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Exopolysaccharides Production by Dairy and Non-Dairy Microorganisms and their Role in Quality and Safety of Dairy Products and Human Health

Hassan, Ashraf
South Dakota State University
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To produce a cocktail of enzymes capable of removing biofilmTo investigate whether EPS-positive dairy strains producing low and highbiofilm on saliva-coated hydroxyapatite aid or inhibit the colonization and biofilm development by the dental pathogen S. mutans.To study role of Bacillus in quality and stability of dairy products.To utilize acid whey in production of xanthan.

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Objective 1:In vitro biofilm formation Five pieces of 2 x 2 cm of RO membranes will be used in biofilm formation. Filter sterilized whey will be used to study the effect of EPS produced by starter cultures on biofilm formation. Whey will be inoculated with EPS-positive or negative genetic variants of lactic acid bacteria and a low biofilm forming hemolytic Bacillus ssp. isolated from used membranes in our laboratory. After incubation for 24 h at 37 oC, cells in biofilm will be detached by stomacher and counted.Application of phage enzymesThe pH of unpasteurized cheese whey will be adjusted to 6.5 followed by filtration through 0.22 um filters to remove phage resistant bacteria in the sample. Aliquots (0.1 ml) of an overnight culture, of all isolates from biofilm and feed, grown in broth will be placed in sterile test tube. Sterile 0.05 ml of 1 molar calcium chloride will be added to each tube followed by 1 ml of whey filtrate. After incubation at 37 C for 15 min soft agar of an appropriate medium will be added (2.5 ml to each tube). After mixing, tube contents will be poured on the surface of set bottom agar plates of the same medium. Plates will be incubated at 37C and observed for the appearance of plaques. Two well isolated plaques will be transferred to 0.5 ml of broth containing 0.2% chloroform. Plaques will be tested for containing only one single type of phage by repeated transfer of the lysates to new plates. Final lysates will be stored at 4°C for further use. For preparation of phage stocks, 0.5 ml of phage lysate and 0.1 ml of 1 M calcium chloride solution will be added to 100 ml of culture grown in broth and incubated at 37 oC. Lysis will be monitored by measuring the optical density. Lysate will be centrifuged at 100,000 g for 20 min. to yield phage-free enzymes. The effect of crude depolymerases on microbial detachment will be evaluated.

Objective 2: Biofilm formation by EPS-positive and negative strains will be assayed according to the model described by Guggenheim et al. (2001) and Furiga et al. (2008). Biofilms will be developed by EPS negative and positive strains on hydroxyapatite (HA) discs (Clarkson Chromatography Products Inc., Williamson, PA, USA) coated with human clarified, filter-sterilized saliva. EPS from the 3 EPS-positive strains producing highest biofilm and 3 strains producing lowest biofilm will be isolated and purified as described by Ayala-Hernández et al. (2008) using repeated precipitation with chilled acetone after protein precipitation with TCA. Purified EPS will be characterized at the Complex Carbohydrate Research Center, the University of Georgia according to the method described by Ayala-Hernández et al. (2008).Objective 3:Preparation of vegetative cells-free cultureBacillus cultures grown on MOPS medium at 37 C for 4 days will be heat treated at 85 C for 10 min to kill vegetative cells.Spore countingSpores will be counted with a hemocytometer or plating on TSA. Spores will be stored in sterile distilled water at -80 C until used.Counting lactic acid bacteria and Bacillus in a mixed cultureThe growth of all Bacillus isolates will be tested in HiCrome™ Bacillus Agar (Sigma Aldrich). M17 and MRS with added antibiotics will be used for lactic starter. Antibiotic sensitivity test will be conducted to select the ones that are active against Bacillus but not starter.Monitoring growth of vegetative cells and germination of spores during fermentationand storageYogurt and sour cream mix (UHT milk and cream will be used for its preparation) will be inoculated with acid adapted vegetative cells (2 h in medium adjusted to pH 5.5 with filter sterilized 85% lactic acid) or spores (prepared by thermal and non-thermal methods) of combined cultures of Bacillus isolated from Daisy Brand product or environment. Equal volume of different cultures of Bacillus will be adjusted to a count of 108 CFU/ml of phosphate buffer saline. The final population of Bacillus mixture will be 105 CFU/ml of the product mix. The mix will be incubated at the appropriate temperature (21 or 37 C) until the desired final pH is attained. During incubation samples will be taken every hour from yogurt mix and 3 hour from sour cream mix. Counts of starter and Bacillus and pH will be monitored during fermentation. During storage for 3 weeks at 4 C, lipolysis, proteolysis, rheology, texture profile analysis, and peptides profile (HPLC) will be evaluated.Objective 4:StrainsTwo different EPS-producing cultures and two EPS-non-producing cultures of each of Streptococcus thermophilus and Lactobacillus delbrueckii ssp bulgaricus (from Chr Hansen, Denmark) will be used in this study. Two mother cultures of X. campestris not fermenting lactose (ATCC 13951 and NRRL-B1459) and their lactose fermenting variants (ATCC 55258 and ATCC®31922) will be used in this study.MediaMRS, M17, and Yeast Malt agar (containing 3 g yeast extract, 3 g malt extract, 5 g peptone, 10 g glucose, and 20 g agar per L) will be used for enumeration of Lactobacillus, Streptococcus, and Xanthomonas respectively. In mixed cultures, MRS adjusted to pH 5.5 and incubated under anaerobic conditions will be used to selectively enumerate Lactobacillus. Differences in cell morphology will be used to differentiate between S. thermophilus and Xanthomonas. Xanthomounas colonies are yellow in color. Two different fermentation media will be used: cottage cheese whey and Greek yogurt whey. Whey will be obtained from General Mills, Dean Foods, or Schreiber and stored frozen until utilized.Testing ability of lactic cultures and X. campestris to grow on Greek yogurt and cottage cheese wheyOvernight cultures of lactic acid bacteria grown on the appropriate growth media (MRS for Lactobacillus and M17 for S. thermophilus) will be used to inoculate Greek yogurt and cottage cheese whey. Yeast Malt agar will be used in the preparation of X. campestris inocula. The cell count of all cultures used to inoculate the fermentation media will be adjusted to 108 CFU/ml. The pH of the fermentation media will not be controlled. Fermentation media will be incubated overnight at 32 C. The pH and bacterial count will be determined in the beginning and at the end of the fermentation period. In case of poor growth, the medium will be supplemented with yeast extract (0.1 to 0.5%) or whey protein concentrate 80 (0.1 to 0.5%).Exopolysaccharides productionGreek yogurt and cottage cheese whey will be used for the production of EPS. A 19.5 L sterilizable-in-place benchtop fermentor (Bioflow415 New Brunswick, Enfield, CT) will be used. The pH will be maintained at 6.8. The agitation (100-500 rpm) and aeration (0-2.9 vvm) levels used will be determined in preliminary experiments. All fermentations will be carried out at 32 C for 72 h. Two different methods will be used to eliminate bacteria cells: centrifugation and pasteurization. The time temperature combination needed to completely kill the fermenting microorganisms in the viscous fermentate will be determined. Alternatively, the fermentate will be centrifuged at 10000 xg for 30 min at 4 C to separate cells (Mesomo et al. 2009). The fermentate will be then ultrafiltered, dried in an oven at 50 C for 24 h, freeze dried, and stored in sealed plastic bags.Quantification of exopolysaccharides in the fermentation mediaThe amount of Xanthan gum will be estimated according to the methods described by Mesomo et al. (2009) and Fu and Tseng (1990). The fermenting media will be diluted 2 to 10 fold with distilled water and centrifuged at 12,000 xg for 20 min to remove cells. The EPS in the supernatant will be precipitated with 70% chilled ethanol at -20 C overnight. The solution will be centrifuged at 20,000 x g for 30 min. The pellet will be suspended in distilled water, extensively dialyzed against sterile ultrapure water, dried in the oven at 50C for 48 h, and weighed.

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Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens