M.E. Johnson

Contribution of Lactococcus lactis Cell Envelope Proteinase Specificity to Peptide Accumulation and Bitterness in Reduced-Fat Cheddar Cheese†

ABSTRACT Bitterness is a flavor defect in Cheddar cheese that limits consumer acceptance, and specificity of the Lactococcus lactis extracellular proteinase (lactocepin) is widely believed to be a key factor in the development of bitter cheese. To better define the contribution of this enzyme to bitterness, we investigated peptide accumulation and bitterness in 50% reduced-fat Cheddar cheese manufactured with single isogenic strains of Lactococcus lactis as the only starter. Four isogens were developed for the study; one was lactocepin negative, and the others produced a lactocepin with group a, e, or h specificity. Analysis of cheese aqueous extracts by reversed-phase high-pressure liquid chromatography confirmed that accumulation of αS1-casein (f 1-23)-derived peptides f 1-9, f 1-13, f 1-16, and f 1-17 in cheese was directly influenced by lactocepin specificity. Trained sensory panelists demonstrated that Cheddar cheese made with isogenic starters that produced group a, e, or h lactocepin was significantly more bitter than cheese made with a proteinase-negative isogen and that propensity for bitterness was highest in cells that produced group h lactocepin. These results confirm the role of starter proteinase in bitterness and suggest that the propensity of some industrial strains for production of the bitter flavor defect in cheese could be altered by proteinase gene exchange or gene replacement.

Production of cheddar cheese using a Lactococcus lactis ssp. Cremoris SK11 derivative with enhanced aminopeptidase activity

Abstract A Lactobacillus helveticus CNRZ32 DNA fragment encoding aminopeptidase N (PepN) was cloned into pIL253 to construct pSUW34. The cell-free extract general aminopeptidase activity of Lactococcus lactis ssp. cremoris SK11 transformed with the pSUW34 construct was increased approximately 100-fold relative to L. lactis ssp. cremoris SK11 wild type and pIL253 transformants. Cheddar cheese was manufactured with L. lactis ssp. cremoris HP in combination with either SK11(pIL253) or SK11(pSUW34). A 99% reduction in viable SK11 derivatives occurred within 2 weeks of ripening. The SK11(pSUW34)/HP cheese had approximately 1000-fold greater aminopeptidase activity and elevated levels of TCA and PTA soluble nitrogen. The increased accumulation of particular free amino acids in the SK11(pSUW34)/HP cheese varied between 15% (methionine) and 230% (lysine). Both the SK11(pSUW34)/HP and SK11(pIL253)/HP cheeses were non-bitter and had no significant sensory differences. Thus, the increased free amino acid pool in the SK11(pSUW34)/HP cheese did not result in accelerated flavor development.

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in Cheddar cheese

ABSTRACT Metabolism of aromatic amino acids by lactic acid bacteria is an important source of off-flavor compounds in Cheddar cheese. Previous work has shown that α-keto acids produced from Trp, Tyr, and Phe by aminotransferase enzymes are chemically labile and may degrade spontaneously into a variety of off-flavor compounds. However, dairy lactobacilli can convert unstable α-keto acids to more-stable α-hydroxy acids via the action of α-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus caseid-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that α-keto acid dehydrogenase activity was significantly higher in pTRKH2:dhic transformants than in wild-type cells. Reduced-fat Cheddar cheeses were made with Lactococcus lactis starter only, starter plus L. casei ATCC 334, and starter plus L. casei ATCC 334 transformed with pTRKH2:dhic. After 3 months of aging, the cheese chemistry and flavor attributes were evaluated instrumentally by gas chromatography-mass spectrometry and by descriptive sensory analysis. The culture system used significantly affected the concentrations of various ketones, aldehydes, alcohols, and esters and one sulfur compound in cheese. Results further indicated that enhanced expression of d-hydroxyisocaproic acid dehydrogenase suppressed spontaneous degradation of α-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

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.

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.

Fate of Listeria monocytogenes during the manufacture of mozzarella cheese

Mozzarella cheese was made from a mixture of pasteurized whole and skim milk which was inoculated to contain 104-105 CFU Listeria monocytogenes (strain Ohio, California, or V7) per ml. Temperature of milk was maintained at 40°C (104°F) for 30 min when curd became resilient and the pH reached 5.90-5.93. Populations of L. monocytogenes changed at different rates during the various phases of making Mozzarella cheese. During the early stages of curd formation, numbers of L. monocytogenes were ca. 4-fold greater in curd than in whey. Numbers of L. monocytogenes in freshly cut curd were 25 to 38% greater than in inoculated milk. Cooking curd at 40°C for ca. 30 min caused a decrease of ca. 38% as compared to numbers of the pathogen in curd after cutting. During cheddaring of curd, numbers of L. monocytogenes increased by ca. 25%, over numbers at the end of cooking. Placing of curd in hot water [77°C (170°F)] and stretching for 3-4 min caused complete demise of the pathogen, as determined by our methods. The curd temperature during stretching was 58 to 65°C (136 to 149°F). Results of cold enrichments were all negative for stretched and brined curd. L. monocytogenes failed to survive during the making of Mozzarella cheese as done in this study.

Technology of Manufacturing Reduced-Fat Cheddar Cheese

For many consumers the thought of a reduced-fat cheese conjures up notions of a bland cheese with either a firm, rubbery body or a soft, pasty one. But more often than not reduced-fat cheeses are described as having a very undesirable taste as well as the body defects. A quality reduced-fat cheese deteriorates in quality after only a few months of storage. Unfortunately, reduced-fat cheese with such a short shelf-life may be purchased well after body and flavor defects have developed. Although sales of reducedfat cheese have not approached the level that many cheese makers had hoped, interest remains high. Our research project was initiated to determine the manufacturing practices that could lead to desirable body and flavor. We did not start from “ground zero” in as much as there is a considerable base of knowledge about defects in cheese body and flavor. Also, the principles of cheese manufacturing, chemistry and flavor development are constant regardless of the variety or name that is attached to the cheese.

Qualitative and quantitative analysis of proteins and peptides in milk products by capillary electrophoresis

Milk protein is an important component of the human diet throughout much of the world. The ability to assess the relative composition and integrity of milk proteins or peptides in dairy foods or food ingredients is important because these molecules have a profound effect on product functionality and quality. This communication describes two capillary electrophoretic methods that are useful for the analysis of proteins and casein‐derived peptides in cheese and milk products. One technique, which uses a buffer containing citrate/phosphate (pH 3.3), 4 M urea, and a polymeric additive in a coated capillary, is useful for qualitative and quantitative analysis of proteins and peptides in milk, cheese, and whey products. The second method employs a citrate/phosphate buffer (pH 2.8) and a bare silica capillary, and is well suited for the analysis of small, casein‐derived peptides in aqueous cheese extracts.

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.

Insoluble calcium content and rheological properties of Colby cheese during ripening

Colby cheese was made using different manufacturing conditions (i.e., varying the lactose content of milk and pH values at critical steps in the cheesemaking process) to alter the extent of acid development and the insoluble and total Ca contents of cheese. Milk was concentrated by reverse osmosis (RO) to increase the lactose content. Extent of acid development was modified by using high (HPM) and low (LPM) pH values at coagulant addition, whey drainage, and curd milling. Total Ca content was determined by atomic absorption spectroscopy, and the insoluble (INSOL) Ca content of cheese was measured by the cheese juice method. The rheological and melting properties of cheese were measured by small amplitude oscillatory rheometry and UW-Melt Profiler, respectively. There was very little change in pH during ripening even in cheese made from milk with high lactose content. The initial (d 1) cheese pH was in the range of 4.9 to 5.1. The INSOL Ca content of cheese decreased during the first 4 wk of ripening. Cheeses made with the LPM had lower INSOL Ca content during ripening compared with cheese made with HPM. There was an increase in melt and maximum loss tangent values during ripening except for LPM cheeses made with RO-concentrated milk, as this cheese had pH <4.9 and exhibited limited melt. Curd washing reduced the levels of lactic acid produced during ripening and resulted in significantly higher INSOL Ca content. The use of curd washing for cheeses made from high lactose milk prevented a large pH decrease during ripening; high rennet and draining pH values also retained more buffering constituents (i.e., INSOL Ca phosphate), which helped prevent a large pH decrease.