Ashraf Hassan

ADSA Foundation Scholar Award: Possibilities and Challenges of Exopolysaccharide-Producing Lactic Cultures in Dairy Foods

Exopolysaccharides (EPS) from lactic acid bacteria are a diverse group of polysaccharides exhibiting various functional properties. Two forms of EPS are produced by lactic acid bacteria: capsular and unattached. Capsular EPS does not cause ropiness nor does production of unattached EPS ensure ropiness. The functions of EPS in dairy products are not completely understood. This is for 2 main reasons: the major variations among exopolysaccharides even from the same group of micro-organisms, which makes it difficult to apply information from one EPS to others, and the lack of availability of techniques with the ability to observe the microstructure and distribution of the highly hydrated EPS in fermented dairy products. The introduction of relatively new microscopic techniques such as confocal scanning laser microscopy and cryo-scanning electron microscopy made it possible to directly observe the distribution of fully hydrated EPS in dairy products. Recently, EPS produced by nonropy strains have drawn the attention of the dairy industry. This is because of the ability of some nonropy strains to produce large capsular and unattached EPS that would improve the texture of dairy products without causing the undesirable slippery mouthfeel produced by the ropy strains. Factors affecting functions of EPS are their molecular characteristics and ability to interact with milk proteins. Studying the interaction between EPS and milk proteins is complex because EPS are gradually produced during fermentation, unlike polysaccharides added directly to milk to stabilize the fermented product. The concentration and possibly molecular characteristics of EPS and protein characteristics such as charge and hydrophobicity change during fermentation. Consequently, the interaction of EPS with proteins might also change during fermentation. Exopolysaccharides provide functions that benefit reduced-fat cheeses. They bind water and increase the moisture in the nonfat portion, interfere with protein-protein interactions and reduce the rigidity of the protein network, and increase viscosity of the serum phase. This review discusses the production of capsular EPS and their role in structure formation in fermented milk, the mechanism of ropiness formation, and applications of EPS-producing cultures in reduced-fat cheeses.

Rheological, sensorial, and chemopreventive properties of milk fermented with exopolysaccharide-producing lactic cultures.

The objective of this research was to evaluate the rheological, sensorial, and chemopreventive properties of milk fermented with different exopolysaccharide (EPS)-producing lactic cultures. Reconstituted skim milk (11% wt/vol) was fermented with single strains of EPS-producing and non-EPS-producing cultures. Whey that collected on the surface of undisturbed fermented milks and after cutting was measured. All EPS-producing cultures reduced the amount of whey present on the surface of the undisturbed samples, whereas only 3 out of 5 strains reduced syneresis measured after cutting. All EPS-producing cultures except a strain of Lactobacillus delbrueckii ssp. bulgaricus reduced viscoelastic moduli in fermented milk. There was a linear correlation between ropiness and smoothness. In the chemoprevention study, 140 male Fisher rats were divided into 7 groups of 20 each. Rats in 6 groups were fed diets supplemented with fermented milks each made with a single strain of EPS-producing or non-producing cultures, whereas rats in group 7 (control) were fed a diet supplemented with milk acidified with glucono-delta-lactone (GDL). All rats were injected with azoxymethane (15 mg/kg, subcutaneous) at wk 7 and 8 of age to induce tumors and fed their respective diets ad libitum throughout the study. After 30 wk of initiation, all rats were anesthetized with ether, and their intestinal tissues were isolated and washed with cold normal saline. The number and size of tumors in the colon and small intestine were recorded. Rats fed diets supplemented with fermented milk made with 2 EPS-positive and 1 EPS-negative strains had significantly lowered incidence of colon tumor and colon tumor multiplicity. Cyclooxygenase-2 enzyme activity (the enzyme implicated in colon tumor development) was significantly lower in the colon tissue of rats fed diets containing milk fermented with 4 EPS-producing and 1 non-producing cultures than that in rats fed diets supplemented with GDL-acidified milk. Different EPS-positive cultures produced fermented milks with distinct rheological characteristics and levels of ropiness. No relationship was found between rheological properties or level of ropiness of fermented milk and its chemopreventive effect.

Microscopic observation of multispecies biofilm of various structures on whey concentration membranes1

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-, 12-, and 14-mo-old membranes were allowed to air dry at room temperature (22 degrees C) for 24h followed by sputter coating with a 5-nm layer of gold and microscopic observations. Scanning electron microscopy images revealed that the hydrophilic layer, used to prevent membrane plugging, was not evenly distributed on the surface. Although this hydrophilic layer seemed to prevent the attachment of proteins, it supported biofilm formation. Three different structures of multispecies biofilm were observed on the retentate side of the membrane: 1) a mono layer, 2) a 3-dimensional structure of a dense matrix of extracellular polymeric substances where different types of bacterial cells were embedded, and 3) cell aggregates. In some of the biofilms, a smooth layer (shell) covered cell aggregates. In the 6-mo-old membranes, part of the shell layer was broken off. Biofilms as observed on the RO membrane were described as having a hill-and-valley type of structure, with hills showing a mushroom-like appearance and valleys comprising dense matrices of extracellular polymers with embedded bacterial cells. Fluorescence microscopy showed live cells on the surface of the biofilm. It is concluded that both cells in the deep layers of biofilm and surface cells may resist cleaning and sanitation. The extent of biofilm formation and the presence of live cells on RO membranes after regular clean in place cycles indicate the need for a more effective cleaning regimen customized for dairy separation systems.

Characteristics of reduced fat Cheddar cheese made from ultrafiltered milk with an exopolysaccharide-producing culture

In a previous study, ultrafiltration (UF) at 1.2x reduced residual chymosin activity and bitterness in exopolysaccharide (EPS)-positive reduced fat Cheddar cheese. The objective of this research was to study the effect of this level of concentration on the textural and functional characteristics of the reduced fat cheese. Ultrafiltration (1.2x) did not affect the hardness, cohesiveness, adhesiveness, chewiness, and gumminess of EPS-positive cheese. The 6-month old UF cheeses were springier than non-UF cheeses. However, the springiness of the EPS-positive cheese made from UF milk was much lower than that of the EPS-negative cheeses. Texture of the EPS-negative cheese was more affected by UF than that of the EPS-positive cheese. Differences were seen in the extent of flow between UF and non-UF cheeses at 1 and 3-months but not after 6 months ripening. Ultrafiltration increased the elastic modulus in the 6-month old EPS-positive cheeses. Higher body and texture scores were given to EPS-positive cheeses than the EPS-negative ones. Sensory panelists found the body of the UF and non-UF cheeses to be similar.

Starter cultures and cattle feed manipulation enhance conjugated linoleic acid concentrations in Cheddar cheese

Conjugated linoleic acid (CLA) is a fatty acid (FA) that provides several health benefits to humans. The feeding of fish oil-supplemented diets to dairy cows has been extensively studied as a means to improve the CLA content in milk. Several studies have also been conducted on the ability of many microorganisms to produce CLA by utilizing substrates containing linoleic acid. In the present study, the dietary manipulated milk was used in combination with the CLA-producing culture to manufacture Cheddar cheese. The two diets fed to cattle were control and treatment diets to obtain control and treatment milk, respectively. The treatment diet containing fish oil (0.75% of dry matter) was fed to 32 dairy cows grouped in a pen for 18 d to increase the total CLA content in milk. Treatment milk had a CLA content of 1.60 g/100g of FA compared with 0.58 g/100g of FA in control milk obtained by feeding the control diet. A 2 × 2 factorial design with 3 replicates was used to test the combined effect of the CLA-producing starter culture of Lactococcus lactis (CI4b) versus a commercial CLA nonproducing cheese starter as the control culture, and type of milk (control vs. treatment milk) on CLA content in Cheddar cheese. Chemical composition (moisture, salt, fat, and protein) was not affected by the type of culture used. However, the age of the cheese affected the sensory properties and microbiological counts in the different treatments. Ripening with the CI4b culture was found to be effective in further enhancing the CLA content. The CI4b cheeses made from control milk and treatment milk contained 1.09 and 2.41 (±0.18) g of total CLA/100g of FA after 1 mo of ripening, which increased to 1.44 and 2.61 (±0.18) g of total CLA/100g of FA after 6 mo of ripening, respectively. The use of treatment milk resulted in an increase in the CLA isomers (trans-7,cis-9+cis-9,trans-11, trans-9,cis-11+cis-10,trans-12, trans-10,cis-12, cis-9,cis-11, trans-11,cis-13, cis-11,cis-13, trans-11,trans-13, and trans-9,trans-11). The CI4b culture specifically increased cis-11,cis-13 and trans-10,cis-12 isomers in cheese. The total CLA content in cheese was significantly higher when the CI4b culture was used compared with CLA nonproducing culture cheeses made from control milk and treatment milk after 1 mo [1.09 and 2.14 (±0.18) g of total CLA/100g of FA] and 6 mo [0.99 and 2.05 (±0.18) g of total CLA/100g of FA] of ripening, respectively. The results indicated that the combination of a CLA-producing starter culture and milk from cattle fed fish oil-supplemented diets (0.99 g of CLA/100g of FA) could enhance levels of total CLA in Cheddar cheese by up to 2.6 times compared with cheese made from control milk with CLA nonproducing starter culture (2.61 g of CLA/100g of FA) after 6 mo.

Inhibition of phage infection in capsule-producing Streptococcus thermophilus using concanavalin A, lysozyme and saccharides

Lactic cultures that produce capsular polysaccharides are widely used in the dairy industry. However, little information is available on their phage-cell interactions. Concanavalin A (Con A), lysozyme, and saccharides were investigated for their ability to modify phage-cell interactions in such a manner as to inhibit phage infection. The ability of phage to infect cells was determined by measuring acid production in Elliker broth. Acid production by capsule-producing Streptococcus thermophilus was inhibited less by bacteriophage when cells were pretreated with Con. A than was acid production by a capsule-free variant. The presence of 0.5 mg/ml lysozyme in Elliker broth significantly reduced phage infection. However, there was no increased effect when lysozyme and Con A were combined in the growth medium. The addition of 5 g/L of glucosamine to Elliker broth also inhibited phage infection. The results of this study indicate that it is possible to reduce phage infection of capsule-forming S. thermophilus by blocking or modifying phage adsorption sites.

Lactococcus lactis subsp. cremoris strain JFR1 attenuates Salmonella adhesion to human intestinal cells in vitro

Lactococcus lactis subsp. cremoris JFR1 has been studied in reduced fat cheese due to its ability to produce exopolysaccharides (EPS) in situ, contributing to improved textural and organoleptic properties. In this study, the effect of strain JFR1 on virulence gene expression and attachment of Salmonella to HT-29 human colon carcinoma cells was investigated. Overnight cultures of L. lactis subsp. cremoris JFR1 containing EPS, grown in M17 media with 0.5% glucose supplementation, decreased attachment as well as down regulated virulence gene expression in Salmonella enterica subsp. enterica when tested on HT-29 cells. However, EPS isolated from milk fermented with L. lactis subsp. cremoris JFR1 did not affect Salmonella virulence gene expression or attachment to HT-29 cells. These results suggest that EPS does not contribute to the attachment of Salmonella to human intestinal cells. However, the possibility that the isolation process may have affected the structural features of EPS cannot be ruled out.