Use of high-pressure processing and low-temperature storage to extend the performance shelf life of 2 types of string cheese.

The manufacturing method of string cheese is similar to mozzarella, but the hot curd is extruded through narrow tubes or pipes, which align the protein fibers that provide the characteristic ability for consumers to pull strings from this cheese. Firmness is another important performance attribute for consumers who just bite into the string cheese without peeling off strings. There have only been a few studies on string cheese, but it is known that stringiness and firmness decrease during prolonged storage, which is a particular challenge for exporting string cheese. We explored 2 treatments to try to retain the stringiness and firmness of string cheese for longer storage periods. The techniques used were high-pressure processing (HPP; 600 MPa for 3 min) and reduced storage temperature (0°C). In other cheese varieties, these techniques have helped extend the performance shelf life. We tested these techniques using the 2 main types of commercial string cheese: direct acid string cheese (DASC) and cultured string cheese (CSC), which were obtained from 2 different manufacturing facilities. The DASC had higher fat (∼2.2%) and higher pH values (∼0.2 units) compared with the CSC. The CSC had higher protein content (∼3.4%), higher insoluble calcium content (∼8 mg insoluble Ca/g protein) and higher texture profile analysis (TPA) hardness values (∼4 N) compared with the DASC. Due to the compositional differences, the 2 varieties were statistically analyzed separately for all other attributes. In both cheese types, HPP caused an immediate reduction in stringiness, some solubilization of insoluble calcium, and a slight increase in the cheese pH values. High-pressure processing also caused a slight increase in TPA hardness of the CSC samples until 14 d (possibly due to a slight increase in cheese pH). The use of the 0°C storage temperature reduced proteolysis and helped retain firmness during storage. Low-temperature storage could help extend the performance shelf life of string cheese by a couple of months, but HPP was not suitable, as the process caused an immediate reduction in stringiness due to the disruption of the matrix induced by the HPP treatment.

Slower proteolysis in Cheddar cheese made from high-protein cheese milk is due to an elevated whey protein content.

A growing number of companies within the cheese-making industry are now using high-protein (e.g., 4-5%) milks to increase cheese yield. Previous studies have suggested that cheeses made from high-protein (both casein and whey protein; WP) milks may ripen more slowly; one suggested explanation is inhibition of residual rennet activity due to elevated WP levels. We explored the use of microfiltration (MF) to concentrate milk for cheese-making, as that would allow us to concentrate the casein while varying the WP content. Our objective was to determine if reducing the level of WP in concentrated cheese milk had any impact on cheese characteristics, including ripening, texture, and nutritional profile. Three types of 5% casein standardized and pasteurized cheese milks were prepared that had various casein:true protein (CN:TP) ratios: (a) control with CN:TP 83:100, (b) 35% WP reduced, 89:100 CN:TP, and (c) 70% WP reduced, 95:100 CN:TP. Standardized milks were preacidified to pH 6.2 with dilute lactic acid during cheese-making. Composition, proteolysis, textural, rheological, and sensory properties of cheeses were monitored over a 9-mo ripening period. The lactose, total solids, total protein, and WP contents in the 5% casein concentrated milks were reduced with increasing levels of WP removal. All milks had similar casein and total calcium levels. Cheeses had similar compositions, but, as expected, lower WP levels were observed in the cheeses where WP depletion by MF was performed on the cheese milks. Cheese yield and nitrogen recoveries were highest in cheese made with the 95:100 CN:TP milk. These enhanced recoveries were due to the higher fraction of nitrogen being casein-based solids. Microfiltration depletion of WP did not affect pH, sensory attributes, or insoluble calcium content of cheese. Proteolysis (the amount of pH 4.6 soluble nitrogen) was lower in control cheeses compared with WP-reduced cheeses. During ripening, the hardness values and the temperature of the crossover point, an indicator of the melting point of the cheese, were higher in the control cheese. It was thus likely that the higher residual WP content in the control cheese inhibited proteolysis during ripening, and the lower breakdown rate resulted in its higher hardness and melting point. There were no major differences in the concentrations of key nutrients with this WP depletion method. Cheese milk concentration by MF provides the benefit of more typical ripening rates.

Effect of substituting whey cream for sweet cream on the textural and rheological properties of cream cheese.

In the manufacture of cream cheese, sweet cream and milk are blended to prepare the cream cheese mix, although other ingredients such as condensed skim milk and skim milk powder may also be included. Whey cream (WC) is an underutilized fat source, which has smaller fat droplets and slightly different chemical composition than sweet cream. This study investigated the rheological and textural properties of cream cheeses manufactured by substituting sweet cream with various levels of WC. Three different cream cheese mixes were prepared: control mix (CC; 0% WC), cream cheese mixes containing 25% WC (25WC; i.e., 75% sweet cream), and cream cheese mixes with 75% WC (75WC; i.e., 25% sweet cream). The CC, 25WC, and 75WC mixes were then used to manufacture cream cheeses. We also studied the effect of WC on the initial step in cream cheese manufacture (i.e., the acid gelation process monitored using dynamic small amplitude rheology). Acid gels were also prepared with added denatured whey proteins or membrane proteins/phospholipids (PL) to evaluate how these components affected gel properties. The rheological, textural, and sensory properties of cream cheeses were also measured. The WC samples had significantly higher levels of PL and insoluble protein compared with sweet cream. An increase in the level of WC reduced the rate of acid gel development, similar to the effect of whey phospholipid concentrate added to mixes. In cream cheese, an increase in the level of added WC resulted in significantly lower storage modulus values at temperatures <20°C. Texture results, obtained from instrumental and sensory analyses, showed that high level of WC resulted in significantly lower firmness or hardness values and higher stickiness compared with cream cheeses made with 25WC or CC cream cheeses. The softer, less elastic gels or cheeses resulting from the use of high levels of WC are likely due to the presence of components such as PL and proteins from the native milk fat globule membrane. The use of low levels of WC in cream cheese did not alter the texture, whereas high levels of WC could be used if manufacturers want to produce more spreadable products.

Behavior of stabilizers in acidified solutions and their effect on the textural, rheological, and sensory properties of cream cheese.

Stabilizers are routinely added during cream cheese manufacture to help prevent syneresis during storage. We investigated how different types of stabilizers affected the texture, rheology, and sensory properties of cream cheese. Cream cheeses were manufactured with 0.33% xanthan gum (XG), locust bean gum (LBG), guar gum (GG), or a combination (CBN) of these 3 stabilizers (0.11% of each). Rheological properties of solutions of the individual stabilizers and their combination (equal amounts) were also determined under conditions similar to the aqueous phase of cream cheese (0.6% gum, 1.8% NaCl, and pH 5). Dynamic small amplitude rheological properties of the cream cheeses were measured during heating from 5 to 80°C at the rate of 1°C/min and cooling at the same rate (because most cream cheese is hot packed/filled before cooling). Measured rheological parameters included storage modulus (G') and loss tangent. Hardness of cream cheeses was determined by texture profile analysis. Quantitative spectrum descriptive sensory analysis was also performed. Distinct differences were observed between the rheological properties of solutions of the individual stabilizers and the CBN containing all the stabilizers. Results showed that CBN solution formed a strong, thermally reversible gel due to synergistic interaction between stabilizers, whereas XG solution formed a weak gel that was not greatly affected by temperature. Solutions of LBG and GG behaved rheologically as entangled polymer solutions. In the high-temperature (>35°C) region, cream cheeses made with XG and CBN showed higher G' values compared with other cream cheeses. The G' values were higher for XG- and CBN-stabilized cream cheeses than LBG- and GG-stabilized cream cheeses at several temperature regions during the cooling cycle. The CBN-stabilized cream cheeses had higher hardness values than the cream cheeses manufactured with the individual stabilizers. Differences were observed between the sensory attributes of cream cheeses stabilized with CBN and those made with individual stabilizers. At low temperatures, the higher hardness and G' values of CBN-stabilized cream cheeses could be due to synergistic interaction between XG and galactomannans. The higher elasticity of XG-stabilized cream cheeses at high temperatures could be due to its higher thermal stability. This study showed that the stabilizers added during manufacture of cream cheese affected its texture, rheological, and sensory properties.

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.

Effects of the depletion of whey proteins from unconcentrated milk using microfiltration on the yield, functionality, and nutritional profile of Cheddar cheese.

Some European dairies use low concentration factor microfiltration (MF) in their cheese plants. Removal of whey protein (WP) from milk before cheesemaking using microfiltration without concentration provides the opportunity to produce a value-added by-product, milk-derived whey. However, few studies have focused on the effects on cheese properties caused by the depletion of WP from cheese milk. Most studies have concentrated cheese milk using MF in addition to depletion of WP. In our approach, cheese milk was not concentrated during WP depletion using MF. We wanted to quantify residual WP levels in cheese made from MF milk and to explore whether WP depletion from milk would influence functionality, nutritional profile, and cheese quality during ripening. Casein (CN) contents for all milks were kept at ∼2.5%, to eliminate the confounding factor of concentration of CN, which was observed in some previous MF studies. Cheese milks had similar ratios of CN to fat. Three standardized milks were produced with various CN:true protein (TP) ratios: (a) control with a CN:TP ratio of 83:100, (b) 35% WP depletion, 89:100 CN:TP, and (c) 70% WP depletion, 95:100 CN:TP. Cheddar cheeses were made from MF milk with various WP depletion levels and aged for 9 mo, and their functionality was evaluated during ripening. We found no major differences in cheese composition or pH values between samples. Cheese yield, solids recovery, and nitrogen recovery were slightly higher in the 95:100 CN:TP cheeses compared with the control. These enhanced recoveries reflect that MF-treated milk started with a higher fraction of CN-based protein solids, rather than WP solids. The standardized milk from the 95:100 CN:TP treatment also had a slightly higher fat content compared with the control, likely helping to increase cheese yield. Rheological properties of cheeses during heating were similar between treatments. Hardness initially decreased with age for all cheeses due to proteolysis or solubilization, or both, of calcium phosphate. Maximum loss tangent (LT), an index of cheese meltability, was slightly lower for the control cheese until 30 d of ripening, but after 30 d, all treatments exhibited similar maximum LT values. The temperature where LT = 1 (crossover temperature), an index of softening point during heating, was slightly lower for MF cheese compared with the control cheeses during ripening. Microfiltration treatment had no significant influence on proteolysis. Sensory properties were similar between the cheeses, except for bitterness. Bitterness intensity was slightly lower in the MF cheeses than in the control cheeses and increased in all cheeses during ripening. We detected no major differences in the concentrations of key nutrients or vitamins between the various cheeses. Depletion of WP in cheese milk by MF did not negatively affect cheese quality, or its nutritional profile, and resulted in similar cheesemaking yields.

Effects of processing conditions on the texture and rheological properties of model acid gels and cream cheese.

Manufacture of cream cheese involves the formation of an initial acid-induced gel made from high-fat milk, followed by a series of processing steps including shearing, heating, and dewatering that complete the conversion of the acid gel into a complex cheese product. We investigated 2 critical parameters for their effect on the initial gel: homogenization pressure (HP) of the high-fat cheese milk, and fermentation temperature (FT). The impact of a low (10 MPa) and high (25 MPa) HP, and low (20°C) and high (26°C) FT were investigated for their effects on rheological and textural properties of acid-induced gels. Intact acid gels were sheared and heated to 80°C, and then their rheological properties were analyzed to help understand the effect of shearing/heating processes on the gel characteristics. The effect of HP on fat globule size distribution and the amount of protein not involved in emulsion droplets (i.e., in the bulk phase) were also studied. For cream cheese trials, a central composite experimental design was used to explore the effect of these 2 parameters (HP and FT) on the texture, rheology, and sensory properties of experimentally manufactured cream cheese. Storage modulus (G') and hardness values of cream cheeses were obtained from small amplitude oscillatory rheology tests and texture profile analysis, respectively. Quantitative spectrum descriptive sensory analysis was also performed. Consistency of acid gels (measured using a penetration test) increased with an increase in FT and with an increase in HP. Although stiffer acid-induced gels were formed at high FT, after the heating and shearing processes the apparent viscosity of the samples formed at high FT was lower than those formed at low FT. For the cream cheeses, significant prediction models were obtained for several rheological and textural attributes. The G' values at 8°C, instrumental hardness, and sensory firmness attributes were significantly correlated (r > 0.84); all these attributes significantly decreased with an increase in FT, and HP was not a significant parameter in the prediction models developed for these attributes. Significant interactions were observed between the HP and FT terms for these prediction models. Higher HP increased the amount of protein adsorbed at interface of fat globules but decreased bulk phase protein content (which may be important for crosslinking this gelled emulsion system). At higher FT temperature, coarser gel networks were likely formed. The combined effect of a coarser acid gel network at high FT, and less bulk phase casein available for crosslinking the acidified emulsion gel with an increase in HP, could have contributed to the lower stiffness/firmness observed in cream cheese made under conditions of both high FT and high HP. Stickiness of cream cheese greatly increased under conditions of high FT and high HP, whereas the sensory attributes cohesiveness of mass and difficulty to dissolve decreased. This study helped to better understand the complex relationships between the initial acid-induced gel phase and properties of the (final) cream cheese.

Investigating the properties of high-pressure-treated, reduced-sodium, low-moisture, part-skim Mozzarella cheese during refrigerated storage.

We proposed that the performance and sensory properties of reduced-Na, low-moisture, part-skim (LMPS) Mozzarella cheese could be extended by the application of high hydrostatic pressure (HHP) to cheese postmanufacture and thereby decrease microbial and enzymatic activity. Fermentation-produced camel chymosin was also used as a coagulant to help reduce proteolysis during storage. Average composition of the LMPS Mozzarella cheeses was 48.6 ± 0.6% moisture, 22.5 ± 0.4% fat, 24.5 ± 0.6% protein, and 1.0 ± 0.1% NaCl. Blocks of cheeses were divided into 3 groups randomly after manufacture and stored at approximately 4°C for 20 wk. The control group was not HHP treated. Two weeks after manufacture, 2 groups of cheese samples were treated with HHP at 500 or 600 MPa for 3 min and then returned to storage at approximately 4°C. Analysis was performed during 20 wk of storage after cheese manufacture. Texture profile analysis (TPA) and dynamic low-amplitude oscillatory rheology were used to monitor cheese functionality. Quantitative descriptive analysis was conducted with 9 trained panelists using a 15-point scale to evaluate texture and flavor attributes of unmelted cheese as well as cheeses melted on pizzas. Pressure treatments at 500 and 600 MPa resulted in approximately 1 and 2 log reduction in the numbers of starter culture, respectively, compared with the control when measured 1 d after HHP treatment. Starter numbers continued to decrease in all cheeses over the 20 wk of storage, but the decrease was larger in the HHP-treated cheeses. Even though the initial numbers of nonstarter lactic acid bacteria were the same in all cheeses, the numbers of these bacteria increased faster in the control cheeses. High-pressure treatment of LMPS Mozzarella cheese resulted in an initial (1 d after HHP treatment) increase in pH, but by 2 wk after HHP treatment there was no statistical difference in pH values between control and HHP-treated samples. Immediately after treatment, HHP-treated cheeses exhibited significantly lower TPA and sensory (unmelted) hardness. However, by 14 wk after pressure treatment, the 600-MPa HHP-treated cheese had significantly higher TPA compared with control or 500-MPa HHP-treated cheeses. Sensory panels also indicated that by 14 wk after HHP treatment, the 600-MPa treated samples were significantly firmer than the control or 500-MPa treated cheeses. Compared with control cheese, cheeses treated at 600 or 500 MPa exhibited lower water-soluble nitrogen values at 6 and 10 wk after pressure treatment, respectively. By 10 wk after pressure treatment, the levels of intact αS1-casein were significantly higher in all HHP-treated cheeses compared with the control. Pizza sensory panels indicated that 600-MPa treated cheese was significantly chewier and exhibited lower blister quantity and higher strand thickness compared with control cheeses. High hydrostatic pressure treatment of low-Na, LMPS Mozzarella cheese could result in the extension of its desired baking characteristics when the cheese is stored at refrigerated temperature.

A 100-Year Review: Cheese production and quality.

In the beginning, cheese making in the United States was all art, but embracing science and technology was necessary to make progress in producing a higher quality cheese. Traditional cheese making could not keep up with the demand for cheese, and the development of the factory system was necessary. Cheese quality suffered because of poor-quality milk, but 3 major innovations changed that: refrigeration, commercial starters, and the use of pasteurized milk for cheese making. Although by all accounts cold storage improved cheese quality, it was the improvement of milk quality, pasteurization of milk, and the use of reliable cultures for fermentation that had the biggest effect. Together with use of purified commercial cultures, pasteurization enabled cheese production to be conducted on a fixed time schedule. Fundamental research on the genetics of starter bacteria greatly increased the reliability of fermentation, which in turn made automation feasible. Demand for functionality, machinability, application in baking, and more emphasis on nutritional aspects (low fat and low sodium) of cheese took us back to the fundamental principles of cheese making and resulted in renewed vigor for scientific investigations into the chemical, microbiological, and enzymatic changes that occur during cheese making and ripening. As milk production increased, cheese factories needed to become more efficient. Membrane concentration and separation of milk offered a solution and greatly enhanced plant capacity. Full implementation of membrane processing and use of its full potential have yet to be achieved. Implementation of new technologies, the science of cheese making, and the development of further advances will require highly trained personnel at both the academic and industrial levels. This will be a great challenge to address and overcome.

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.

Potential of Lactobacillus curvatus LFC1 to produce slits in Cheddar cheese.

Defects in Cheddar cheese resulting from undesired gas production are a sporadic problem that results in significant financial losses in the cheese industry. In this study, we evaluate the potential of a facultatively heterofermentative lactobacilli, Lactobacillus curvatus LFC1, to produce slits, a gas related defect in Cheddar cheese. The addition of Lb. curvatus LFC1 to cheese milk at log 3 CFU/ml resulted in the development of small slits during the first month of ripening. Chemical analyses indicated that the LFC1 containing cheeses had less galactose and higher levels of lactate and acetate than the control cheeses. The composition the cheese microbiota was examined through a combination of two culture independent approaches, 16S rRNA marker gene sequencing and automated ribosomal intergenic spacer analysis; the results indicated that no known gas producers were present and that high levels of LFC1 was the only significant difference between the cheese microbiotas. A ripening cheese model system was utilized to examine the metabolism of LFC1 under conditions similar to those present in cheeses that exhibited the slit defect. The combined cheese and model system results indicate that when Lb. curvatus LFC1 was added to the cheese milk at log 3 CFU/ml it metabolized galactose to lactate, acetate, and CO2. For production of sufficient CO2 to result in the formation of slits there needs to be sufficient galactose and Lb. curvatus LFC1 present in the cheese matrix. To our knowledge, facultatively heterofermentative lactobacilli have not previously been demonstrated to result in gas-related cheese defects.

Low-sodium Cheddar cheese: Effect of fortification of cheese milk with ultrafiltration retentate and high-hydrostatic pressure treatment of cheese.

Low-sodium cheeses often exhibit an acidic flavor due to excessive acid production during the manufacturing and the initial stage of ripening, which is caused by ongoing starter culture activity facilitated by the low salt-in-moisture levels. We proposed that this excessive starter-induced acidity could be prevented by the fortification of cheese milk with ultrafiltration (UF) retentates (to increase curd buffering), and by decreasing microbial activity using the application of high-hydrostatic pressure (HHP) treatment (that is, to reduce residual starter numbers). Camel chymosin was also used as a coagulant to help reduce bitterness development (a common defect in low-sodium cheeses). Three types of low-Na (0.8% NaCl) Cheddar cheeses were manufactured: non-UF fortified, no HHP applied (L-Na); UF-fortified (cheese milk total solids = 17.2 ± 0.6%), no HHP applied (L-Na-UF); and UF-fortified, HHP-treated (L-Na-UF-HHP; 500 MPa for 3 min applied at 1 d post-cheese manufacture). Regular salt (2% NaCl) non-UF fortified, non-HHP treated (R-Na) cheese was also manufactured for comparison purposes. Analysis was performed at 4 d, 2 wk, and 1, 3, and 6 mo after cheese manufacture. Cheese functionality during ripening was assessed using texture profile analysis and dynamic low-amplitude oscillatory rheology. Sensory Spectrum and quantitative descriptive analysis was conducted with 9 trained panelists to evaluate texture and flavor attributes using a 15-point scale. At 4 d and 2 wk of ripening, L-Na-UF-HHP cheese had ~2 and ~4.5 log lower starter culture numbers, respectively, than all other cheeses. Retentate fortification of cheese milk and HHP treatment resulted in low-Na cheeses having similar insoluble calcium concentrations and pH values compared with R-Na cheese during ripening. The L-Na-UF cheese exhibited significantly higher hardness values (measured by texture profile analysis) compared with L-Na cheese until 1 mo of ripening; however, after 1 mo, all low-Na cheeses exhibited similar hardness values, which were significantly lower than R-Na cheese. Pressure treatment significantly increased maximum loss tangent (meltability) from rheology testing and decreased melt temperature. Sensory results indicated only very slight bitterness (<2.5 out of 15-point scale) was detected in all cheeses during the 6 mo of ripening. The L-Na-UF-HHP cheese did not significantly differ in bitterness and acidity from R-Na cheese during ripening. Pressures treatment of cheese at 500 MPa and cheese milk retentate fortification could be used to improve the quality of low-Na cheese.

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.

Antibody elution with 2-me/SDS solution: Uses for multi-layer immunohistochemical analysis of wholemount preparations of human colonic myenteric plexus.

Indirect immunofluorescence is usually restricted to 3-5 markers per preparation, limiting analysis of coexistence. A solution containing 2-mercaptoethanol and sodium dodecyl sulfate (2-ME/SDS) can elute indirect immunofluorescence labelling (i.e. primary antisera followed by fluorophore-conjugated secondary antisera) and has been used for sequential staining of sections. The aim of this study was to test whether 2-ME/SDS is effective for eluting indirect immunofluorescent staining (with primary antisera visualised by fluorophore-coupled secondary antisera) in wholemount preparations. We also analysed how 2-ME/SDS may work and used this understanding to devise additional uses for immunofluorescence in the nervous system. 2-ME/SDS appears to denature unfixed proteins (including antisera used as reagents) but has much less effect on antigenicity of formaldehyde-fixed epitopes. Moieties linked by strong biotin-streptavidin bonds are highly resistant to elution by 2-ME/SDS. Two primary antisera raised in the same species can be applied without spurious cross-reactivity, if a specific order of labelling is followed. The first primary antiserum is followed by a biotinylated secondary, then a tertiary of fluorophore-conjugated streptavidin. The preparation is then exposed to 2-ME/SDS, which has minimal impact on labelling by the first primary/secondary/tertiary combination. However, when this is followed by a second primary antiserum (raised in the same species), followed by a fluorophore-conjugated secondary antiserum, the intervening 2-ME/SDS exposure prevents cross-reactivity between primary and secondary antisera of the two layers. A third property of 2-ME/SDS is that it reduces lipofuscin autofluorescence, although it also raises background fluorescence and strongly enhances autofluorescence of erythrocytes. In summary, 2-ME/SDS is easy to use, cost-effective and does not require modified primary antisera. It can be used as the basis of a multi-layer immunohistochemistry protocol and allows 2 primary antisera raised in the same species to be used together.

Effect of various high-pressure treatments on the properties of reduced-fat Cheddar cheese.

A major problem with reduced-fat cheese is the difficulty in attaining the characteristic flavor and texture of typical full-fat versions. Some previous studies have suggested that high hydrostatic pressure (HHP) can accelerate the ripening of full-fat cheeses. Our objective was to investigate the effect of HHP on reduced-fat (~7.3% fat) Cheddar cheese, with the goal of improving its flavor and texture. We used a central composite rotatable design with response surface methodology to study the effect of pressure and holding time on the rheological, physical, chemical, and microbial characteristics of reduced-fat Cheddar cheese. A 2-level factorial experimental design was chosen to study the effects of the independent variables (pressure and holding time). Pressures were varied from around 50 to 400 MPa and holding times ranged from 2.5 to 19.5 min. High pressure was applied 1 wk after cheese manufacture, and analyses were performed at 2 wk, and 1, 3, and 6 mo. The insoluble calcium content as a percentage of total Ca in cheeses were not affected by pressure treatment. Pressure applications ≥ 225 MPa resulted in softer cheese texture during ripening. Pressures ≥ 225 MPa increased melt, and resulted in higher maximum loss tangent values at 2 wk. Pressure treatment had a greater effect on cheese microbial and textural properties than holding time. High-pressure-treated cheeses also had higher pH values than the control. We did not observe any significant difference in rates of proteolysis between treatments. In conclusion, holding times of around 5 min and pressures of ≥ 225 MPa could potentially be used to improve the excessively firm texture of reduced-fat cheese.

Microbiology of Cheddar cheese made with different fat contents using a Lactococcus lactis single-strain starter.

Flavor development in low-fat Cheddar cheese is typified by delayed or muted evolution of desirable flavor and aroma, and a propensity to acquire undesirable meaty-brothy or burnt-brothy off-flavor notes early in ripening. The biochemical basis for these flavor deficiencies is unclear, but flavor production in bacterial-ripened cheese is known to rely on microorganisms and enzymes present in the cheese matrix. Lipid removal fundamentally alters cheese composition, which can modify the cheese microenvironment in ways that may affect growth and enzymatic activity of starter or nonstarter lactic acid bacteria (NSLAB). Additionally, manufacture of low-fat cheeses often involves changes to processing protocols that may substantially alter cheese redox potential, salt-in-moisture content, acid content, water activity, or pH. However, the consequences of these changes on microbial ecology and metabolism remain obscure. The objective of this study was to investigate the influence of fat content on population dynamics of starter bacteria and NSLAB over 9 mo of aging. Duplicate vats of full fat, 50% reduced-fat, and low-fat (containing <6% fat) Cheddar cheeses were manufactured at 3 different locations with a single-strain Lactococcus lactis starter culture using standardized procedures. Cheeses were ripened at 8°C and sampled periodically for microbiological attributes. Microbiological counts indicated that initial populations of nonstarter bacteria were much lower in full-fat compared with low-fat cheeses made at all 3 sites, and starter viability also declined at a more rapid rate during ripening in full-fat compared with 50% reduced-fat and low-fat cheeses. Denaturing gradient gel electrophoresis of cheese bacteria showed that the NSLAB fraction of all cheeses was dominated by Lactobacillus curvatus, but a few other species of bacteria were sporadically detected. Thus, changes in fat level were correlated with populations of different bacteria, but did not appear to alter the predominant types of bacteria in the cheese.

Humidity control can compensate for the damage induced in protein crystals by alien solutions.

The use of relative humidity control of protein crystals to overcome some of the shortcomings of soaking ligands (i.e. inhibitors, substrate analogs, weak ligands) into pre-grown apoprotein crystals has been explored. Crystals of PurE (EC 4.1.1.21), an enzyme from the purine-biosynthesis pathway of Bacillus anthracis, were used as a test case. The findings can be summarized as follows: (i) using humidity control, it is possible to improve/optimize the diffraction quality of crystals soaked in solutions of organic solvent (DMSO, ethanol) containing ligands/inhibitors; (ii) optimization of the relative humidity can compensate for the deterioration of the diffraction pattern that is observed upon desalting crystals grown in high salt; (iii) combining desalting protocols with the addition of PEG it is possible to achieve very high concentrations of weak ligands (in the 5-10 mM range) in soaking solutions and (iv) fine control of the relative humidity of crystals soaked in these solutions can compensate for the deterioration of crystal diffraction and restore `high-resolution' diffraction for structure-based and fragment-based drug design. It is suggested that these experimental protocols may be useful in other protein systems and may be applicable in academic or private research to increase the probability of obtaining structures of protein-ligand complexes at high resolution.

Effect of selected Hofmeister salts on textural and rheological properties of nonfat cheese.

Three Hofmeister salts (HS; sodium sulfate, sodium thiocyanate, and sodium chloride) were evaluated for their effect on the textural and rheological properties of nonfat cheese. Nonfat cheese, made by direct acidification, were sliced into discs (diameter=50 mm, thickness=2 mm) and incubated with agitation (6 h at 22°C) in 50 mL of a synthetic Cheddar cheese aqueous phase buffer (pH 5.4). The 3 HS were added at 5 concentrations (0.1, 0.25, 0.5, 0.75, and 1.0 M) to the buffer. Post-incubation, cheese slices were air dried and equilibrated in air-tight bags for 18 h at 5°C before analysis. Small amplitude oscillatory rheology properties, including the dynamic moduli and loss tangent, were measured during heating from 5 to 85°C. Hardness was determined by texture profile analysis. Acid-base buffering was performed to observe changes in the indigenous insoluble (colloidal) calcium phosphate (CCP). Moisture content decreased with increasing HS concentration. Cheeses incubated in high concentrations of SCN(-) softened earlier (i.e., loss tangent=1) compared with other HS treatments. Higher melting temperature values were observed for cheeses incubated in high concentrations of SO(4)(2-). Hardness decreased in cheeses incubated in buffers with high concentrations of SCN(-). The indigenous CCP profile of nonfat cheese was not greatly affected by incubation in Cl(-) or SCN(-), whereas buffers with high concentrations of SO(4)(2-) reduced the acid-base buffering contributed by CCP. The use of high concentrations (1.0M) of SCN(-) for incubation of cheeses resulted in a softer protein matrix at high temperatures due to the chaotropic effect of SCN(-), which weakened hydrophobic interactions between CN. Cheese samples incubated in 1.0M SO(4)(2-) buffers exhibited a stiffer protein matrix at high temperatures due to the kosmotropic effect of SO(4)(2-), which helped to strengthen hydrophobic interactions in the proteins during the heating step. This study showed that HS influenced the texture and rheology of nonfat cheese probably by altering the strength of hydrophobic interactions between CN.

Effect of different curd-washing methods on the insoluble Ca content and rheological properties of Colby cheese during ripening.

A curd-washing step is used in the manufacture of Colby cheese to decrease the residual lactose content and, thereby, decrease the potential formation of excessive levels of lactic acid. The objective of this study was to investigate the effect of different washing methods on the Ca equilibrium and rheological properties of Colby cheese. Four different methods of curd-washing were performed. One method was batch washing (BW), where cold water (10°C) was added to the vat, with and without stirring, where curds were in contact with cold water for 5 min. The other method used was continuous washing (CW), with or without stirring, where curds were rinsed with continuously running cold water for approximately 7 min and water was allowed to drain immediately. Both methods used a similar volume of water. The manufacturing pH values were similar in all 4 treatments. The insoluble (INSOL) Ca content of cheese was measured by juice and acid-base titration methods and the rheological properties were measured by small amplitude oscillatory rheology. The levels of lactose in cheese at 1 d were significantly higher in CW cheese (0.06-0.11%) than in BW cheeses (∼0.02%). The levels of lactic acid at 2 and 12 wk were significantly higher in CW cheese than in BW cheeses. No differences in the total Ca content of cheeses were found. Cheese pH increased during ripening from approximately 5.1 to approximately 5.4. A decrease in INSOL Ca content of all cheeses during ripening occurred, although a steady increase in pH took place. The initial INSOL Ca content as a percent of total Ca in cheese ranged from 75 to 78% in all cheeses. The INSOL Ca content of cheese was significantly affected by washing method. Stirring during manufacturing did not have a significant effect on the INSOL Ca content of cheese during ripening. Batch-washed cheeses had significantly higher INSOL Ca contents than did CW cheeses during the first 4 wk of ripening. The maximum loss tangent values (meltability index) of CW cheese at 1 d and 1 wk were significantly higher compared with those of BW cheeses. In conclusion, different curd washing methods have a significant effect on the levels of lactose, lactic acid, meltability, and INSOL Ca content of Colby cheese during ripening.

Standardization of milk using cold ultrafiltration retentates for the manufacture of Swiss cheese: effect of altering coagulation conditions on yield and cheese quality.

Fortification of cheesemilk with membrane retentates is often practiced by cheesemakers to increase yield. However, the higher casein (CN) content can alter coagulation characteristics, which may affect cheese yield and quality. The objective of this study was to evaluate the effect of using ultrafiltration (UF) retentates that were processed at low temperatures on the properties of Swiss cheese. Because of the faster clotting observed with fortified milks, we also investigated the effects of altering the coagulation conditions by reducing the renneting temperature (from 32.2 to 28.3°C) and allowing a longer renneting time before cutting (i.e., giving an extra 5min). Milks with elevated total solids (TS; ∼13.4%) were made by blending whole milk retentates (26.5% TS, 7.7% CN, 11.5% fat) obtained by cold (<7°C) UF with part skim milk (11.4% TS, 2.5% CN, 2.6% fat) to obtain milk with CN:fat ratio of approximately 0.87. Control cheeses were made from part-skim milk (11.5% TS, 2.5% CN, 2.8% fat). Three types of UF fortified cheeses were manufactured by altering the renneting temperature and renneting time: high renneting temperature=32.2°C (UFHT), low renneting temperature=28.3°C (UFLT), and a low renneting temperature (28.3°C) plus longer cutting time (+5min compared to UFLT; UFLTL). Cutting times, as selected by a Wisconsin licensed cheesemaker, were approximately 21, 31, 35, and 32min for UFHT, UFLT, UFLTL, and control milks, respectively. Storage moduli of gels at cutting were lower for the UFHT and UFLT samples compared with UFLTL or control. Yield stress values of gels from the UF-fortified milks were higher than those of control milks, and decreasing the renneting temperature reduced the yield stress values. Increasing the cutting time for the gels made from the UF-fortified milks resulted in an increase in yield stress values. Yield strain values were significantly lower in gels made from control or UFLTL milks compared with gels made from UFHT or UFLT milks. Cheese composition did not differ except for fat content, which was lower in the control compared with the UF-fortified cheeses. No residual lactose or galactose remained in the cheeses after 2 mo of ripening. Fat recoveries were similar in control, UFHT, and UFLTL but lower in UFLT cheeses. Significantly higher N recoveries were obtained in the UF-fortified cheeses compared with control cheese. Because of higher fat and CN contents, cheese yield was significantly higher in UF-fortified cheeses (∼11.0 to 11.2%) compared with control cheese (∼8.5%). A significant reduction was observed in volume of whey produced from cheese made from UF-fortified milk and in these wheys, the protein was a higher proportion of the solids. During ripening, the pH values and 12% trichloroacetic acid-soluble N levels were similar for all cheeses. No differences were observed in the sensory properties of the cheeses. The use of UF retentates improved cheese yield with no significant effect on ripening or sensory quality. The faster coagulation and gel firming can be decreased by altering the renneting conditions.