Thesis (Ph.D.)--University of Western Sydney, 2007. / A thesis submitted to the University of Western Sydney, College of Health and Science, Centre for Plant and Food Science, in fulfilment of the requirements for the degree of Doctor of Philosophy. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Two factors that are important in the production of the typical body and texture of cheddar cheese are acid development and milk fat content. When either of these is absent or present in less-than-normal amounts, the resulting cheese shows marked defects of curdiness and firmness.

This study sought to determine whether a recently isolated slow-growing nonstarter lactic acid bacterium, Lactobacillus wasatchii sp. nov., could be implicated in late gassy defect in Cheddar cheese. I demonstrated that Lb. wasatchii grows readily in the laboratory under cheese-like stress conditions of 5% salt and pH 5.2, and has the potential to survive pasteurization. Lactobacillus wasatchii can co-utilize ribose and galactose to maximize its growth. Due to being an obligatory heterofermentative, Lb. wasatchii produces CO2 whenever it ferments a hexose such as galactose. A second investigation extended these findings by examining the growth and gas forming characteristics of Lb. wasatchii in Cheddar cheese. The optimum growth of Lb. wasatchii and highest levels of gas production were observed in cheese supplemented with ribose plus galactose, and stored at 12°C rather than 6°C. Lactobacillus wasatchii also grew readily and produced gas in Cheddar cheese even without added ribose and galactose, which corresponds with the ability of Lb. wasatchii to grow on starter cell lysate. A challenge still remains of how to easily enumerate Lb. wasatchii in cheese with a higher background population of other nonstarter lactic acid bacteria. The third set of experiments explored the consequences on growth and gas production of Lb. wasatchii in Cheddar cheese made with Streptococcus thermophilus. Using St. thermophilus in cheesemaking results in galactose accumulation, which Lb. wasatchii then can utilize for growth, causing release of CO2 with the end result of having blown Cheddar cheese. Results showed Lb. wasatchii or similar nonstarter lactic acid bacteria are likely to be particularly problematic in cheesemaking involving starter or adventitious St. thermophilus. From these observations, it was concluded that Lb. wasatchii is a contributor to late gassy defect in Cheddar cheese and may be widely present as part of the nonstarter lactic bacteria population but has been undetected up until now. The late gassy defect is more pronounced at temperatures used for accelerated ripening of cheese and when there are substantial residual levels of galactose in the cheese. Thus, researchers and cheese manufacturers now must consider slow-growing obligatory heterofermentative nonstarter lactic acid bacteria when dealing with late gassy defect in cheese.

Recent studies suggest aromatic amino acid catabolism by starter lactococci and flavor adjunct bacteria have a significant impact on off-flavor development during Cheddar cheese ripening. We hypothesized that a flavor adjunct bacterium, Brevibacterium linens BL2, produces off-flavor compounds from aromatic amino acid metabolism that will have a detrimental impact on cheese flavor. The mechanism of tryptophan (Trp) catabolism in Brevibacterium linens BL2, was investigated in a chemically defined medium during incubation in laboratory conditions (no carbohydrate, pH 6.50, 220 rpm, 25°C) and cheese-like conditions (no carbohydrate, 4% NaCl, static incubation, l5°C). In laboratory conditions, metabolic studies and enzyme assays confirmed that Trp was converted to kynurenine and anthranilic acid. However, cells incubated in cheese-like conditions did not utilize Trp, indicating that these enzymes are not likely to be involved in formation of Trp compounds associated with off-flavors in Cheddar cheese. In an attempt to verify the metabolic activity of the cells during incubation by monitoring the amino acid metabolism in chemically defined medium inoculated with B. linens BL2, a capillary electrophoresis-laser-induced fluorescence method was developed that could separate, detect, and quantitate 18 amino acids within 38 min. The data indicated that B. linens BL2 was metabolically active. Presumably, the cells will be metabolically active and metabolize amino acids in cheese as well. The ability to determine the Trp metabolic activity of B. linens BL2 in cheese, and to quantify Trp catabolic compounds in cheese during ripening, requires a quantitative extraction procedure. An analytical method was developed to extract and quantify aromatic amino acids and Trp catabolites from cheese using capillary electrophoresis. Methanol was used to extract Cheddar cheese made with Lactococcus lactis S3 alone and in combination with B. linens BL2 to quantitatively determine the influence of BL2 on the occurrence of aromatic catabolites. All cheeses contained aromatic amino acids, indole acetic acid, and indole. The concentration and time taken for development of these compounds were significantly decreased or delayed by the addition of B. linens BL2. After 6 months of aging, the concentrations of Trp catabolites were significantly lower in cheese made with B. linens BL2. Addition of BL2 did not directly contribute to off-flavors derived from Trp catabolism in Cheddar cheese. Therefore, the hypothesis was rejected.

The purpose of this dissertation was to study factors affecting moisture distribution in 290-kilogram stirred-curd Cheddar cheese blocks cooled in stainless steel hoops. Uneven moisture distribution within blocks may create cheese with variable texture and flavor, which can be extremely costly to the producer. The effects of temperature, pH, and vacuum treatment on moisture distribution were investigated. Temperature, pH, moisture, and pressure profiles were presented. Also, comparisons were made between temperature profiles of 290- kilogram stirred-curd Cheddar cheese blocks cooled in stainless steel and in plywood hoops, as well as between temperature profiles of 66-kilogram Swiss cheese blocks cooled in cardboard and in plastic boxes. Moisture transferred from high to low temperature in the cheese blocks. Moisture may have transferred in response to thermally induced curd moisture-holding capacity gradients in the cheese blocks. Moisture also may have transferred in the cheese blocks by a mechanism similar to thermo-osmosis of liquids in porous solids. The cheese in the plywood or cardboard insulating materials cooled more uniformly than the cheese in the stainless steel or plastic containers. More uniform cooling of the cheese produced more uniform moisture distribution in the cheese blocks. Recommendations were made to help the cheesemaker produce cheese with even moisture distribution.

A suitable microtome sampling technique was used to sample cheese for analysis using FTIR spectroscopy. Well-separated fat- and protein-related bands were obtained in the spectra of Cheddar and Mozzarella cheese samples using this method. The absorbance intensity of the spectra was proportional to the thickness of the sample. The intensity of absorbance and fat- and protein-related bands increased with an increase in the fat and protein content in the sample. Strong and well-separated bands at 1744, 1450, 1240, 1170, and 1115 cm-1 arising mainly from fat content were observed using this method. Bands observed at 1650 and 1540 cm-1 were attributed to the protein present in the cheese. Bands at 1615-1639, 1640-1648, 1650-1658, and 1660-1688 cm-1 corresponding to β-sheet, random coil, helix, and the turns/sheet portion of the secondary structure were observed int he range of the amide I band. Characteristics of spectra for full-fat (FFCC) and reduced-fat Cheddar cheese (RFCC) during ripening were investigated. The absorbance of bands at 1116-1240 from C-C, C-O, C-N stretch, 1461 cm-1 from C-N bend (scissoring), 1744 cm-1 from ester carbonyl groups (fat A), 2850-2930 cm-1 from C-H stretch (fat B), 1650 and 1540 cm-1 from protein amide I and II varied druing cheese aging. Bands at 1116 and1240 cm-1 arising from C-O, C-N, and C-C stretch changed slightly during cheese aging. A correlation coefficient of 0.97 for bands between 1744 and 1167 cm-1 arising from fat, and that of 0.93 at 1650 and 1540 cm-1 arising from protein, respectively, showed that one of these fat or protein groups was highly related to the other. A correlation coefficient of greater than 0.80 among the bands of fat and protein groups indicated a strong interaction in those bands. Correlation of ripening time and absorbance at bands corresponding to each function group was analyzed. A ripening index model was obtained by correlating ripening time with predominant reactive group absorbance peaks. An R2 of 0.83 and 0.59 was obtained for full-fat and reduced fat Cheddar cheese, respectively. Texture development and its correlation with FTIR spectra data for FFCC and RFCC during aging were also studied. RFCC had a higher value of hardness, gumminess, and chewiness than its full-fat counterpart. The values decreased during the early stages of ripening and then increased with time. The change in hardness, adhesiveness, and springiness was expressed as a function of the change in absorbance of the FTIR spectra using multiple regression analysis. An R2 value of 0.67, 0.54, and 0.75 was obtained for full-fat Cheddar cheese, and a value of 0.51, 0.59, and 0.54 was obtained for reduced-fat Cheddar cheese for the respective texture parameters.

The manufacture of cheddar cheese is greatly dependent on bacterial growth for acid production. The quality of cheese depends upon the type and extent of microbial activity. An important function of the lactic fermenting bacteria is the production of acid resulting from cellular metabolism. If little or no acid is production of acid resulting from cellular metabolism. If little or no acid is produced the resulting cheese will have an inferior body, flavor, and texture and may even cause the cheese to be used as grinders. The major causes for inhibited lactic bacterial growth are poor starter handling procedures, antibiotic in mild coming from cows treated for mastitis, quaternary ammonium compounds used in plant sanitation, and bacteriophage. The occurrence of bacteriophage contamination and the increased use of quaternary ammonium compounds and antibiotics have caused a serious problem in cheese manufacturing. Purpose of project. The purpose of this project is to determine if cheddar cheese, comparable to normal cheese, can be made from slow or non-acid milk with the use of added lactic acid. Lactic acid will be used for the purpose of substituting for the acid that is normally produced by bacterial metabolism in the manufacture of cheddar cheese.

A modification of a linear diffusion test for measuring milk clotting enzymes at concentrations of 1 x 10^-4 to 1 x 10^-1 chymosin units/ ml was developed to permit quantitative assay of porcine pepsin in Cheddar cheese with a standard deviation of 6%. The amount of porcine pepsin retained in Cheddar cheese curd was dependent on pH of milk at setting. Milk at pH 6.6, 6.4, 6.2, and 6.0 was set with porcine pepsin, acidified with lactic acid and glucono-~-lactone and made into Cheddar cheese. After pressing the corresponding curd contained 0, 3.64+0 .12 %, 4.79+0.52%, and 5.94+0.30% of the pepsin activity added to the milk. Polyacrylamide gel electrophoresis of cheese revealed increasing degradation of the ~s-casein band with increasing residual pepsin in the curd. However, some degradation of the ~s-casein band was evident in curd set at pH 6.6 which showed no residual pepsin. Curd was made by ultrafiltration from whole milk followed by acidification to pH 5.2 with hydrochloric acid and glucono-6- lactone and vacuum evaporated to 39% moisture at 40 C (no clotting enzymes and no starter) . Degradation of the ~s- casein in this curd was similar to that observed in pepsin cheese set at pH 6.6. All degradation in ~s-casein cannot be attributed to milk clotting enzymes or starter bacteria. Porcine pepsin does not contribute to protein digestion in cheese curd during ripening unless the milk is below pH 6.6 at setting.

Background: Manufacturers continue to look for methods to save time in aging cheese. Complex reactions occur during cheese ripening that produce characteristic flavors and aromas. The addition of exogenous lipase enzymes is one accelerated ripening method that has been studied. Our objective was to document and compare the flavor profiles of cheddar cheeses created with either microbial lipase or animal lipase. Materials and Methods: The experiment followed a conventional formulation to create experimental batches of cheddar cheese. Kid goat lipase and three microbial lipase treatments were compared against a control. Other animal lipases were evaluated in preliminary research but were omitted in the final experiment due to bitter and unfavorable flavor development. Objective cheese parameters evaluated were texture, moisture, volatile, and free fatty acid (FFA) analysis. We observed subjective sensory properties through quantitative descriptive analysis. Results: The results of the current study showed that the addition of exogenous lipases caused minimal changes in cheese moisture and a reduction in firmness in cheese samples. Various levels of microbial and animal lipase in Cheddar cheese showed significant differences in the FFA profile of the aged cheese. Microbial lipases tended to liberate more medium and long-chain fatty acids while animal lipases tended to liberate more short-chain fatty acids. Mucor javanicus lipase was an exception among microbial lipases and liberated relatively greater amounts of short-chain fatty acids. The addition of exogenous lipase had significant impacts on cheese volatiles. Acetic acid levels decreased in the lipase-treated samples and there were changes in odd-numbered FFA. In terms of sensory analysis, the lipase-treated cheeses showed an increase in FFA flavor notes. Conclusions: If a microbial lipase were to replace kid goat lipase in the production of Cheddar cheese, the results of the current study show that Mucor javanicus microbial lipase would result in the closest free fatty acid profile and sensory characteristics to that of kid goat gastric lipase. Future studies could investigate the addition of animal lipases in cheese that traditionally makes use of exogenous lipases, such as Blue cheese, Emmental Parmesan, Romano, Provolone, and Feta cheeses.