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Role of Exopolysaccharides-Producing Cultures in Biofilm Formation and as a Texturizing Agent


<p>Objective 1. To optimize manufacturing conditions for the production of 50% reduced fat and low fat Cheddar cheeses with characteristics similar to those in the full fat types using EPS-producing cultures. </p>
<p>Objective 2. To develop novel functional ingredients from whey containing exopolysaccharides </p>
<p>Objective 3. To study the formation of biofilms on whey RO membranes as a cause of membrane fouling.</p>

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<p>NON-TECHNICAL SUMMARY:<br/> Low fat cheese is gaining popularity due to consumer awareness of the health benefit of low fat diets. Fat reduction results in inferior cheese texture. Some strains of lactic acid bacteria produce polymers outside the cell wall (exopolysaccharides, EPS). EPS bind water and interfere with protein-protein interactions which are responsible for cheese rigidity. We successfully applied EPS-producing cultures in making 33% reduced fat cheese (Awad et al., 2005a&b; Hassan and Awad, 2005; Hassan et al., 2005). This cheese did not differ in texture from the full fat counterpart. Consumers are more interested in cheeses with more than 1/3 fat reduction. EPS-producing cultures will be applied to 50% reduced fat and low fat Cheddar cheeses. Protein-carbohydrate conjugates influence the structure and stability of food systems. Many chemical and enzymatic
methods have been developed to improve the functional properties of whey proteins (WP) by forming complexes with polysaccharides. Polysaccharides affect solubility, gelling properties, heat stability, foaming capacity, and emulsifying stability of whey proteins (Briczinski and R. F. Roberts, 2002; Cai et al. 2003). The minimum protein concentration for gelation to occur and the gelation temperature and time decrease in the presence of the polysaccharide, galactomannan (Tavares and Lopes da Silva, 2003). EPS-producing lactic acid bacteria convert lactose into functional polysaccharides. Our previous research showed that the EPS-WP interaction depends on the type of EPS, WP concentration, level of WP denaturation and pH. Fermentation of whey with EPS-producing cultures may provide a new class of "all natural" biopolymer ingredients with novel functionality. To our knowledge, there is very
limited information on the effect of EPS on functionality of WPC. Different means will be used to optimize conditions for production of WPC from viscous whey containing EPS. Membrane processing technologies like reverse osmosis (RO) are increasingly being applied in the US whey processing industry. Membrane fouling is a major operational problem during RO processing that leads to several problems such as loss in throughput capacity, reduction in native design selectivity, complex cleaning and food safety issues, in addition to, increased operational costs. The common causes of membrane fouling include scaling, organic fouling and particle fouling. However, it is yet not clear if biofouling, due to bacterial biofilms, play any role in reduced membrane performance, and if there are any related quality and food safety issues. Biofilm on dairy separation and concentration membranes including
RO will be studied.
<p>APPROACH:<br/> Objective 1: A non EPS-producing commercial Cheddar cheese starter culture will be used for the control cheese. The EPS-producing strain Lactococcus lactis subsp cremoris (JFR) will be used. This strain produced reduced fat Cheddar cheese with similar texture to the full fat counterpart. The standard Cheddar cheese protocol will be used (Awad et al., 2005b). Target fat levels in reduced and low fat cheeses will be 16 and 6% respectively. Cheese composition, proteolysis, and rheological, textural, sensorial and melting properties will be evaluated. Objective 2: We propose to produce and compare WPC from commercial cheese whey (containing no EPS) after fermentation with different EPS-producing and nonproducing cultures. To avoid problems associated with filtration of high viscosity whey, whey will be fermented after ultrafiltration. Preliminary study will
optimize conditions for production of WPC from whey containing EPS. Different UF concentration levels (3x, 5x and 8x) and different outlet air temperatures (75 and 100 C) will be evaluated. The strain producing the highest viscosity in whey (Lactococcus lactis ssp cremoris JFR) will be used in the preliminary study. Cheese whey will be ultrafiltered using a spiral wound membrane (model 1/1: Koch membrane systems Inc, Wilmington, MA) at 55 C , pasteurized at 63 C/30 min and then fermented with one of the EPS-producing or non-producing cultures. The fermented whey will be added to fresh UF whey at 2 or 5%. The 2 and 5% were chosen based on previous work. UF whey will also be fermented by the strain extensively studied in our lab (Lactococcus lactis ssp. cremoris) to a pH value of 5.8. The fermented whey will be spray dried without and with pH adjustment to a value of 7.0 (a common practice
in the whey drying industry). Composition and functional properties of of WPC will be determined. Objective 3: Different encapsulated strains of lactic acid bacteria will be used to form biofilm on RO membranes. Enzymes from phages attacking such strains will be used to study the role of capsules on attachment and biofilm formation. Microbial consortia will be isolated from biofilms formed on RO membranes. Cells of predominant species grown in whey will be tested for capsule production, cell surface charge, hydrophobicity, and slime production. Selective strains will be used to form biofilm on RO membranes. Ten pieces of 2 x 2 cm RO membranes will be submerged in whey inoculated with single or mixed cultures from the consortia isolated from 1 year old RO membranes. Whey will be incubated for 1 week at 37 C. Membranes will be rinsed to remove loosely attached cells and transferred to
fresh whey daily. Biofilm formation on membranes will be evaluated by direct observations with fluorescence microscopy. The live/dead specific dye and lectins (wheat germ agglutinin and concanavalin A) conjugated with a fluorescent dye will be used to differentiate between live and dead cells and specifically label EPS respectively. Scanning electron microscopy will be used to elucidate details of biofilm microstructure.
<p>PROGRESS: 2013/01 TO 2013/09<br/>Target Audience: Food and dairy scientists and food and dairy industry. The research finding was presented at the American Dairy Science Association Annual meeting in Indianapolis, IN (July 2013) and Midwest Dairy Foods Research Center meeting at University of Minnesota in July 2013. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? The project provided training for 2 MS students and one Ph.D. student. How have the results been disseminated to communities of interest? Two Research papers were published in Journal of Dairy Science. Three abstracts were presented at the American Dairy Science Association Annual meetings. Students presented the research findings at those meetings received the third place in oral paper competition and first place in poster oral
competition. What do you plan to do during the next reporting period to accomplish the goals? More research will be conducted to study role of EPS in biofilm formation and stability.
<p>PROGRESS: 2012/01/01 TO 2012/12/31<br/>OUTPUTS: The objective of this work was to compare the physical properties of several commercially available ropy strains of lactic acid bacteria with those of the most successful culture we previously used in reduced fat cheese making (Lactococcus lactis ssp. cremoris JFR). A non-EPS producing culture was also used as a control. Reconstituted (11% w/v) low heat nonfat dry milk was steamed (95C for 30 min), cooled to 30C (for mesophiles) or 37C (for thermophiles), inoculated with the test culture, and incubated until a pH value of 4.6 was attained. Fermented milk was then kept at 4C overnight before analyses. Heat stability, shear stability, viscosity, water holding capacity, flow properties, and viscoelastic properties of fermented milk were determined. The objective of the second research project was to produce whey protein
concentrate (WPC) with modified functionality using exopolysaccharide- (EPS) producing cultures. Two different EPS-producing cultures, Lactococcus lactis ssp. cremoris JFR and Streptococcus thermophilus, producing EPS1 and EPS2 respectively, were used in this study. One EPS-nonproducing commercial cheese culture (DVS 850; Chr. Hansen, Milwaukee, WI) was used as the control. Reconstituted sweet whey powder was used in this study to eliminate variations from fresh whey. Cultures grown overnight in reconstituted WPC (10% wt/vol) were added, directly or after overnight cooling (cooled EPS), at 2% (wt/vol) to 6% (wt/ wt) solution of reconstituted whey. Whey was then high-temperature, short-time pasteurized at 75C for 35 s and ultrafiltered to a volume reduction factor of 5. Ultrafiltered whey (retentate) was spray dried at inlet and outlet air temperatures of 200 and 90C, respectively, to
obtain WPC. <p>PARTICIPANTS:<br/> Dr. Ashraf Hassan, Dr. Vikram Mistry, Dr. Sanjeev Anand, Dr. Lloyd Metzger, Mr. A. Biswas, Mr. G. Deep, and Mr. J. Janevski: Dairy Science Department, South Dakota State University. TARGET AUDIENCES: Dairy and food industry and dairy foods scientists and graduate students. PROJECT MODIFICATIONS: Nothing significant to report during this period.
<p>PROGRESS: 2011/01/01 TO 2011/12/31<br/>OUTPUTS: The objective of this research was to study the role of bacterial cell and dairy separation membrane characteristics on biofilm formation. Eight bacterial strains isolated from membranes obtained from the dairy industry were tested for hydrophobicity, slime formation, and acid and capsule production. The growth curve of individual strains was studied and the OD at 600 nm that gives a log bacterial count of 7.4 was determined for each strain. The bioreactor was assembled and used for in vitro biofilm formation by individual strains. A combination of 3 strains and 4 different types of membranes obtained from a membrane manufacturer was used in this stage of study to form in vitro biofilm under dynamic conditions. The number of cells in biofilm formed on each of the 4 membranes was counted. Activities and Events: MS Thesis
entitled: Application of salt whey in process cheese food made from young cheddar cheese containing exopolysaccharides was approved. Products: A US patent application on utilization of exopolysaccharide-producing cultures in low fat cheese has been submitted. PARTICIPANTS: Dr. Ashraf Hassan (PI): planning and conducting research, data analysis and interpretation, and report writing. Dr. Jongwoo Choi: conducting research. Collaborators: Dr. Sanjeev Anand and Dr. Vikram Mistry. This project provided training to a postdoctoral associate and graduate students. TARGET AUDIENCES: Target Audiences: 1) Dairy and Food Industry, 2) Dairy and Food Scientists. <p>PROJECT MODIFICATIONS:<br/> Nothing significant to report during this reporting period.
<p>PROGRESS:2010/01/01 TO 2010/12/31<br/>OUTPUTS: Study 1. The objective of this study was to evaluate biofilm formation on polyamide reverse osmosis (RO) whey concentration membranes. Biofilms were observed with scanning electron and fluorescence microscopy. For scanning electron microscopy, pieces of 6 and 12, and 14 mo old membranes were allowed to air dry at room temperature (22 C) for 24 h followed by sputter coating with a 5 nm layer of gold and microscopic observations. Study 2. The objective of this study was to maximize the level of salt whey in making process cheese food (PCF). In order to achieve this goal, low salt (0.6%) Cheddar cheese was utilized. Since salt reduction causes undesirable physiochemical changes during extended cheese ripening, young Cheddar cheese was used. In young cheese, the intact casein level is high, thus giving undesirable melting and
emulsifying profile for process cheese manufacture. Therefore, an exopolysaccharides (EPS) producing culture (JFR) was used to reduce cheese rigidity and improve meltability. An EPS-nonproducing culture (DVS) was applied in making the control cheese. In order to obtain similar composition in the EPS-positive and negative Cheddar cheeses, the making protocol was modified in the later cheese to increase its moisture content. This was done using a different cooking profile. Cheese was pressed for 18 hours, and then vacuum sealed and ripened for three weeks at 4 C. The composition of Cheddar cheese was determined to formulate the PCF (JFR-PCF and DVS-PCF). Three week old Cheddar cheeses containing 41.4% moisture, 31% fat and 21.2% protein, were shredded, and stored frozen until used for PCF manufacture. Study 3. The objective of this work is to produce whey protein concentrates with improved
functional properties. Exopolysaccharides (EPS) are high molecular weight polymers secreted by microorganisms into the surrounding environment. Whey protein concentrate is produced from whey using membrane separation techniques that remove sufficient non-protein constituents from whey so that the finished dry product contains not less than 25% protein. Protein-EPS interactions have the potential to improve the functional properties of whey protein concentrates. Whey protein concentrates were prepared from ultrafiltered whey inoculated with 5% of whey fermented with different EPS-producing and nonproducing cultures. Two EPS-producing cultures (Lactococcus lactis ssp. cremoris JFR and Lactobacillus paraplantarum 868) and one EPS-nonproducing culture (DVS 850) were used in this study. Concentrated whey was then dried in a single stage dryer at an outlet air temperature of 75 C. Whey protein
concentrates were analyzed for chemical composition (moisture, protein, fat and ash) and functional properties (solubility, foam overrun, emulsifying capacity, gelling properties, and denaturation). <p>PARTICIPANTS:<br/> Individuals: Dr. Ashraf Hassan (PI). Planning and conducting research, data analysis and interpretation and manuscript and Thesis writing. Dr. Vikram Mistry (PI). Planning research, data interpretation and manuscript writing. Dr. Sanjeev Anand (PI). Planning research, data interpretation and manuscript writing. Dr. Lloyd Metzger (collaborator). Planning research, data interpretation and manuscript writing. Oliver Janvski, Gagan Deep, and Mallika Avadhanula (graduate students). Conducting research, data analysis, interpretation and presentation at scientific meetings and Thesis and manuscript writing. <p>Partner Organizations:<br/> Alexandria University, Egypt. TARGET AUDIENCES: Dairy
and Food Industry. Dairy and Food Science scientists. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Mistry, Vikram; Hassan, Ashraf; Anand, Sanjeev
South Dakota State University
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