Fractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells

Breast Cancer -Current and Alternative Therapeutic Modalities 150 Materials and methodsKefir samples were provided by Liberte Inc. (Brossard, Canada).The large-scale production of kefir involves a two-step fermentation process.The first fermentation is achieved by directly adding kefir grains (2-10%) to milk that has been pasteurized and cooled to 20-25 o C.After a period of fermentation lasting around 24 h, the grains are removed by filtration.The filtrate (kefir mother culture) is added to milk (1-3%), which is further fermented for 24 h and packaged for the consumer market (final kefir commercial product).Samples from three different batches were used.Upon receipt of the samples, they were immediately well stirred, and centrifuged at 4°C, 32,000 x g, for 60 min (Sorvall RC 5C Centrifuge, rotor ss-34, Sorvall Instruments, Wilmington, USA).The supernatant was filtered with a 0.45 µm membrane filter followed by a 0.2 µm filter.The filtrates were stored at -80°C for future use. Macronutrients and mineralsA Flexi-Dry MP lyophilizer (FTS Systems Inc., Stone Ridge, USA) was used for triplicate determination of moisture.Ten grams of homogenized sample was transferred into preweighed aluminium weigh boat, frozen at -80 o C for approximately 1 h and then freeze dried for 48 h.The boat was weighed again and the moisture was calculated.A LECO FP-428 Nitrogen Determination System (LECO Corporation, St. Joseph, USA) was used to determine nitrogen content in triplicate for freeze-dried samples.Crude protein content was calculated using a conversion factor of 6.25.Protein in solution was determined by using Bio-Rad protein assay kit according to the instruction with the kit (Bio-Rad Laboratories, Hercules, USA).Peptide concentrations were analyzed by the method of Church et al. (1983) (opthialdehyde; OPA).Crude fat was analyzed in triplicate with an automatic Soxtec extraction system (Soxtec HT6 Tecator AB, Hoganas, Sweden).Three grams of freeze-dried, well-mixed sample was loaded and analyzed.Proper amount of freeze-dried samples were digested in 70% (w/v) nitric acid (Fisher Scientific, trace metal grade) and minerals (i.e., Ca, Mg, Zn, Fe, Na and K) were determined by using Hitachi Z-8200 Zeeman polarized atomic absorption spectrophotometer (Nissei Sango Ltd., Mississauga, ON, Canada). Organic acidsLactic acid content was analyzed using a Sigma lactate kit assay (Sigma Diagnostics, Cat.No. 735-10, St. Louis, USA).Organic acids were determined by HPLC according to the method of Guzel-Seydim et al. (2000).Five mL of each sample was diluted with 25 mL 0.01 M H 2 SO 4 , vortexed for 1 min followed by centrifugation at 2000 x g for 10 min.Supernatants were collected and filtered through 0.2 µm filter.Volumes of 20 µl of samples and standards were injected into a Beckman Gold HPLC system (Beckman Coulter, Fullerton, USA) equipped with an Aminex HPX-87H (300 mm x 7.8 mm) organic acid column (Bio-Rad Laboratories, Hercules, USA).Degassed 8 mM sulfuric acid (H 2 SO 4 ) was used as the mobile phase.The organic acids oxalate, citrate, malate, succinate, formate and acetate were detected at 215 nm.Organic acids were quantified using external standards (organic acid analysis standard, Bio-Rad Laboratories, Hercules, USA). Molecular weight cut-off fractionation (MWCO)Centriplus centrifugal filter devices were used to get MWCO fractions at 3000 Da (Millipore, Bedford, USA).Ten milliliters whole extract were loaded to the sample reservoir and the www.intechopen.com of cancer death among females.In this book, we discussed various therapeutic modalities from signaling pathways through various anti-tumor compounds as well as herbal medicine for this deadly cancer.We hope that this book will contribute to the development of novel diagnostic as well as therapeutic approaches. How to referenceIn order to correctly reference this scholarly work, feel free to copy and paste the following: Chujian Chen, Hing Man Chan and Stan Kubow (2011).Fractionation and Characterization of Bioactive Components in Kefir Mother Culture that Inhibit Proliferation of Cultured MCF-7 Human Breast-Cancer Cells, Breast Cancer -Current and Alternative Therapeutic Modalities, Prof.

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