Most naturally occurring folate derivatives in foods are highly sensitive to temperature, oxygen, light, and their stability is affected by food processing conditions. Edible polysaccharides (hydrocolloids) were evaluated for folic acid encapsulation, both as single and mixed polymers as a way of increasing folic acid stability. Results obtained from the study demonstrate for the first time dietary incorporation of encapsulated folic acid using Cheddar cheese as the delivery vehicle mitigates against hyperhomocysteinemia and monocyte/macrophage adhesion in mice. / Doctor of Philosophy (PhD)

Please read the abstract in the section, 00front , of this document / Dissertation (M Inst Agrar ( Food Processing))--University of Pretoria, 2008. / Food Science / MInstAgrar / unrestricted

Whey from a commercial cheese plant, taken at draining on five separate days, from cheese made with a Mucor miehei coagulant was cooled within 1 h to 4 C. Portions were adjusted from pH 4.2 to 6.4 at .2 pH intervals and subjected to HTST pasteurization at 73.9, 76.6, and 79.5 C for 25 sec. Milk clotting activity in whey was determined before and after pasteurization. Resistance to heat in-activation increased with decreasing pH. All measurable activity was destroyed above pH 5.4 by pasteurization at 79.5 C, above pH 5.8 at 76.6 C and above pH 6.0 at 73.9 C. Milk clotting activity in Cheddar cheese mad with Mucor miehei remained unchanged for 26 weeks. Four commercial sterile liquid-milk-based consisting of infant formula, concentrated infant formula, nutritionally complete food, and diet food was aseptically inoculated with sterile Mucor miehei protease solutions to concentrations ranging from 5 x 10-3 to 1 x 10-7 chymosin units/ml of product. The samples were stored at 30 C. After 20 weeks there was no change in the nutritionally complete food. The diet food showed slight whey separation and thickening at 1 x 10-4 CU/ml and coagulation at higher concentrations. The infant formula showed definite whey separation and thickening at 1 x 10-4 CU/ml and coagulation and higher concentrations. The concentrated infant formula showed visible thickening at 1 x 10-3 CU/ml and coagulation at higher concentrations.

Low-fat cheese faces great challenges associated with its texture being hard and rubbery, desirable flavors being missing, color being undesirably intense and translucent appearing, and melting being improper. In an effort of improving the quality of low-fat cheeses, several strategies have been tried to accomplish three major objectives, 1) improving the melting and baking properties of low-fat Mozzarella cheese, 2) improving the color of low-fat Cheddar cheese, and 3) investigating the feasibilities of enriching low-fat Cheddar cheese with dietary fibers. For objective 1, 4 batches of low-fat Mozzarella cheese with target fat of 6.0%, 4.5%, 3.0%, and 1.5% were made using a stirred curd method, comminuted in a bowl chopper and mixed with different levels of melted butter (0.0, 1.5, 3.0, and 4.5% (wt/wt), respectively) before pressing. This would made the cheese that had increased free oil, increased melting, and improved baking as the level of added butter increased. The added butterfat was present as free fat along the curd particle junctions as shown by laser scanning confocal microscopy while the fat droplets originating from the milk were distributed within the protein matrix of the cheese. In objective 2, consumer tests and flavor profile analysis were performed on 4 commercial brands of full-fat Cheddar cheese and 9 low-fat Cheddar cheeses manufactured at Utah State University with different colors. Low-fat cheeses were rated different (P < 0.05) for their liking by a consumer panel even though they were all made the same way except for addition of color. The only difference in flavor detected by a trained panel was for a slight variation in bitterness. Using a combination of annatto and titanium dioxide produced a cheese that was rated the highest. Annatto when added singly produced a low-fat cheese that was rated the lowest. Moreover, commercial cheeses were also ranked significantly different for liking and buying preference. For objective 3, several trials were conducted to enrich low-fat cheese with inulin, pectin, polydextrose, or resistant-starch either by incorporating them into cheesemilk, mixing with 15-d aged cheese followed by repressing, or by formulating a W/O/W emulsion with inulin and incorporating the emulsion into the milk prior to cheesemaking. Adding fibers directly to milk resulted in less or no retention of fibers in cheese, whereas fibers added to comminuted cheeses were too crumbly. Adding fiber as a W/O/W emulsion improved fiber retention in the cheese and produced an improved texture of low-fat cheese.

Low-fat Cheddar cheese flavor is different from full-fat Cheddar cheese and is not acceptable to many consumers. This 2-part experiment was designed to examine effects adjunct cultures have on low-fat Cheddar cheese flavor as determined through descriptive analysis and consumer feedback. In Part 1, low-fat (5%) Cheddar cheese was produced in duplicate, using 6 combinations of DVS850, LH32, CR540, CRL431, Emfour, and CR319 bacterial cultures. Due to a previously observed positive effect by sodium gluconate on low-fat cheese flavor, each replicate was split into treatments of 0.0% and 0.8% sodium gluconate. Each of these treatments was then split into ripening temperature treatments: 6°C for 21 ± 1 wk; or 6°C for 3 wk, 10°C for 8 wk, and 6°C for 10 wk. Cheese was tasted first by an informal panel. The 4 treatment combinations for the control cheese and the CR540 (a Lactococcus lactis ssp. and Lactobacillus ssp. blend) cheese, along with all culture combinations containing sodium gluconate and ripened only at 6°C, were selected for descriptive analysis. Some statistically significant differences in culture treatment were observed. Sodium gluconate addition had a positive influence on flavor while elevated ripening temperature resulted in undesirable flavor notes. Low-fat (5%) Cheddar cheese with the CR540 adjunct with and without sodium gluconate was evaluated in a consumer taste panel with commercial full-fat (33% fat) and commercial reduced-fat (25% fat) Cheddar cheese. The low-fat cheeses were not significantly different from the commercial reduced-fat, indicating comparable cheese. Part 2 involved making Cheddar-like cheese with non-Cheddar adjunct cultures, using the same process as Part 1. Sodium gluconate was again added but elevated ripening temperature was not included. Each treatment was also divided into a sodium treatment, full salt (2%) and reduced salt (1.5%). After 2 mo of storage at 6°C, cheese was tasted by an informal panel and found to be bitter because of the starter culture used. A culture was added to the second replicate of the experiment to reduce bitterness. This adjunct was found to be somewhat effective in reducing bitterness but not entirely. Descriptive analysis was performed on the high salt level treatments for both replicates. Some difference was observed among cultures and sodium gluconate treatments; however, no acceptable cheese was produced due to bitterness in both replicates. Sodium treatments were not analyzed.

Cheddar cheese was made by the traditional 4.5-h method from three experimental lots of milk, each standardized to casein/fat ratios of approximately 0.64, 0.67 and 0.70. The effect of casein/fat ratio on milk fat recovery was determined. The effects of milk composition on curd firmness at cutting, cheese composition and resulting yield of cheese were evaluated. Correlations between milk constituents and various cheese components were obtained. Milk fat recovery was unaffected by casein/fat ratios within the limits of 0.64 and 0.71. Average milk fat recovery was 91.58 ± 1.73%. Cheese yield was a function of milk protein, milk fat and cheese moisture; and a modified Van Slyke equation predicted cheese yield better than the original equation within the limits of casein/fat ratio studied. Strong negative correlations were observed between casein/fat ratio and cheese fat and cheese fat in the dry matter whereas positive correlations were observed between casein/fat ratio and cheese protein. At constant protein levels curd firmness increased directly with the amount of fat in cheese milk.

In many countries of the world and in some parts of the United States milk is produced which has a high bacterial contamination. Such milk of undesirable quality is frequently delivered to factories engaged in the manufacture of cheddar cheese. This milk commonly contains large numbers of lactic acid-producing bacteria or other types of microorganisms which cause objectionable flavors and textural defects in the cheese. The improvement of the quality of milk supply under some conditions is a matter of great difficulty so that the manufacture of inferior quality milk into cheese is a problem often encountered. In the United States pasteurization of milk is used to reduce the bacterial content and give the cheese maker control over the manufacturing process. Public health officials favor pasteurization as a protection against pathogens; however, in many areas of the world pasteurization is not available. Although pasteurization of milk for cheddar cheese offers certain advantages such as destruction of pathogenic bacteria which may be present, and control of certain undesirable fermentations, experience has shown that pasteurized milk cheese develops flavor slowly and, even with extended ripening, does not have as satisfactory a flavor as good raw milk cheese. The slow ripening usually is attributed to the destruction by heat of certain essential bacteria and enzymes normally present in milk. Pasteurization, however, destroys many enzymes indigenous to milk as well as some beneficial organisms; consequently, cheese made from pasteurized milk ripens more slowly than cheese made from raw milk. For years, leading dairy technologists have been laboring assiduously but quite unsucessfully to produce cheese free from undesirable organisms yet comparable in flavor and in the rapidity of ripening to the best quality of raw milk cheese. Pursuant to these objectives a number of methods such as replenishing the enzymes in milk destroyed by pasteurization, the use of select ripening cultures, and the use of mixtures of various percentages of raw and pasteurized milk have been tried but without complete success. These objectionable features of pasteurization led to interest in another method such as the treatment of milk with edible hydrogen peroxide to control fermentation by means of its germicidal and inhibitory action. This comparative study was conducted to determine the effect of the germicidal properties of hydrogen peroxide in treating raw milk for cheddar cheese making in relation to the flora, quality, and ripening of the cheese. This study was concerned with the remedial measures which can be applied to milk to overcome some defects in the cheese. The antiseptic and germicidal properties of hydrogen peroxide are well known. A study involving the use of hydrogen peroxide and catalase has many possibilities in the dairy industry, and the practical aspects of this problem are numerous. Some phases are herewith indicated: 1. If hydrogen peroxide could be used to improve the general quality of cheddar cheese, it would be a boon to the industry and should have a value in the manufacture of cheddar cheese for shelf curing purposes, canning, processing, and for natural ripening in transparent packages. 2. It was believed that the use of hydrogen peroxide and catalase would increase the safety of raw milk cheese. (Kernsman, 1934, found that 0.1 percent of hydrogen peroxide killed E. coli, E. typhi and staphilococcus.) 3. If hydrogen peroxide could be used for destroying organisms harmful in milk and thus for preventing undesirable fermentation, yet leave intact more of the natural enzymes than is possible in accepted pasteurization procedures, the cheese treated with hydrogen peroxide and catalase might ripen faster than pasteurized-milk cheese and have a finer and more pronounced flavor. 4. If approved by public health authorities in the United States, treating milk with hydrogen peroxide would be a simple method of reducing bacterial content in small communities and rural areas. Such procedure would be very practical in preventing growth of bacteria in milk produced under unsanitary conditions. 5. If the use of hydrogen peroxide could be proved practicable, a beneficial program in most countries and especially in the Middle East where dairy equipment and pasteurizers are not readily available and where the production of unsanitary milk predominates might be established. 6. Since this process does not require special equipment it might prove economical and might become, in the future, a useful method of reducing the bacterial content of milk and preserving some of the natural characteristics of the raw milk for cheese making.

Residual rennin in cheese whey and curd was measured by using a special sensitive substrate. The substrate was made by reconstituting 6 g NDM in 500 ml of buffer containing 0.2 M CaCl2, 0.5 M cacodylic acid and 0.2 M triethanolamine at pH 5.7. Cheese curd was blended into a 1:7 slurry (1 part curd, 7 parts water), and 1.67% sodium chloride was added to the why and slurry to liberate residual rennin from casein. The residual rennin in cheese whey and slurry were determined simultaneously with an identical sample containing known rennin activity. Samples with known activity were prepared by destroying the residual rennin in unknown samples after which a known amount of rennin was added back to a standard. The examine the effectiveness of this method for measuring rennin activity in whey or slurry, a recovery test was developed to measure rennin activities in the whey and curd made by centrifuging rennet-coagulated milk. The average total recovery from 15 replications was 101.6 ± 2.4%. It was found that pH was a main factor affecting rennin distribution between whey and curd. The amount in the curd increased with decreasing pH at setting. Adding 0.02% CaCl2 to milk was of little effect. The results showed that 68.1 6 ± 6.6% of the residual rennin was found in Cheddar cheese whey after dipping and 17.2 6 ± 2.6% of the residual rennin was in the curd after milling.

La présente étude visait à évaluer le potentiel de cultures lactiques bioprotectrices de même que leurs métabolites à activité antimicrobienne (bactériocines) pour le contrôle de Clostridium tyrobutyricum dans du fromage de type Cheddar et la prévention des défauts de texture et de saveur qui lui sont associés.Trois cent quarante et une souches de bactéries lactiques ont été criblées in vitro pour leur capacité à produire des molécules antimicrobiennes actives contre C. tyrobutyricum ATCC 25755. Trois isolats identifiés comme étant des Lactococcus lactis ssp. lactis ont montré une activité inhibitrice significative contre C. tyrobutyricum. L’opéron codant pour la production de nisine A a été identifié chez ces trois isolats alors que la production de nisine a été confirmée par des tests de diffusion sur gélose contre C.tyrobutyricum. Des essais d’interaction et de biocompatibilité entre ces souches productrices de nisine A et des ferments industriels pour fromage Cheddar ont permis de définir un ferment protecteur mixte comprenant un Lactococcus lactis ssp lactis CUC-H, lactococcus lactis ssp cremoris CUC222 et Lactococcus lactis ssp lactis 32 producteur de nisine A. D’autre part, de la nisine A a été produite et purifiée à partir du surnageant de culture de la souche Lactococcus lactis ssp lactis 32 puis encapsulée dans des billes constituées d'alginate et d'amidon non gélatinisé. Le ferment mixte protecteur de même que la nisine purifiée encapsulée ont été testés pour leur efficacité à inhiber C. tyrobutyricum dans un caillé modèle de fromage Cheddar en utilisant deux concentrations de sel (1.3 et 2%), pendant deux semaines à 30°C et pendant un mois à 4°C. Les résultats obtenus avec les échantillons de caillé modèle ont été validés lors d’une production à l’échelle pilote de fromage Cheddar. Les résultats ont montré que C. tyrobutyricum n'a pas été détectée dans les échantillons entreposés à 4°C contenant de la nisine encapsulée à partir de la 3e semaine en présence de 1.3% de sel et de la deuxième semaine en présence de 2% de sel. Lorsque le ferment protecteur est utilisé, une diminution d'environ 0.6 log₁₀ de C. tyrobutyricum a été observée à partir de la deuxième semaine dans le caillé modèle entreposé à 4°C. D’autre part, l'analyse de chromatographie en phase liquide à haute performance (CLHP) des caillés a montré que Lactococcuslactis ssp lactis 32 était capable de produire in situ de la nisine à partir de la deuxième semaine.Les résultats de l’analyse métagénomique montrent que l'abondance relative du genre Clostridium a diminuée dans les caillés fromagers en présence aussi bien de la souche protectrice que de la nisine encapsulée, comparativement aux fromages témoins. Ces résultats confirment que l’ajout de Lactococcus lactis ssp. lactis 32 en tant que souche protectrice productrice de nisine permet de contrôler le développement de C. tyrobutyricum dans les caillés modèles de fromage cheddar. Les résultats obtenus dans le fromage Cheddar produit à l’échelle pilote ont montré une réduction de 1log₁₀ de C. tyrobutyricum dans les groupes traités aussi bien avec la nisine encapsulée qu’avec la souche protectrice. De plus, l’analyse de l’activité protéolytique des fromages a montré une augmentation significative de la fraction d’azote soluble dans l'eau en présence de la souche protectrice ou de la nisine encapsulée. En revanche, les teneurs en azote des fractions TCA et PTA étaient plus élevées dans le groupe contenant de la nisine encapsulée. En conclusion, les deux stratégies utilisées dans le cadre de cette étude basées sur l’utilisation de lanisine purifiée encapsulée ou de la souche L. lactis ssp lactis 32 productrice de nisine semblent être efficaces à différents niveaux pour le contrôle de C. tyrobutyricum dans du fromage Cheddar. La présence de nisine dans la matrice fromagère semble également avoir des effets significatifs sur l’activité protéolytique globale de la matrice fromagère. Ce résultat suggère des effets potentiels sur la vitesse de maturation des fromages ainsi que sur leurs caractéristiques organoleptiques et sensorielles. / The present study aimed to assess the potential of bioprotective lactic acid cultures as well as their metabolites with antimicrobial activity (bacteriocins) for the control of Clostridium tyrobutyricum in Cheddar-type cheese and the prevention of texture and flavour side effects.Three hundred forty-one strains of Lactococci have been screened in vitro for their ability to produce antimicrobial molecules active against C. tyrobutyricum ATCC 25755. Three isolates identified as Lactococcus lactis ssp. lactis showed significant inhibitory activity against C. tyrobutyricum. The gene coding for the production of nisin-A has been identified in these three isolates while the production of nisin has been confirmed by agar diffusion test against C. tyrobutyricum ATCC 25755. Interaction and biocompatibility assays between these nisin-A producing strains and industrial starter for Cheddar cheese allowed to define a mixed protective starter comprising a Lactococcus lactis ssp lactis CUC-H,a Lactococcus lactis ssp cremoris CUC 222 and Lactococcus lactis ssp lactis 32 as a nisin-A producer. Nisin-A was also purified, then encapsulated in vesicles made of alginate and non-gelatinized starch.The effectiveness of the protective mixed starter as well as the encapsulated nisin were tested for their effectiveness in inhibiting C. tyrobutyricum in a Cheddar cheese slurry using two different salt concentrations (1.3 and 2%), for two weeks at 30 °C or for one month at 4 °C. The results obtained with the cheese slurry samples were validated during a pilot scale production of Cheddar cheese.The results showed that C. tyrobutyricum was not detected in the samples containing encapsulated nisin from third week in the presence of 1.3% salt and from second week in the presence of 2% salt at 4 °C. When the protective starter is used, a progressive decrease in the number of C. tyrobutyricum, of approximately 0.6 log₁₀, was observed from the second week in cheese slurry stored at 4 °C. High performance liquid chromatography (HPLC) analysis indicated that the protective culture was capable of producing nisin in situ since the second week.The results of the metagenomic analysis showed that the relative abundance of the genus Clostridium decreased in cheese samples in the presence of both the protective strain and the encapsulated nisin, compared to the control. These results confirm that the addition of Lactococcus lactis ssp. lactis 32 asa nisin-A producing strain helps in controlling C. tyrobutyricum in cheddar cheese slurries. In Cheddar cheese produced at a pilot scale, a reduction of 1.0 log₁₀ in the number of C. tyrobutyricum was obtained in cheeses treated with both the encapsulated nisin and with the protective strain. In addition, analysis of the proteolytic activity of cheeses showed a significant increase in the water-soluble nitrogen (WSN) fraction in the presence of the protective strain or of the encapsulated nisin. On the other hand, the TCA- soluble nitrogen and PTA- soluble nitrogen fractions were higher in the encapsulated nisin group. In conclusion, the two strategies used in this study based on the use of encapsulated nisin or ofNisin-producing Lactococcus lactis appear to be effective at various extend for the control of C.tyrobutyricum in Cheddar cheese. The presence of nisin in the cheese matrix also seems to have significant effects on the overall proteolytic activity of the cheese matrix. This result suggests potential effects on the ripening speed of cheeses as well as on their intrinsic organoleptic and sensory characteristics.

Les ferments utilisés dans la fabrication du fromage Cheddar, généralement composés de combinaisons de souches de Lactococcus lactis ssp. cremoris, ont un impact important sur la qualité du produit final. Une dynamique d’association est susceptible de s’installer entre les souches présentes. L’objectif de cette étude visait à distinguer les différents groupes de souches de L. lactis ssp. cremoris sélectionnées et à quantifier les proportions de chaque groupe de souches utilisées comme ferment afin d’étudier leur comportement lors d’une fabrication de fromage Cheddar. Lors de l’analyse MLST, le gène nusA s’est démarqué des autres car il a permis de séparer les 23 souches en six groupes intéressants. Une méthode PMA-qPCR spécifique a donc été développée avec ce gène pour cible. Cette technique a été utilisée pour l’évaluation des proportions des souches en combinaisons pendant une simulation de production fromagère. Cette méthode pourrait avantageusement être transférée à l’industrie puisqu’elle est accessible, rapide et reproductible. / Starters used in Cheddar cheesemaking have a major impact on the quality of the final product. These starters are usually composed of Lactococcus lactis subsp. cremoris strain combinations and interactions between strains can be present. The main goal of this study was to discern groups of L. lactis subsp. cremoris and to quantify proportions of each strain group with the objective of studying their behavior during Cheddar cheesemaking. The MLST study showed that the nusA gene classifies strains into six interesting groups. Therefore, a specific PMA-qPCR method was developped with this target gene. The technique was used to quantify strain proportions during cheesemaking simulation. This method could be transferred to the industry because it is easy to use, fast and reproductible.