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.

Cheddar Cheese samples (good cheese, weak cheese, cheese made with pasteurized milk, cheese made with heat-shocked milk, cheese from production plant A, cheese from production plant B, cheese made with adjunct culture, and cheese made without adjunct culture), were evaluated during the ripening stage. Proteolysis was studied by a fractionation scheme, resulting in an insoluble fraction analyzed by urea polyacrylamide gel electrophoresis (Urea-PAGE), and a soluble fraction which was further investigated through water soluble nitrogen (WSN), trichloroacetic acid soluble nitrogen (TCA-SN) and phosphotungstic acid soluble nitrogen (PTA-SN) analyzed by total Kjeldahl nitrogen content (TKN). Reversed phase high performance liquid chromatography (RP-HPLC) was used to study the peptide profile of the water soluble fraction. Lipolyisis was studied by levels of individual free fatty acids determined through gas chromatography-flame ionization detection (GC-FID) after isolation employing solid phase extraction (SPE). Volatile sulfur compounds were studied using head space solid phase micro-extraction (SPME) coupled with gas chromatography-pulsed flame photometric detection (PFPD). It was found that Urea-PAGE is capable to differentiate samples according their age, but cannot discriminate samples regarding the treatment assessed, quality or origin of the samples. However, measurements of total Kjeldahl Nitrogen (TKN) of the WSN, TCA-SN, and PTA-SN fractions, and the principal component analysis of the RP-HPLC peptide profile of the WSN fraction, revealed differences in the rate and pattern of proteolysis for each one of the manufacturing cases. Good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture isolated from plant TCCA cheese, and cheese made with heat-shocked milk developed higher level of total nitrogen for the WSN, TCA-SN and PTA-SN fractions, indicating that primary and secondary proteolysis were faster for these samples. This is supported by a PCA model with three principal components that account for the 80-83% of the variability of the data from the RP-HPLC peptide profile analysis, which discriminates the samples according to age and manufacturing practice. In addition, FFA profiles demonstrated higher levels of low and medium chain free fatty acids for good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture, and cheese made with heat-shocked milk samples, which suggest faster lipolysis during ripening. The Volatile Sulfur Compounds (VSC) analysis showed higher levels of DMS and MeSH and lower levels of H2S, suggesting faster catabolism of sulfur containing amino acids in good cheese, cheese produce in plant TCCA, cheese made in plant CRP with adjunct culture, and cheese made with heat-shocked milk. / Graduation date: 2013

Thesis (D. Tech.) -- Central University of Technology, Free State, 2010 / Branded dairy products have lately become a global trend. As a result of this, the origin of the milk used in the manufacturing of branded cheeses must be declared by the producer, since it is known that these products are highly adulterated with foreign milk. In South Africa, branded Ayrshire Cheddar cheese has become highly popular due to its unique organoleptic properties and in light of claims that it ripens much faster than cheese made from other milk (not including Ayrshire). This study was therefore directed to investigate the unique properties of branded Ayrshire Cheddar cheese versus Cheddar cheese manufactured from a mixture of other breeds’ milk (not including Ayrshire milk) and to establish a catabolite profile for each cheese type. The outlay of the thesis was constructed into six chapters each with its own outcomes. The first chapter focused on the variations between the two Cheddar cheese batches (produced from Ayrshire and other breeds’ milk) with regards to organic acid, selected chemical parameters and starter microbiotia. In the following three chapters mathematical models were developed that would predict organic-; fatty and amino acid fluxtuations respectively in the cheese made from Ayrshire and other milk. In the last chapter two artificial neural networks were designed with the two starter organisms, Lactococcus lactis and Streptococcus thermophilus as variable indicator respectively. Thirty-two cheese samples of each batch (pure Ayrshire (4) / mix breed with no Ayrshire (4)) were ripened and samples were analysed under the same conditions on the following days after production: 2, 10, 22, 36, 50, 64, 78, and 92. In the subsequent chapters, the following analysis were done on each day of analysis: organic acid by means of high performance liquid chromatography (HPLC); fatty acids by means of Gas Chromatography Mass Spectometry (GCMS); amino acids by means of GC-MS; microbial analysis by means of traditional methods, total DNA extraction and polymerase chain reaction (PCR); and standard chemical analysis for moisture, NaCl and pH. In the first research chapter, the minimum and maximum (min/max) values, standard deviations and proposed rel X values of organic acids were evaluated in Ayrshire and the mixed-breed Cheddar cheese, and showed that isovaleric acid is the organic acid with the least variation relative to concentration in both cheeses and it was assumed that this organic acid is the most effective indicator of cheese uniformity. Clear differences in organic acids, chemical variables and starter micro-organisms were also evident in the two cheese batches. Results obtained from the regression models which was defined for each organic -; amino - and fatty acid by means of mathematical equations can be used by the manufacturer to achieve i.e. the selection of cheese for specialist lines, the early exclusion of defective cheeses, and the establishment of brand origin (Ayrshire vs. mixed-breed Cheddar cheeses). The regression graphs also illustrate unique flux patterns in Ayrshire and the mixed-breed in terms of organic -, fatty -, and amino acid content. In the last chapter, the discrimination between the two batches was respectively done via artificial neural network (ANN) modelling of Lactococcus lactis and Streptococcus thermophilus as indicator organisms. The ANN consisted of a multilayered network with supervised training arranged into an ordered hierarchy of layers, in which connections were allowed only between nodes in immediately adjacent layers. The construction thereof allowed for two output nodes, connected to an input layer consisting of two nodes to which the inputs were connected. In both cheeses the results from the ANN showed acceptable classification of the cheeses based on the counts of L. lactis and S. thermophilus.

Changes in cheese physical properties during aging are related to proteolysis by coagulant type, culture enzymes, and non-starter lactic acid bacteria (NSLAB). Storage temperature also affects aging rate. Cultures are important for flavor development , but less is understood about their role in melting and textural properties. Our objective was to make Cheddar cheese using different cultures, to age it at 6 and 13°C, and measure physical and proteolytic properties over 12 mo to determine whether changes in texture and melting correlated with the extent of proteolysis that occurred during aging. Cheese was manufactured using Lactococcus lactis starter culture either alone or combined with one or both of Lac Lc. Lactis or Lactobacillus helveticus adjunct cultures . Three replicates of cheese were made using 1500 lb of milk. Cheese composition was 35.5 ± 1.0% moisture, 52.5 ± 2.5% FDB, 1.65 ± 0.05% salt, and pH 5.2 ± 0.1. All cheeses were initially stored at 6°C, then half moved to l3°C after 21 d. Texture profile analysis was performed using 25% and 60% compression and melting measured using a Meltmeter at 65°C. The data were analyzed based on culture and temperature over 12-mo storage time. The overall hardness decreased, while the cohesiveness decreased for all treatments. Extent of melting was significantly correlated with hardness (r = 0.62), cohesiveness (r = 0.40), and inversely with adhesiveness (r = 0.24). Correlations with adhesiveness and cohesiveness were not linear. Proteins were extracted from cheese at 1 wk, 1, 2, 4, 6, 9, and 12 mo of aging using 500 mM sodium citrate solution containing 1% sodium chloride. Purified extracts were then applied to a high-performance liquid chromatography CS reverse phase column and large hydrophobic peptides and protein peaks monitored at 214 nm. Melting was inversely correlated with the amount of intact ɑs1-caserienm remaining in the cheese (r = -0.54) and directly correlated with what was thought to be ɑs1-casein (f 24 - 199) (r = 0.56).

The effects of whey-based and milk -based starter media, and low concentrations (0.02% each) of citrate, phosphate and calcium upon various milk coagulation properties were measured. Samples inoculated with milk-based starter medium had shorter coagulation times, faster rate of curd formation, and greater final curd firmness than those inoculated with externally neutralized whey-based starter. Starter medium treatment was statistically interacted with calcium for coagulation time and rate of curd formation. Citrate addition caused longer coagulation times, slower rates of curd formation, and weaker final coagula. Citrate interacted with calcium and phosphate for several of the parameters measured. Addition of phosphate did not affect any of the measured parameters but was involved in several significant two-factor interactions as was calcium addition.

This study was conducted to evaluate several forms of equations for predicting Cheddar cheese yields based on the fat and protein content of milk and moisture content of cheese. Production and quality control data from a Cheddar cheese plant for one entire year was used. This included the pounds of milk that went into each vat of cheese, yield of cheese from each vat, cheese moisture from each vat, and fat and protein percentages of the milk. Seven models were derived to predict the yield of Cheddar cheese. The seven models were statistically fitted to the data by applying the Marquardt non-linear least squares method of iteration. These were compared with the commonly used Van Slyke and Price formula, with casein estimated as a percentage of total protein. The differences among the eight models were small.

The use of slower acid-producing starter bacteria for the production of lower fat Cheddar cheese has lead to milder flavor Cheddar cheeses that lack intense Cheddar notes. The metabolism of methionine leads to the production of methanethiol, which is one of the desirable Cheddar cheese flavor compounds. The influence of NaCl and reduced pH was determined for aminopeptidase, lipase/ esterase, and methanethiol-producing capability in selected lactic acid bacteria and brevibacteria in simulated cheese-like conditions. The activity of each enzyme decreased with NaCl addition and pH reduction to approximate a Cheddar cheese environment (5% NaCl and pH 5.2). The mechanism for methanethiol production by the starter and adjunct bacteria was also investigated. Different enzyme systems were found to be responsible for methanethiol production in starter lactococci, lactobacilli, and brevibacteria. In the lactococci, enzymes that acted primarily on cystathionine were responsible for methanethiol production from methionine. Lactobacilli also contained cystathionine-degrading enzymes, but these enzymes have properties different from the lactococcal enzymes. Brevibacterium linensBL2 lacked cystathionine-degrading enzymes, but was capable of the direct conversion of methionine to methanethiol. L-Methionine γ-lyase from B. linens BL2 was purified to homogeneity, and was found to catalyze the α, γ elimination of methionine resulting in the production of methanethiol, α-ketobutyrate, and ammonia. Characterization of the pure enzyme demonstrated that it is pyridoxal phosphate dependent, which is active at salt and pH conditions existing in ripening Cheddar cheese. The addition of either B. linens BL2 or L-methionine γ-lyase to aseptic cheese curd slurries increased methanethiol and total volatile sulfur compound production. In an attempt to increase methanethiol production and Cheddar cheese flavor in reduced-fat Cheddar cheese, B. linens BL2 was added as a starter adjunct to 60% reduced-fat cheese. Sensory evaluation of the cheese indicated that B. linens BL2 improved the flavor of 60% reduced-fat Cheddar cheese. This suggests that the addition of B. linens BL2 is an alternative to the addition of lactic acid bacteria to improve Cheddar cheese flavor via the metabolism of methionine.

A Spiral Plater and a Microtiter system were used to isolate and evaluate cultures for a paired strain culture program. Bacteriophage and temperature sensitivity data of 43 Streptococcus cremoris strains were introduced into a computer cluster program to pair the least similar strains. Selected pairs were challenged with phage. Resistant mutants were developed. Characteristics of proteinase positive and proteinase negative variants were examined. Proteinase positive isolates produced more changes in pH, cell mass and more generations in milk than their counter-parts. Paired proteinase negative cultures produced more change in pH and cell mass and more generations in milk than single strains. Whey based medium under pH control was superior to commercial internal pH control medium for proteinase negative culture propagation. Proteinase negative isolates achieved 90% of the cell mass obtained by their counterparts in nonfat dry milk-yeast medium. Proteinase negative starter culture endured significantly higher phage titers than proteinase positive cells. Proteinase negative variants sustained activity comparable with phage-free controls when challenged for seven cycles with high phage titers. Proteinase positive cells had impaired activity after the second cycle. Pairing of proteinase positive strains was advantageous for phage protection. Erythromycin, streptomycin and penicillin adversely affected the activity of both cell types, yet proteinase positive cells were significantly more inhibited. Pairing neither variant enhanced activity. Cheddar cheese was exclusively manufactured with 2% inoculum of proteinase negative cultures compared to 1.5% usage of the proteinase positive paired strains. Cheese quality and cheese making times were normal. Over 4200 consecutive vats of Cheddar cheese were made in 1982 employing one pair of proteinase positive culture. Acid control and cheese quality were improved. The cheese making times were more uniform. Smaller inocula volumes could successfully be used for bulk starter in cheese plants utilizing pH controlled starter propagation. A needle/syringe system for inoculating starter tanks provided better protection against contamination during inoculation.

Differences in texture at any particular stage of ripening depend upon differences in the basic structure and the extent to which the basic structure is modified by physical parameters. Thus, very young cheeses of the same variety differ in texture because of variations in pH and in salt, moisture, and fat content. How well a cheese melts and shreds depend on its texture and physical parameters. Streptococcus thermophilus MR1C produces an exopolysaccharide (EPS) that is tightly associated with the bacterial cell wall. Addition of S. thermophilus MR1C to the cheese make will increase the moisture of the cheese 2-3% and thus affect the texture, melt, and shreddability of that cheese. To determine the effect of S. thermophilus MR1C on the texture, melt, and shreddability of cheese, two stirred-curd cheeses with equivalent physical parameters using BPS-producing S. thermophilus MR1C or non-BPS-producing S. thermophilus DM10 adjunct cultures were produced. Because MR1C cheese would increase moisture, the curd size, wash water temperature, and pH at salting had to be altered in order to make the physical parameters the same for both cheeses. The MR1C cheese was harder and had a higher fracture stress than the DM10 cheese. The MRlC cheese was also more adhesive, but only for one of the two trials. Even with adjustments in the method of manufacture, the MR1C cheese still had a slightly higher SM and pH, which may be partly responsible for the differences between the two cheeses. There were no differences between the MRlC cheese and the DM1 0 cheese in shreddability as determined by fines, stickiness, and gumminess. Cheese produced without a streptoccus adjunct culture was more cohesive and had fewer fines than the MRIC or DM10 cheese.

Graduation date: 1993