5µg/ml. For the bioassay, the stock solu- SEM images. trophotometer (JascoV-670 UV-VIS-NIR tions of lipid fractions were sterilized byuse fil- Spectrophotometer Tokyo, Japan). Total tration
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