Effect of Free or Encapsulated Recombinant Aminopeptidase of Lactobacillus rhamnosus S93 on Acceleration of Cheddar Cheese Ripening

The use of recombinant aminopeptidase (PepN) from Lactobacillus rhamnosus S93 in free or encapsulated form was investigated to shorten the duration of Cheddar cheese ripening. Proteolysis was determined by measuring the soluble nitrogen as phosphotungstic acid (PTA-N) derivatives and free amino acids (FAA) over a 6-month period. The experimental cheeses received higher scores for sensory properties than the control cheese. The amounts of PTA-N and total FAA in the cheese with the encapsulated enzyme after 2 months of ripening were close to those of the control cheese after 6 months, suggesting the acceleration in proteolysis by about 4 months.     

Use of Recombinant Chymosin in the Manufacture of Cheddar and Colby Cheese

Recombinant chymosin purified from E. coli K12 (Genencor Inc.) was examined for its suitability to manufacture Cheddar cheese. Commercial calf rennet or recombinant chymosin with and without Bacillus subtilis cellular homogenate was used to determine if enzyme sources affected Cheddar cheese quality or yield. Cheese was evaluated at 3 and 6 mo of age. Purified enzyme was mixed with cellular extracts from B. subtilis at approximately one and four times the protein concentration in the initial homogenates. Cellular materials did not affect clot times, cheese quality, or inhibit cheese acid development. No differences were noted between the yields of Colby cheese manufactured with calf rennet and recombinant chymosin. Initial Cheddar cheese manufactured with recombinant chymosin were excessively bitter, typical of cheese retaining excessive amounts of proteases. Dilution of purified prochymosin preparations increased total enzymic activity 2.7 times. Evidently, the purified recombinant preparations contained some oligomers that became active after being incorporated into the cheese mass, causing bitter flavors to develop during aging. Diluted enzyme preparations were used successfully in the manufacture of Cheddar cheese.     

Application of encapsulated enzymes to accelerate cheese ripening

The suitability of gellan, κ-carrageenan and a high-melting-fat-fraction of milk fat (HMFF) to encapsulate protease enzymes (Flavourzyme) and impact in accelerating Cheddar cheese ripening were studied. The rates of enzyme entrapment were 48.2%, 55.6%, and 38.9% for gellan, κ-carrageenan and HMFF, respectively. The enzyme capsules were incorporated into milk during cheese manufacture. The moisture content of cheeses with added gum capsules was higher than control cheeses. Casein (β) degradation was monitored by High-Performance Capillary Electrophoresis. All cheeses treated with encapsulated enzyme showed higher rates of proteolysis than the control cheese throughout the ripening period. The rate of proteolysis was greater with cheeses made incorporating κ-carrageenan capsules containing protease. Cheese texture and sensory quality were not significantly influenced by the type of encapsulating material (gum or milk fat). Differences in textural and sensory quality between treated and control cheeses were consistent with release of protease enzymes from capsules.     

Effect of galactose metabolising and non-metabolising strains of Streptococcus thermophilus as a starter culture adjunct on the properties of Cheddar cheese made with low or high pH at whey drainage

Cheddar cheese was made using control culture (Lactococcus lactis subsp. lactis), or with control culture plus a galactose-metabolising (Gal⁺) or galactose-non-metabolising (Gal⁻) Streptococcus thermophilus adjunct; for each culture type, the pH at whey drainage was either low (pH 6.15) or high (pH 6.45). S. thermophilus affected the levels of residual lactose and galactose, and the volatile compound profile and sensory properties of the mature cheese (270 d) to an extent dependent on the drain pH and phenotype (Gal⁺ or Gal⁻). For all culture systems, reducing drain pH resulted in lower levels of moisture and lactic acid, a higher concentration of free amino acids, and higher firmness. The results indicate that S. thermophilus may be used to diversify the sensory properties of Cheddar cheese, for example from a fruity buttery odour and creamy flavour to a more acid taste, rancid odour, and a sweaty cheese flavour at high drain pH.     

High-Pressure Treatment and Freezing of Raw Goat Milk Curd for Cheese Manufacture: Effects on Cheese Characteristics

High-pressure treatment of raw goat milk curd was investigated as an alternative to thermal treatment of milk in cheese manufacture, and curd freezing as a procedure to surmount the seasonality of goat milk production. Experimental cheeses were made by mixing (70:30) fresh cow milk curd with frozen curd from pasteurized goat milk (PGC), frozen curd from raw goat milk (RGC), or frozen pressurized curd from raw goat milk (PRGC). Control cheese was made from a mixture (70:30) of pasteurized cow and goat milk. RGC cheese showed the highest counts of staphylococci, Gram-negative bacteria and coliforms, whereas PRGC cheese had maximum aminopeptidase activity, esterase activity, and overall proteolysis. Control cheese exhibited the highest dry matter content and peptide levels, the lowest concentration of free amino acids, the highest concentration of volatile compounds such as free fatty acids, alcohols and esters, and the firmest texture. Differences in sensory characteristics between experimental and control cheeses were of minor importance. High-pressure treatment of curd allowed the production of cheese of bacteriological quality similar to that of control cheese made using pasteurized milk, while curd freezing did not alter the sensory characteristics of experimental cheeses with respect to control cheese.     

Influence of reuterin-producing Lactobacillus reuteri coupled with glycerol on biochemical,physical and sensory properties of semi-hard ewe milk cheese

The biochemical, physical and sensory characteristics of ewe milk cheeses made with reuterin-producing Lactobacillus reuteri and glycerol (substrate for reuterin production) were assessed. Cheese made with lactococci starter (CTRL), cheese made with starter and L. reuteri (SLR), and cheese made with starter, L. reuteri and 30 mM glycerol (SLR-G) were manufactured. L. reuteri reached counts above 7 log cfu/g on day 1. Lactococci survival was enhanced in SLR cheese without affecting cheese pH, dry matter, proteolysis, concentration of most free amino acids (FAA), textural and most color parameters, or sensory characteristics. In situ production of reuterin by L. reuteri was only detected in SLR-G cheese, decreasing LAB counts although acidification remained unaffected. SLR-G cheese showed higher values of cell free aminopeptidase activity, overall proteolysis and FAA, particularly glutamic acid, than CTRL and SLR cheeses. The addition of L. reuteri-glycerol resulted in lower hardness and elasticity values in SLR-G cheese and influenced its L*, a* and b* color parameters. However, these changes, which were detected by instrumental analysis, did not affect the sensory scores for texture and color quality of SLR-G cheese, and it received the highest scores for taste quality. Our results suggest that L. reuteri-glycerol may provide a suitable system to release the antimicrobial reuterin in cheese without affecting negatively its sensory characteristics.     

Effect of pasture versus indoor feeding systems on quality characteristics,nutritional composition,and sensory and volatile properties of full-fat Cheddar cheese

The purpose of this study was to investigate the effects of pasture-based versus indoor total mixed ration (TMR) feeding systems on the chemical composition, quality characteristics, and sensory properties of full-fat Cheddar cheeses. Fifty-four multiparous and primiparous Friesian cows were divided into 3 groups (n = 18) for an entire lactation. Group 1 was housed indoors and fed a TMR diet of grass silage, maize silage, and concentrates; group 2 was maintained outdoors on perennial ryegrass only pasture (GRS); and group 3 was maintained outdoors on perennial ryegrass/white clover pasture (CLV). Full-fat Cheddar cheeses were manufactured in triplicate at pilot scale from each feeding system in September 2015 and were examined over a 270-d ripening period at 8°C. Pasture-derived feeding systems were shown to produce Cheddar cheeses yellower in color than that of TMR, which was positively correlated with increased cheese β-carotene content. Feeding system had a significant effect on the fatty acid composition of the cheeses. The nutritional composition of Cheddar cheese was improved through pasture-based feeding systems, with significantly lower thrombogenicity index scores and a greater than 2-fold increase in the concentration of vaccenic acid and the bioactive conjugated linoleic acid C18:2 cis-9,trans-11, whereas TMR-derived cheeses had significantly higher palmitic acid content. Fatty acid profiling of cheeses coupled with multivariate analysis showed clear separation of Cheddar cheeses derived from pasture-based diets (GRS or CLV) from that of a TMR system. Such alterations in the fatty acid profile resulted in pasture-derived cheeses having reduced hardness scores at room temperature. Feeding system and ripening time had a significant effect on the volatile profile of the Cheddar cheeses. Pasture-derived Cheddar cheeses had significantly higher concentrations of the hydrocarbon toluene, whereas TMR-derived cheese had significantly higher concentration of 2,3-butanediol. Ripening period resulted in significant alterations to cheese volatile profiles, with increases in acid-, alcohol-, aldehyde-, ester-, and terpene-based volatile compounds. This study has demonstrated the benefits of pasture-derived feeding systems for production of Cheddar cheeses with enhanced nutritional and rheological quality compared with a TMR feeding system.     

Identification of bioactive peptides in commercial Cheddar cheese

This study examined the presence of antimicrobial, antioxidant and antihypertensive peptides in three commercially available Australian Cheddar cheeses. Peptide extracts as well as fractionated peptide extracts were examined. Commercial cheese A peptides exhibited the greatest inhibition against Bacillus cereus and also commercial cheese A fractionated peptides greater than 10 kDa showed the highest inhibition against B. cereus. Commercial cheese A peptides also showed the highest inhibition of 2,2-diphenyl-1-picrylhydrazyl (DPPH), a free radical used to measure antioxidant activity. All cheese fractionated peptides greater than 10 kDa demonstrated higher inhibition of DPPH after fractionation. Antihypertensive peptides were determined by inhibition of the angiotensin-converting enzyme (ACE). Overall, commercial cheese A had the lowest concentration required to inhibit ACE and commercial cheese A fractionated peptides lower than 5 kDa had the lowest inhibition after fractionation. These preliminary findings suggest that peptide extracts of three commercial Australian Cheddar cheeses exhibit antimicrobial, antihypertensive and antioxidant properties.     

Proteolysis development in enzyme-modified Cheddar cheese using natural and recombinant enzymes of Lactobacillus rhamnosus S93

Proteolysis in enzyme-modified cheese was investigated with natural crude enzyme or recombinant aminopeptidase, both derived from Lactobacillus rhamnosus S93 in the presence of a commercial proteinase, Neutrase. For production of enzyme-modified cheeses, a cheese slurry was produced and pre-incubated with Neutrase. Natural enzyme or recombinant aminopeptidase (50 units 200 g⁻¹ slurry) was added alone or in combination to the cheese slurries, which were then incubated anaerobically under vacuum at 37 °C for 1, 3 and 6 d. The greatest levels of phosphotungstic acid soluble nitrogen and free amino acids were observed in the enzyme-modified cheese containing natural enzyme followed by the one treated with a combination of the natural enzyme and recombinant aminopeptidase. The enzyme-modified cheese containing the recombinant aminopeptidase alone resulted in the complete disappearance of proline after 1 d of maturation time.     

Impact of fat reduction on flavor and flavor chemistry of Cheddar cheeses

A current industry goal is to produce a 75 to 80% fat-reduced Cheddar cheese that is tasty and appealing to consumers. Despite previous studies on reduced-fat cheese, information is critically lacking in understanding the flavor and flavor chemistry of reduced-fat and nonfat Cheddar cheeses and how it differs from its full-fat counterpart. The objective of this study was to document and compare flavor development in cheeses with different fat contents so as to quantitatively characterize how flavor and flavor development in Cheddar cheese are altered with fat reduction. Cheddar cheeses with 50% reduced-fat cheese (RFC) and low-fat cheese containing 6% fat (LFC) along with 2 full-fat cheeses (FFC) were manufactured in duplicate. Cheeses were ripened at 8°C and samples were taken following 2 wk and 3, 6, and 9 mo for sensory and instrumental volatile analyses. A trained sensory panel (n = 10 panelists) documented flavor attributes of cheeses. Volatile compounds were extracted by solid-phase microextraction or solvent-assisted flavor evaporation followed by separation and identification using gas chromatography-mass spectrometry and gas chromatography-olfactometry. Selected compounds were quantified using external standard curves. Sensory properties of cheeses were distinct initially but more differences were documented as cheeses aged. By 9 mo, LFC and RFC displayed distinct burnt/rosy flavors that were not present in FFC. Sulfur flavor was also lower in LFC compared with other cheeses. Forty aroma-active compounds were characterized in the cheeses by headspace or solvent extraction followed by gas chromatography-olfactometry. Compounds were largely not distinct between the cheeses at each time point, but concentration differences were evident. Higher concentrations of furanones (furaneol, homofuraneol, sotolon), phenylethanal, 1-octen-3-one, and free fatty acids, and lower concentrations of lactones were present in LFC compared with FFC after 9 mo of ripening. These results confirm that flavor differences documented between full-fat and reduced-fat cheeses are not due solely to differences in matrix and flavor release but also to distinct differences in ripening biochemistry, which leads to an imbalance of many flavor-contributing compounds.     

Permeate Flux During Ultrafiltration of Whey: Influence of Milk Coagulant Used for Cheese Manufacture

A pilot-scale plate and frame UF system was used to fractionate Cheddar cheese whey and study the effects of different commercial milk coagulants on permeate flux. Coagulants used in this study were calf rennet, Mucor pusillus protease, and Mucor miebei protease. Whey UF performance studies were conducted at a commercial Cheddar cheese plant and at Cornell under controlled conditions. Ultrafiltration was done in a continuous mode and initial concentration factor was set at 2× to simulate the first stage of a multistage whey UF system.Permeate flux decline was rapid in the first 30 min of UF for all wheys studied. More important, the type of milk coagulant used in cheese making had a profound effect on permeate flux during whey UF. No differences in the gross composition of the various wheys were correlated with differences in permeate flux. The highest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using coagulant derived from Mucor pusillus. Lowest permeate flux was measured for UF of whey produced during manufacture of Cheddar cheese using calf rennet. Whey from cheese manufactured using Mucor miebei coagulant had flux performance intermediate to Mucor pusillus and calf rennet. The impact of milk coagulants on whey UF process efficiency should be considered by cheese makers.     

A survey of lipolytic and glycolytic end-products in commercial Cheddar enzyme-modified cheese

The concentrations of L- and D-lactic acid and free fatty acids, C4:0 to C18:3, were quantified in a range of commercial enzyme-modified Cheddar cheeses. Lactic acid in Cheddar enzyme-modified cheeses varied markedly depending on the manufacturer. Differences in the ratio of L- to D-lactic acid indicate that cheeses of different age were used in their manufacture or contained varying levels of nonstarter lactic acid bacteria. The level of lipolysis in enzyme-modified cheese was higher than in natural Cheddar cheese; butyrate was the predominant free fatty acid. The addition of exogenous acetate, lactate, and butyrate was also indicated in some enzyme-modified cheeses and may be used to confer a specific flavor characteristic or reduce the pH of the product. Propionate was also found in some enzyme-modified cheese products and most likely originated from Swiss-type cheese used in their manufacture. Propionate is not normally associated with natural Cheddar cheese flavor; however, it may be important in the flavor and aroma of Cheddar enzyme-modified cheese. Levels of lipolysis and glycolysis appear to highly controlled as interbatch variability was generally low. Overall, the production of enzyme-modified Cheddar cheese involves manipulation of the end-products of glycolysis (lactate, propionate, and acetate) and lipolysis to generate products for specific applications.     

Autolysis of selected Lactobacillus helveticus adjunct strains during Cheddar cheese ripening

Cheddar cheeses were manufactured on a pilot scale (500 L vats) with three different Lactobacillus helveticus strains, which showed varying degrees of autolysis, added as adjuncts to the starter. Autolysis of adjunct strains was monitored by reduction in cell numbers, level of intracellular enzymes released into the cheese, and by the consequent changes in the degree of proteolysis and concentration of free amino acids in the cheese. The flavour profiles of the cheeses at 6 months were also determined. Significant variation in viability of the Lb. helveticus strains, which showed a positive correlation with the indicators of autolysis, was observed. However, cheese manufactured with the most autolytic strain did not receive the highest flavour scores. The results indicate that whereas autolysis of adjunct strains is an important factor in Cheddar cheese flavour development, other factors also contribute to the overall flavour improvement observed.     

Evaluation of NaCl,pH, and lactic acid on the growth of Shiga toxin-producing Escherichia coli in a liquid Cheddar cheese extract

A Cheddar cheese model system, Cheddar cheese extract, was used to examine how different levels of known microbial hurdles (NaCl, pH, and lactic acid) in Cheddar cheese contribute to inhibition of bacterial pathogens. This knowledge is critical to evaluate the safety of Cheddar varieties with altered compositions. The range of levels used covered the lowest and highest level of these factors present in low-sodium, low-fat, and traditional Cheddar cheeses. Four pathogens were examined in this model system at 11°C for 6 wk, with the lowest levels of these inhibitory factors that would be encountered in these products. The 4 pathogens examined were Salmonella enterica, Staphylococcus aureus, Listeria monocytogenes, and Shiga toxin-producing Escherichia coli (STEC). None of these organisms were capable of growth under these conditions. The STEC exhibited the highest survival and hence was used to examine which of these inhibitory factors (NaCl, pH, and lactic acid) was primarily responsible for the observed inhibition. The STEC survival was examined in Cheddar cheese extract varying in NaCl (1.2 vs. 4.8%), lactic acid (2.7 vs. 4.3%), and pH (4.8 vs. 5.3) at 11°C for 6 wk. The microbial hurdle found to have the greatest effect on STEC survival was pH. The interactions between pH and levels of protonated lactic acid and anionic lactic acid with STEC survival was also evaluated; only the concentration of protonated lactic acid was determined to have a significant effect on STEC survival. These results indicate that, of the pathogens examined, STEC is of the greatest concern in Cheddar varieties with altered compositions and that pH is the microbial hurdle primarily responsible for controlling STEC in these products.     

Effects of temperature,relative humidity,and protective netting on Tyrophagus putrescentiae (schrank) (sarcoptiformes: Acaridae) infestation,fungal growth,and product quality of cave-aged Cheddar cheese

Research was conducted to evaluate the effects of using food-grade ingredients on cave aged Cheddar cheese as either a surface coating or in nets to prevent infestation by Tyrophagus putrescentiae growth at different environmental conditions. Food grade coating formulations with 1) xanthan gum and propylene glycol (XG+PG) and 2) carrageenan, propylene glycol alginate, and PG (CG+PGA+PG) were made and infused into nets. Jars with cave aged Cheddar cheese cubes that were inoculated with 20 mites were stored in an environmental chamber for 14 d at temperature and relative humidity (RH) combinations of 10, 15, and 20 °C and 75 ± 2 and 85 ± 2% RH. When averaged over RH, mite counts were fewer on control cheese cubes at 10 °C when compared to 15 °C and 20 °C, regardless of whether nets were used or not. However, mites were able to reproduce on untreated cheese cubes at all temperatures. The CG+PGA+PG and XG+PG coatings and nets controlled mite reproduction, as evidenced by harboring less than the initial inoculation level of 20 mites. Sensory results indicated that CG+PGA+PG and XG+PG coated Cheddar cheese at 10 °C and 75% RH and netted Cheddar cheese at 10 or 15 °C and 75% RH did not differ (P > 0.05) from the control with respect to sensory attributes. The treatments at 15 °C and 85% RH and 20 °C caused the cheese to be softer and more bitter than control cheese. In conclusion, the CG+PGA+ 40% PG and XG+40% PG treatments of both coatings and nets inhibited the growth of mites, and the use of nets lessened the impact of food grade coatings on the sensory properties of the Cheddar cheese.     

Factors affecting calcium lactate and liquid expulsion defects in Cheddar cheese

This paper summarizes the results of 2 studies designed to investigate the influence of several manufacturing and curing treatments on the appearance of Cheddar cheese defects. Specifically, 2 defects, calcium lactate crystal formation and the expulsion of free liquid (weeping) were monitored in Cheddar cheese. Both studies were conducted at a commercial cheese manufacturing facility that produces Cheddar in 18.14-kg (40-lb) blocks. In the first study we monitored cheese calcium, both total and soluble during manufacture and early curing. In the second study we measured cheese pH from 3 d through 8 mo, as well as some factors that are influenced by cheese pH. Early cheese pH (3 d to 7 d) patterns were used to select vats of cheese for retail packaging. Mild Cheddar packaged at 30 d postmanufacture and sharp Cheddar packaged at 8 mo postmanufacture from the same vats were monitored for the incidence and severity of the defects. Our results indicated that factors measured in early stages of manufacture and curing (less than 7 d) such as cheese pH at mill, lactic acid concentration, nonprotein nitrogen, and calcium (total and soluble) in cheese did not correlate with the appearance of either calcium lactate or expulsion of free liquid in packaged cheeses. Factors including pH, lactic acid concentrations, and soluble calcium measured during curing (greater than 7 d) of cheese were found to be statistically significant in the development of defects and appeared to be associated with use of specific starter culture groups. In the study, 5 different starter culture groups, each consisting of a 4-strain blend of Lactococcus lactis ssp. cremoris and Lactococcus lactis ssp. lactis, were used to manufacture the cheeses. Cheese manufactured with one particular culture group showed no incidence of calcium lactate crystal formation or weeping during curing and shelf-life of cheeses in this study. This starter group also generated the least amount of pH change in cheese during the first month of curing. From these results we conclude that starter culture group, more than any other factor measured, played an important role in the development of calcium lactate and liquid expulsion defects in Cheddar cheese. Starter culture group appeared to strongly influence cheese pH, lactic acid, and soluble calcium concentrations during curing and storage.     

Effect of anthocyanin-absorbed whey protein microgels on physicochemical and textural properties of reduced-fat Cheddar cheese

Reduced-fat foods have become more popular due to their health benefits; however, reducing the fat content of food affects the sensory experience. Therefore, it is necessary to improve the sensory acceptance of reduced-fat foods to that of full-fat equivalents. The aim of this study was to evaluate the effect of adding whey protein microgels (WPM) with an average diameter of 4 μm, or WPM with adsorbed anthocyanins [WPM (Ant)] on the textural and sensory properties of reduced-fat Cheddar cheese (RFC). Reduced-fat Cheddar cheese was prepared in 2 ways: (1) by adding WPM, designated as RFC+M, or (2) by adding WPM (Ant), designated as RFC+M (Ant). For comparison, RFC without fat substitutes and full-fat Cheddar cheese were also prepared. We discovered that the addition of WPM and WPM (Ant) increased the moisture content, fluidity, and meltability of RFC, and reduced its hardness, springiness, and chewiness. The textural and sensory characteristics of RFC were markedly inferior to those of full-fat Cheddar cheese, whereas addition of WPM and WPM (Ant) significantly improved the sensory characteristics of RFC. The WPM and WPM (Ant) showed a high potential as fat substitutes and anthocyanin carriers to effectively improve the acceptance of reduced-fat foods.