Assessing the ability of nisin A and derivatives thereof to inhibit gram-negative bacteria from the genus Thermus.

Nisin is a bacteriocin that is globally employed as a biopreservative in food systems to control gram-positive, and some gram-negative, bacteria. Here we tested the bioactivity of nisin A-producing Lactococcus lactis NZ9700 and producers of bioengineered variants thereof against representatives of the gram-negative genus Thermus, which has been associated with the pink discoloration defect in cheese. Starting with a total of 73 nisin variant-producing Lactococcus lactis, bioactivity against Thermus was assessed via agar diffusion assays, and 22 variants were found to have bioactivity greater than or equal to that of the nisin A-producing control. To determine to what extent this enhanced bioactivity was attributable to an increase in specific activity, minimum inhibitory concentrations were determined using the corresponding purified form of these 22 nisin A derivatives. From these experiments, nisin M17Q and M21F were identified as peptides with enhanced antimicrobial activity against the majority of Thermus target strains tested. In addition, several other peptide variants were found to exhibit enhanced specific activity against a subset of strains.

Low- and reduced-fat milled curd, direct-salted Gouda cheese: Comparison of lactose standardization of cheesemilk and whey dilution techniques.

Control of acidity is critical for cheese quality, as high acidity can be associated with poor flavor and textural attributes. We investigated an alternative method to control cheese acidity, specifically in low-fat (LF) and reduced-fat (RF) milled curd, direct-salted Gouda cheese, which involved altering the initial lactose content of cheesemilk. In traditional Gouda cheese manufacture, a critical technique to control acidity is whey dilution (WD); that is, partial removal of whey and its replacement with water. Direct standardization of the lactose content of milk during the ultrafiltration process could be a simpler and more effective technique to control cheese acidity. This study compared the effect of traditional WD at 2 different levels, 15 and 30% (WD15 and WD30), with the alternative approach of adjustment of the lactose content of milk using low-concentration-factor ultrafiltration (LCF-UF). The composition, texture, functionality, and sensory properties of these LF and RF Gouda cheeses were evaluated. A milled curd, direct-salted cheese manufacturing protocol was used. Milks used for cheesemaking had a lactose-to-casein (L:CN) ratio of approximately 1.8, which is the typical ratio found in milk, whereas milks prepared with lactose standardization (LS) were made from UF concentrated milks with water added during filtration to achieve a L:CN ratio of approximately 1.1. Cheeses made with LS exhibited lower lactose and lactic acid contents than WD30 and WD15, leading to significantly higher pH values in the cheese. Dynamic small-amplitude oscillatory rheology indicated that use of LS led to cheeses with a lower crossover temperature (melting point) than the cheeses made with WD. Cheeses made with LS had lower insoluble Ca contents, likely caused by the addition of water required to achieve the lower L:CN ratio in these milks. Sensory analysis also indicated that LS cheeses had lower acidity and softer texture. These results suggest that standardization of the L:CN ratio of milk could be a useful alternative to WD (or a curd rinse step) to reduce acidity in cheeses. In addition, LS could be used to help soften texture and increase meltability, if desired in lower-fat cheese types.

Omics-Based Insights into Flavor Development and Microbial Succession within Surface-Ripened Cheese.

In this study, a young Cheddar curd was used to produce two types of surface-ripened cheese, using two commercial smear-culture mixes of yeasts and bacteria. Whole-metagenome shotgun sequencing was used to screen the microbial population within the smear-culture mixes and on the cheese surface, with comparisons of microorganisms at both the species and the strain level. The use of two smear mixes resulted in the development of distinct microbiotas on the surfaces of the two test cheeses. In one case, most of the species inoculated on the cheese established themselves successfully on the surface during ripening, while in the other, some of the species inoculated were not detected during ripening and the most dominant bacterial species, Glutamicibacter arilaitensis, was not a constituent of the culture mix. Generally, yeast species, such as Debaryomyces hansenii and Geotrichum candidum, were dominant during the first stage of ripening but were overtaken by bacterial species, such as Brevibacterium linens and G. arilaitensis, in the later stages. Using correlation analysis, it was possible to associate individual microorganisms with volatile compounds detected by gas chromatography-mass spectrometry in the cheese surface. Specifically, D. hansenii correlated with the production of alcohols and carboxylic acids, G. arilaitensis with alcohols, carboxylic acids and ketones, and B. linens and G. candidum with sulfur compounds. In addition, metagenomic sequencing was used to analyze the metabolic potential of the microbial populations on the surfaces of the test cheeses, revealing a high relative abundance of metagenomic clusters associated with the modification of color, variation of pH, and flavor development. IMPORTANCE Fermented foods, in particular, surface-ripened cheese, represent a model to explain the metabolic interactions which regulate microbial succession in complex environments. This study explains the role of individual species in a heterogeneous microbial environment, i.e., the exterior of surface-ripened cheese. Through whole-metagenome shotgun sequencing, it was possible to investigate the metabolic potential of the resident microorganisms and show how variations in the microbial populations influence important aspects of cheese ripening, especially flavor development. Overall, in addition to providing fundamental insights, this research has considerable industrial relevance relating to the production of fermented food with specific qualities.

Effect of standardizing the lactose content of cheesemilk on the properties of low-moisture, part-skim Mozzarella cheese.

The texture, functionality, and quality of Mozzarella cheese are affected by critical parameters such as pH and the rate of acidification. Acidification is typically controlled by the selection of starter culture and temperature used during cheesemaking, as well as techniques such as curd washing or whey dilution, to reduce the residual curd lactose content and decrease the potential for developed acidity. In this study, we explored an alternative approach: adjusting the initial lactose concentration in the milk before cheesemaking. We adjusted the concentration of substrate available to form lactic acid. We added water to decrease the lactose content of the milk, but this also decreased the protein content, so we used ultrafiltration to help maintain a constant protein concentration. We used 3 milks with different lactose-to-casein ratios: one at a high level, 1.8 (HLC, the normal level in milk); one at a medium level, 1.3 (MLC); and one at a low level, 1.0 (LLC). All milks had similar total casein (2.5%) and fat (2.5%) content. We investigated the composition, texture, and functional and sensory properties of low-moisture, part-skim Mozzarella manufactured from these milks when the cheeses were ripened at 4°C for 84d. All cheeses had similar pH values at draining and salting, resulting in cheeses with similar total calcium contents. Cheeses made with LLC milk had higher pH values than the other cheeses throughout ripening. Cheeses had similar moisture contents. The LLC and MLC cheeses had lower levels of lactose, galactose, lactic acid, and insoluble calcium compared with HLC cheese. The lactose-to-casein ratio had no effect on the levels of proteolysis. The LLC and MLC cheeses were harder than the HLC cheese during ripening. Maximum loss tangent (LT), an index of cheese meltability, was lower for the LLC cheese until 28d of ripening, but after 28d, all treatments exhibited similar maximum LT values. The temperature where LT=1 (crossover temperature), an index of softening point during heating, was higher for MLC and LLC cheese at 56 and 84d of ripening. The LLC cheese also had lower blister color and less stretch than MLC and HLC cheese. Adjusting the lactose content of milk while maintaining a constant casein level was a useful technique for controlling cheese pH, which affected the texture, functionality, and sensory properties of low-moisture, part-skim Mozzarella cheese.

Microbiological, physico-chemical and proteolytic changes in a Spanish blue cheese during ripening (Valdeón cheese).

The aim of this work was to study the microbiological, physico-chemical and proteolytic changes in Valdeón blue-veined cheese during ripening. Eight replicas of cheese were produced and a total of 48 cheeses were analysed. Lactic acid bacteria, mainly lactococci, were the predominant flora during the early stages of ripening, gradually being replaced by moulds and yeasts (8 log units). Enterococci and Enterobacteriaceae counts were very low or zero. This variety was characterised by a total solids content of 61.80g per 100g(-1) of cheese, a salt/moisture ratio of 8.92g salt per 100g(-1) moisture, a pH of 6.4-7.6 and a water activity of 0.917. At the end of ripening, primary and secondary proteolysis were very high, which resulted in an almost total degradation of αs1- and ß-casein (approximately 90%). The peptide profile of the aqueous soluble extracts at pH 4.6 showed great complexity during ripening.

Effect of camel chymosin on the texture, functionality, and sensory properties of low-moisture, part-skim Mozzarella cheese.

The objective of this study was to compare the effect of coagulant (bovine calf chymosin, BCC, or camel chymosin, CC), on the functional and sensory properties and performance shelf-life of low-moisture, part-skim (LMPS) Mozzarella. Both chymosins were used at 2 levels [0.05 and 0.037 international milk clotting units (IMCU)/mL], and clotting temperature was varied to achieve similar gelation times for each treatment (as this also affects cheese properties). Functionality was assessed at various cheese ages using dynamic low-amplitude oscillatory rheology and performance of baked cheese on pizza. Cheese composition was not significantly different between treatments. The level of total calcium or insoluble (INSOL) calcium did not differ significantly among the cheeses initially or during ripening. Proteolysis in cheese made with BCC was higher than in cheeses made with CC. At 84 d of ripening, maximum loss tangent values were not significantly different in the cheeses, suggesting that these cheeses had similar melt characteristics. After 14 d of cheese ripening, the crossover temperature (loss tangent = 1 or melting temperature) was higher when CC was used as coagulant. This was due to lower proteolysis in the CC cheeses compared with those made with BCC because the pH and INSOL calcium levels were similar in all cheeses. Cheeses made with CC maintained higher hardness values over 84 d of ripening compared with BCC and maintained higher sensory firmness values and adhesiveness of mass scores during ripening. When melted on pizzas, cheese made with CC had lower blister quantity and the cheeses were firmer and chewier. Because the 2 types of cheeses had similar moisture contents, pH values, and INSOL Ca levels, differences in proteolysis were responsible for the firmer and chewier texture of CC cheeses. When cheese performance on baked pizza was analyzed, properties such as blister quantity, strand thickness, hardness, and chewiness were maintained for a longer ripening time than cheeses made with BCC, indicating that use of CC could help to extend the performance shelf-life of LMPS Mozzarella.

Effect of exopolysaccharide produced by isogenic strains of Lactococcus lactis on half-fat Cheddar cheese.

Fat-reduced cheeses often suffer from undesirable texture, flavor, and cooking properties. Exopolysaccharides (EPS) produced by starter strains have been proposed as a mechanism to increase yield and to improve the texture and cooking properties of reduced-fat cheeses. The objective of this work was to assess the influence of an exopolysaccharide on the yield, texture, cooking properties, and quality of half-fat Cheddar cheese. Two pilot-scale half-fat Cheddar cheeses were manufactured using single starters of an isogenic strain of Lactococcus lactis ssp. cremoris (DPC6532 and DPC6533) that differed in their ability to produce exopolysaccharide. Consequently, any differences detected between the cheeses were attributed to the presence of the exopolysaccharide. The results indicated that cheeses made with the exopolysaccharide-producing starter had an 8.17% increase in actual cheese yield (per 100 kg of milk), a 9.49% increase in moisture content, increase in water activity and water desorption rate at relative humidities

Impact of chymosin- and plasmin-mediated primary proteolysis on the growth and biochemical activities of lactobacilli in miniature Cheddar-type cheeses.

Strongly proteolytic starters seem to improve the growth of nonstarter lactobacilli during cheese ripening, but no information is available on the impact of the nonmicrobial proteases usually active in cheese on their development. In the current study, the influence of chymosin- and plasmin-mediated proteolysis on the growth and biochemical activities of lactobacilli during ripening of miniature Cheddar-type cheeses, manufactured under controlled microbiological conditions, was studied. Two experiments were performed; in the first, residual chymosin activity was inhibited by the addition of pepstatin, and in the second, plasmin activity was increased by adding more enzyme, obtained in vitro through the activation of plasminogen induced by urokinase. Cheeses with or without a Lactobacillus plantarum I91 adjunct culture and with or without added pepstatin or plasmin solution were manufactured and ripened for 60 d. The addition of the adjunct culture resulted in enhancement of secondary proteolysis, evidenced by an increase in the total content of free amino acids (FAA) and modifications of the individual FAA profiles. Reduction in residual chymosin activity caused a decrease in primary and secondary proteolysis, characterized by the absence of alpha(s1)-casein hydrolysis and reduced production of peptides and FAA, respectively. The increase in plasmin activity accelerated primary proteolysis but no enhancement of secondary proteolysis was observed. Chymosin- and plasmin-mediated proteolysis did not influence the growth and biochemical activities of adventitious or adjunct lactobacilli, indicating that it is not a limiting factor for the development and proteolytic-peptidolytic activities of lactobacilli in the cheese model studied.

Viability and contribution to proteolysis of an adjunct culture of Lactobacillus plantarum in two model cheese systems: cheddar cheese-type and soft-cheese type.

AIMS: The influence of the cheese-making process, ripening conditions and primary starter on the viability and proteolytic activity of an adjunct culture of Lactobacillus plantarum I91 was assessed in two miniature cheese models, representative of Cremoso Argentino and Cheddar cheeses. METHODS AND RESULTS: Cheeses with and without adjunct culture were made under controlled microbiological conditions and sampled during ripening for physicochemical and microbiological analyses. The addition of lactobacilli neither contributed to acid production nor caused changes to the composition of the cheeses. The strain studied exhibited good development and survival and showed a similar growth pattern in both cheese matrices. The adjunct culture caused changes to secondary proteolysis of both cheese types, which were evidenced by modification of peptide profiles and the increase in the levels of some individual amino acids as well as the total content of free amino acids. The changes observed were consistent with the acceleration of proteolysis in the two cheese models assayed. CONCLUSION: Lactobacillus plantarum I91 has desirable and robust technological properties, which makes it a suitable adjunct culture for cheese-making. SIGNIFICANCE AND IMPACT OF THE STUDY: Other cultures and environmental conditions prevailing in the food may affect the viability of adjunct cultures and its biochemical activities; this is the first report describing the successful performance of an adjunct culture of Lact. plantarum I91 in two different model cheese systems.

Measurement of the oxidation-reduction potential of cheddar cheese.

The objective of this study was to develop a method to measure the oxidation-reduction (redox) potential of hard cheeses such as cheddar and to investigate the impact on this parameter of measurement temperature, and factors associated with electrochemical cell design such as distance between reference and working electrodes and depth into the cheese of the platinum electrodes. For this purpose, a novel, self-sealing, platinum working electrode was constructed which was thin and flexible enough to be inserted directly into the cheese sample. A calomel electrode was used as the reference electrode and the circuit was completed with a 3 M KCl salt bridge. The physical orientation of electrodes, such as distance between reference electrode and working electrode, had a substantial effect on equilibrium time for redox potential measurement. The time required for redox potential to reach equilibrium was 2 d in cheddar cheese and the optimum distance between the platinum and calomel electrodes was 2.5 cm. The fastest equilibration time was obtained when the working electrode was inserted 5 or 6 cm into the cheese. Temperature also had an important effect on redox potential. The shortest time to reach equilibrium of potential was at room temperature (20 degrees C), but it was not practical to keep cheese at this temperature for a period of 2 d. Therefore, redox measurement at 12 degrees C was recommended in spite of the longer equilibration time compared with room temperature. The results of this study suggest that the novel platinum working electrode allows reproducible measurement of the oxidation-reduction potential of cheddar cheese.

Influence of emulsifying salts on the textural properties of nonfat process cheese made from direct acid cheese bases.

The objective of this study was to investigate the influence of several types of emulsifying salts (ES) on the texture of nonfat process cheese (NFPC). Improperly produced nonfat cheese tends to exhibit several problems upon baking including stickiness, insufficient or excessive melt, pale color upon cooling, formation of a dry skin (skinning) often leading to dark blistering, and chewy texture. These attributes are due to the strength and number of interactions between and among casein molecules. We propose to disrupt these interactions by using suitable emulsifying salts (ES). These ES chelate Ca and disperse caseins. Stirred curd cheese bases were made from skim milk using direct acidification with lactic acid to pH values 5.0, 5.2, and 5.4, and ripened for 1 d. Various levels of trisodium citrate (TSC; 0.5, 1, 1.5, 2, 2.5, 3, and 5%), disodium phosphate (DSP; 1, 2, 3, and 4%), or trisodium phosphate (TSP; 1, 2, 3, and 4%) were blended with the nonfat cheese base. Cheese, ES, and water were weighed into a steel container, which was placed in a waterbath at 98 degrees C and then stirred using an overhead stirrer for 9 min. Molten cheese was poured into plastic containers, sealed, and stored at 4 degrees C for 7 d before analysis. Texture and melting properties were determined using texture profile analysis and the UW-Melt-profiler. The pH 5.2 and 5.4 cheese bases were sticky during manufacture and had a pale straw-like color, whereas the pH 5.0 curd was white. Total calcium contents were approximately 400, 185, and 139 mg/100 g for pH 5.4, 5.2, and 5.0 cheeses, respectively. Addition of DSP resulted in NFPC with the lowest extent of flow, and crystal formation was apparent at DSP levels above 2%. The NFPC manufactured from the pH 5.0 base and using TSP had reduced melt and increased stickiness, whereas melt was significantly increased and stickiness was reduced in NFPC made with pH 5.4 base and TSP. However, for NFPC made from the pH 5.4 cheese and with 1% TSP, the pH value was >6.20 and crystals were observed within a few days. Use of TSC increased extent of flow up to a maximum with the addition of 2% ES for all 3 types of cheese bases. Addition of high levels of TSC to the pH 5.2 and 5.4 cheese bases resulted in increased stickiness. Similar pH trends for attributes such as extent of flow, hardness, and adhesiveness were observed for both phosphate ES but no consistent pH trends were observed for the NFPC made with TSC. These initial trials suggest that the pH 5.0 cheese base was promising for further research and scale-up to pilot-scale process cheese making, because cheeses had a creamy color, reasonable melt, and did not have high adhesiveness when TSC was used as the ES. However, the acid whey produced from the pH 5.0 curd could be a concern.

The effect of natural cheddar cheese ripening on the functional and textural properties of the processed cheese manufactured therefrom.

Cheddar cheese ripened at 8 degrees C was sampled at 7, 14, 28, 56, 112, and 168 d and subsequently used for the manufacture of processed cheese. The cheddar cheese samples were analyzed throughout ripening for proteolysis while the textural and rheological properties of the processed cheeses (PCs) were studied. The rate of proteolysis was the greatest in the first 28 d of cheddar cheese ripening but began to slow down as ripening progressed from 28 to 168 d. A similar trend was observed in changes to the texture of the PC samples, with the greatest decrease in hardness and increase in flowability being in the first 28 d of ripening. Confocal scanning laser microscopy showed that the degree of emulsification in the PC samples increased as the maturity of the cheddar cheese ingredient increased from 7 to 168 d. This increased emulsification resulted in a reduction in the rate of softening in the PC in samples manufactured from cheddar cheese bases at later ripening times. Multivariate data analysis was performed to summarize the relationships between proteolysis in the cheddar cheese bases and textural properties of the PC made therefrom. The proportion of alpha(s)(1)-casein (CN) in the cheddar cheese base was strongly correlated with hardness, adhesiveness, fracturability, springiness, and storage modulus values for the corresponding PC. Degradation of alpha(s) (1)-CN was the proteolytic event with the strongest correlation to the softening of PC samples, particularly those manufactured from cheddar cheese in the first 28 d of ripening.

A survey of the microbial and chemical composition of seven semi-ripened Provola dei Nebrodi Sicilian cheeses.

AIMS: The microbial and chemical composition of seven different semi-ripened (45 days) Provola dei Nebrodi Sicilian cheese samples were assessed in order to investigate the diversity of the microbial population in cheese made from different geographical areas throughout Sicily. METHODS AND RESULTS: The samples, which were obtained from seven different Provola dei Nebrodi manufacturers, were assessed using selective media. Interestingly, concentrations of presumptive lactobacilli represented over 90% of the total microbial population. In total, 105 presumptive Lactobacillus isolates were characterized to determine the relatedness of the isolates between the seven different cheeses. Randomly amplified polymorphic DNA polymerase chain reaction (RAPD PCR) analysis of the 105 presumptive lactobacilli indicated the presence of 22 distinct isolates. Further investigation of the isolates using pulsed field gel electrophoresis (PFGE) following restriction with the enzyme ApaI revealed the presence of 19 distinct macrorestriction patterns and the presence of between one and four distinct isolates per cheese sample (out of a total of 15 isolates per cheese randomly taken from Lactobacillus selective media plates). Analysis of the 16S rDNA sequence of each genetically distinct isolate demonstrated the dominance of the Lactobacillus casei species in all cheese samples assessed. Lactobacillus delbrueckii and Pediococcus pentosaceus species were also detected. The concentration of free amino acids, used to estimate the extent of proteolysis in each cheese, ranged from 59 to 433 mg 100 g(-1) cheese. CONCLUSIONS: Microbiological assessment of the cheeses demonstrated the dominance of Lactobacillus species after 45 days of ripening with levels ranging from 8.3 to 9.4 log CFU g(-1). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new information on the diversity of lactobacilli within an artisanal Sicilian cheese, enabling the identification of 17 strains of Lact. casei, one strain of Lact. delbrueckii and Ped. pentosaceus through the combined use of RAPD PCR, PFGE and 16S rDNA sequencing.

Molecular identification and typing of natural whey starter cultures and microbiological and compositional properties of related traditional Mozzarella cheeses.

Cell numbers of presumptive lactic acid bacteria varied markedly between 7 natural whey starter cultures (NWSC) used for producing traditional cows' milk Mozzarella cheeses in the Apulia region of Southern Italy. Taxonomic identification revealed a large diversity at species level, including mesophilic and thermophilic lactobacilli, lactococci, streptococci and enterococci. Randomly Amplified Polymorphic DNA (RAPD-PCR), analysis showed the biodiversity among the strains and, for lactobacilli, some relationships with provenience of the natural starter. Cell numbers of presumptive lactic acid bacteria in the corresponding Mozzarella cheeses were similar or higher than those found in the corresponding NWSC. RAPD-PCR analyses showed that most of the strains in cheese originated from the starter. The gross composition varied markedly between the 7 Mozzarella cheeses and ranged from 53-64% moisture, 17-23% protein, 13-20% fat and 0.50-1.61% salt. The values of pH for several samples were above 6.0. As shown by urea-PAGE of the pH 4.6-insoluble nitrogen fractions, cheese samples were characterized by differences in alpha(S1)- and beta-casein hydrolysis. Cheeses also differed with respect to secondary proteolysis as shown by Principal Component Analysis (PCA) of data from RP-HPLC of the pH 4.6-soluble, pH 4.6-70% ethanol-soluble and 70% ethanol-insoluble nitrogen fractions. These differences were attributed to the different microbial composition of the NWSC. Strain selection and optimization of a protocol for producing a natural whey starter culture to be used by dairy factories of the Apulia region appears to be a pre-requisite to standardize the major traits distinguishing this cheese variety.

Microbiology, biochemistry, and volatile composition of Tulum cheese ripened in goat's skin or plastic bags.

Tulum cheeses were manufactured from raw ewe's milk and ripened in goat's skin bags (tulums) or plastic containers to understand the effect of ripening container on the chemical composition, biochemistry, microbiology, and volatile composition of Tulum cheeses during 150 d of ripening. Chemical compositions of the cheeses ripened in tulums were significantly different and the moisture contents decreased rapidly in those cheeses because of the porous structure of the tulum. Higher microbial counts were detected in the cheeses ripened in plastic than in cheeses ripened in tulums. Differences in nitrogenous compounds and total free AA of the cheeses were not significant. Total concentrations of free AA in cheeses increased with age and Glu, Ala, Val, Leu, and Phe were the most abundant AA in the cheeses. Urea-PAGE of pH 4.6-insoluble fractions of the cheeses during ripening showed similar degradation patterns in all cheeses. Peptide profiles by reversed-phase HPLC of pH 4.6- and ethanol-soluble or ethanol-insoluble fractions of the cheeses revealed only minor differences in the concentrations of some peptides among the cheeses; however, age-related changes in peptide concentrations were significantly different among the cheeses. Cheeses were analyzed at 90 d of ripening for volatile compounds by solid-phase microextraction gas chromatography-mass spectrometry. One hundred volatile components were identified, including 11 acids, 16 esters, 12 methyl ketones, 7 aldehydes, 22 alcohols, 7 sulfur compounds, 6 terpenes, and 19 miscellaneous compounds. The main components were short-chain fatty acids, 2-butanone, diacetyl, and primary alcohols. Quantitative differences in several volatile compounds were evident among the cheeses. Cheeses ripened in tulums or plastic had similar aroma patterns, but the concentrations of some components were different.

A model system for studying the effects of colloidal calcium phosphate concentration on the rheological properties of Cheddar cheese.

A novel model system was developed for studying the effects of colloidal Ca phosphate (CCP) concentration on the rheological properties of Cheddar cheese, independent of proteolysis and any gross compositional variation. Cheddar cheese slices (disks; diameter = 50 mm, thickness = 2 mm) were incubated in synthetic Cheddar cheese aqueous phase solutions for 6 h at 22 degrees C. Control (unincubated) Cheddar cheese had a total Ca and CCP concentration of 2.80 g/100 g of protein and 1.84 g of Ca/100 g of protein, respectively. Increasing the concentration of Ca in the synthetic Cheddar cheese aqueous phase solution incrementally in the range from 1.39 to 8.34 g/L significantly increased the total Ca and CCP concentration of the cheese samples from 2.21 to 4.59 g/100 g of protein and from 1.36 to 2.36 g of Ca/100 g of protein, respectively. Values of storage modulus (index of stiffness) at 70 degrees C increased significantly with increasing concentrations of CCP, but the opposite trend was apparent at 20 degrees C. The maximum in loss tangent (index of meltability/flowability) decreased significantly with increasing concentration of CCP, and there was no significant effect on the temperature at which the maximum in loss tangent occurred (68 to 70 degrees C). Fourier transform mechanical spectroscopy showed the frequency dependence of all of the cheese samples increased with increasing temperature; however, solubilization of CCP increased the frequency dependence of the cheese matrix only in the high temperature region (i.e., >35 degrees C). These results support earlier studies that hypothesized that the concentration of CCP strongly modulates the rheological properties of cheese.

Influence of starters on chemical, biochemical, and sensory changes in Turkish White-brined cheese during ripening.

Turkish White-brined cheese was manufactured using Lactococcus strains (Lactococcus lactis ssp. lactis NCDO763 plus L. lactis ssp. cremoris SK11 and L. lactis ssp. lactis UC317 plus L. lactis ssp. cremoris HP) or without a starter culture, and ripened for 90 d. It was found that the use of starters significantly influenced the physical, chemical, biochemical, and sensory properties of the cheeses. Chemical composition, pH, and sensory properties of cheeses made with starter were not affected by the different starter bacteria. The levels of soluble nitrogen fractions and urea-PAGE of the pH 4.6-insoluble fractions were found to be significantly different at various stages of ripening. Urea-PAGE patterns of the pH 4.6-insoluble fractions of the cheeses showed that considerable degradation of alpha(s1)-casein occurred and that beta-casein was more resistant to hydrolysis. The use of a starter culture significantly influenced the levels of 12% trichloroacetic acid-soluble nitrogen, 5% phosphotungstic acid-soluble nitrogen, free amino acids, total free fatty acids, and the peptide profiles (reverse phase-HPLC) of 70% (vol/vol) ethanol-soluble and insoluble fractions of the pH 4.6-soluble fraction of the cheeses. The levels of peptides in the cheeses increased during the ripening period. Principal component and hierarchical cluster analyses of electrophoretic and chromatographic results indicated that the cheeses were significantly different in terms of their peptide profiles and they were grouped based on the use and type of starter and stage of ripening. Levels of free amino acid in the cheeses differed; Leu, Glu, Phe, Lys, and Val were the most abundant amino acids. Nitrogen fractions, total free amino acids, total free fatty acids, and the levels of peptides resolved by reverse phase-HPLC increased during ripening. No significant differences were found between the sensory properties of cheeses made using a starter, but the cheese made without starter received lower scores than the cheeses made using a starter. It was found that the cheese made with strains NCDO763 plus SK11 had the best quality during ripening. It was concluded that the use of different starter bacteria caused significant differences in the quality of the cheese, and that each starter culture contributed to proteolysis to a different degree.

Proteolysis in miniature cheddar-type cheeses manufactured using extracts from the crustacean Munida as coagulant.

Miniature (20 g) Cheddar-type cheeses were manufactured using enzymes extracted from the crustacean Munida or chymosin as coagulant. Cheeses were ripened at 8 degrees C and samples were collected for analysis after 2, 6 and 12 weeks. Proteolysis was assessed by urea-polyacrylamide gel electrophoresis, which showed that cheeses manufactured with the Munida extracts had a higher extent of degradation of beta-casein than cheeses made using chymosin as coagulant. Patterns of proteolysis were also obtained by reverse-phase high-performance liquid chromatography (RP-HPLC) and matrix assisted laser desorption ionisation-time of flight (MALDI-ToF) mass spectrometry. In general, the products of proteolysis were more complex in cheese made using the Munida extracts than in cheese made by chymosin as coagulant. Statistical analysis of results clearly discriminated the cheeses on the basis of coagulant used. Molecular mass of peptides found in cheese made using Munida extracts were similar to those of peptides commonly detected in cheeses made using chymosin as coagulant.

Chymosin-mediated proteolysis, calcium solubilization, and texture development during the ripening of cheddar cheese.

Full fat, milled-curd Cheddar cheeses (2 kg) were manufactured with 0.0 (control), 0.1, 1.0, or 10.0 micromol of pepstatin (a potent competitive inhibitor of chymosin) added per liter of curds/whey mixture at the start of cooking to obtain residual chymosin levels that were 100, 89, 55, and 16% of the activity in the control cheese, respectively. The cheeses were ripened at 8 degrees C for 180 d. There were no significant differences in the pH values of the cheeses; however, the moisture content of the cheeses decreased with increasing level of pepstatin addition. The levels of pH 4.6-soluble nitrogen in the 3 cheeses with added pepstatin were significantly lower than that of the control cheese at 1 d and throughout ripening. Densitometric analysis of urea-PAGE electro-phoretograms of the pH 4.6-insoluble fractions of the cheese made with 10.0 micromol/L of pepstatin showed complete inhibition of hydrolysis of alpha(S1)-casein (CN) at Phe23-Phe24 at all stages of ripening. The level of insoluble calcium in each of 4 cheeses decreased significantly during the first 21 d of ripening, irrespective of the level of pepstatin addition. Concurrently, there was a significant reduction in hardness in each of the 4 cheeses during the first 21 d of ripening. The softening of texture was more highly correlated with the level of insoluble calcium than with the level of intact alpha(S1)-CN in each of the 4 cheeses early in ripening. It is concluded that hydrolysis of alpha(S1)-CN at Phe23-Phe24 is not a prerequisite for softening of Cheddar cheese during the early stages of ripening. We propose that this softening of texture is principally due to the partial solubilization of colloidal calcium phosphate associated with the para-CN matrix of the curd.

Evaluation of bitterness in Ragusano cheese.

The appearance of undesirable bitter taste in Ragusano cheese was investigated by comparing the composition of 9 bitter cheeses with that of 9 reference cheeses of good quality by means of chemical, electrophoretic, and chromatographic analyses. Rates of proteolysis were significantly affected in cheeses of different quality. Primary proteolysis, as measured by pH 4.6-soluble N, was significantly greater in bitter cheeses compared with reference samples. Urea-PAGE profiles showed an almost complete breakdown of caseins in bitter cheeses and the further degradation of primary peptides into smaller compounds not detectable by this technique. Cheeses with defects had significantly lower levels of secondary proteolysis as reflected by the percentage of pH 4.6-soluble N soluble in 12% trichloroacetic acid and the amounts of total free amino acids. Peptides separated by reversed phase-HPLC revealed that the large and significant differences in peptide profiles of the soluble fractions between bitter and reference cheeses were mainly due to a much higher proportion of hydrophobic peptides in the former. The occurrence of bitterness in Ragusano cheese was therefore attributable to unbalanced levels of proteolysis and peptidolysis. Extensive degradation of caseins and primary peptides by activities of proteases produced large amounts of small- and medium-sized hydrophobic peptides that were not adequately removed by peptidases of microflora and therefore accumulated in cheese potentially contributing to its bitter taste. The presence of these compounds in bitter cheeses was related to high salt-in-moisture and low moisture contents that limited the enzymatic activities of microflora important in secondary proteolysis. Combining salt-in-moisture and the ratio of hydrophobic-to-hydrophilic soluble peptides resulted in the best logistic partial least squares regression model predicting cheese quality. Although bitterness is known to be rarely encountered in cheese at salt-in-moisture levels >5.0, all of the bitter cheeses analyzed in this study had salt-in-moisture levels much greater than this value. According to the logistic model, a risk of bitterness development may exist for cheeses with a midrange (5 to 10%) salt-in-moisture content but with an inadequate level of secondary proteolysis.