Mammals, milk, molecules, and micelles.

After a brief description of my family background and school days, my professional career as a dairy scientist is described under three headings: research, teaching, and writing. My research activities fall into four areas: biochemistry of cheese, fractionation and characterization of milk proteins, heat stability of milk, and dairy enzymology. Finally, I offer some advice to young scientists.

Characterization of Italian cheeses ripened under nonconventional conditions.

Four Italian cheeses (Casciotta di Urbino, Barricato San Martino, Vento d'Estate, and Ubriaco di Raboso) nonconventionally ripened under different plant materials (walnut leaves, herbs, hay, and wine by-products, respectively) were compared for compositional, microbiological, biochemical, and volatile profile characteristics. Mean values for gross composition were rather similar. Because primary starters were not used for manufacture, the endogenous lactic acid bacteria were mainly present (7.0 to 9.0 log10 cfu/g). Except for Lactobacillus paracasei and Leuconostoc mesenteroides, which were commonly identified in 3 cheeses, Lactococcus lactis, Enterococcus sanguinicola, Lactobacillus brevis, Enterococcus durans/Enterococcus faecium, Lactobacillus plantarum, and Weissella cibaria/Weissella confusa were variously found in the 4 cheeses. Random amplification of polymorphic DNA-PCR analysis showed the biodiversity among the strains, and the species of lactobacilli were in part grouped according to their origin. As shown by the principal component analysis of reverse-phase fast protein liquid chromatography data for the pH 4.6-soluble fractions and by the determination of free AA, the secondary proteolysis of Barricato San Martino and Vento d'Estate mainly differed from the other 2 cheeses. Purge-and-trap and solid-phase microextraction were coupled with gas chromatography-mass spectrometry to determine volatile compounds. Vento d'Estate showed the highest levels of almost all chemical classes, and Casciotta di Urbino was characterized by a very low level of volatile components. Esters, ketones, and terpenes were the chemical classes that mainly differentiated the cheeses. Several volatile compounds seemed to be released directly from the plant materials used for ripening, especially terpenes for Vento d'Estate cheese. The lowest level of volatile free fatty acids was found in Casciotta d'Urbino, in which rennet paste was not used during manufacture. The highest concentration of free fatty acids, especially butyric and caproic acids, was found in Vento d'Estate cheese.

Defined multi-species semi-liquid ready-to-use sourdough starter.

This paper aimed at setting up a protocol for the manufacture of a defined multi-species semi-liquid ready-to-use sourdough starter consisting of lactic acid bacteria (LAB) selected on the basis of their capacity of rapid acidification and adaptation to several technological factors. Preliminarily, 56 strains of sourdough LAB were screened based on the acidification kinetics during sourdough fermentation. The influence of temperature (25-37 degrees C), NaCl (2%, w/w, of wheat flour), initial cell number (10(7)-10(9) cfu g(-1)), dough yield (150-180), substrate for cell cultivation (mMRS or SDB media) and phase of cell growth (exponential or stationary phases) on the kinetics of acidification was also determined. Lactobacillus casei DPPMA27, Weissella confusa DPPMA20 and Lactobacillus fructivorans DPPMA8 were selected and used to produce a defined multi-species semi-liquid sourdough starter. Fermentation was carried out in a 2l batch fermentation bioreactor (12 h at 37 degrees C), under controlled pH, by cultivating cells in a medium made of water (60%), flour and other nutrients (40%). The semi-liquid sourdough starter was stored for 30 days under different conditions and used for dough fermentation (dough yield 160). The microbial composition, acidifying activity and fermentation end-products were kept constant during 21 days of storage. The findings of this study may be useful to increase the use of sourdough in bakery industries.

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

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

Angiotensin I-converting-enzyme-inhibitory and antibacterial peptides from Lactobacillus helveticus PR4 proteinase-hydrolyzed caseins of milk from six species.

Sodium caseinates prepared from bovine, sheep, goat, pig, buffalo or human milk were hydrolyzed by a partially purified proteinase of Lactobacillus helveticus PR4. Peptides in each hydrolysate were fractionated by reversed-phase fast-protein liquid chromatography. The fractions which showed the highest angiotensin I-converting-enzyme (ACE)-inhibitory or antibacterial activity were sequenced by mass spectrum and Edman degradation analyses. Various ACE-inhibitory peptides were found in the hydrolysates: the bovine alpha(S1)-casein (alpha(S1)-CN) 24-47 fragment (f24-47), f169-193, and beta-CN f58-76; ovine alpha(S1)-CN f1-6 and alpha(S2)-CN f182-185 and f186-188; caprine beta-CN f58-65 and alpha(S2)-CN f182-187; buffalo beta-CN f58-66; and a mixture of three tripeptides originating from human beta-CN. A mixture of peptides with a C-terminal sequence, Pro-Gly-Pro, was found in the most active fraction of the pig sodium caseinate hydrolysate. The highest ACE-inhibitory activity of some peptides corresponded to the concentration of the ACE inhibitor (S)-N-(1-[ethoxycarbonyl]-3-phenylpropyl)-ala-pro maleate (enalapril) of 49.253 micro g/ml (100 micro mol/liter). Several of the above sequences had features in common with other ACE-inhibitory peptides reported in the literature. The 50% inhibitory concentration (IC(50)) of some of the crude peptide fractions was very low (16 to 100 micro g/ml). Some identified peptides were chemically synthesized, and the ACE-inhibitory activity and IC(50)s were confirmed. An antibacterial peptide corresponding to beta-CN f184-210 was identified in human sodium caseinate hydrolysate. It showed a very large spectrum of inhibition against gram-positive and -negative bacteria, including species of potential clinical interest, such as Enterococcus faecium, Bacillus megaterium, Escherichia coli, Listeria innocua, Salmonella spp., Yersinia enterocolitica, and Staphylococcus aureus. The MIC for E. coli F19 was ca. 50 micro g/ml. Once generated, the bioactive peptides were resistant to further degradation by proteinase of L. helveticus PR4 or by trypsin and chymotrypsin.

Characterization of sourdough lactic acid bacteria based on genotypic and cell-wall protein analyses.

AIMS: To evaluate the effectiveness of two independent methods in differentiating a large population of lactic acid bacteria (LAB) isolated from wheat flours and sourdoughs and to correlate eventual differences/similarities among strains with their geographical origin and/or process parameters. METHODS AND RESULTS: One hundred fifty strains belonging to Lactobacillus spp. and Weissella spp., plus eight type strains, one for each species, and two unidentified isolates, were characterized by randomly amplified polymorphic DNA (RAPD) and SDS-PAGE of cell-wall proteins. The RAPD analysis separated the eight type strains but did not always assign all the strains of a species to the same group, while SDS-PAGE cell-wall protein profiles were species-specific. Frequently, strains isolated from sourdoughs of the same geographical origin or produced by similar raw material/process parameters showed similar RAPD and/or cell-wall profiles. CONCLUSIONS: The combined use of the RAPD and cell-wall protein analysis represents a useful tool to classify large adventitious microbial populations and to discriminate the diversity of the strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a typing of a large collection of flour/sourdough LAB and provides evidence of the advantage of using two independent methods in the classification and traceability of microorganisms.

Influence of transglutaminase treatment on some physico-chemical properties of milk.

Transglutaminase (TGase) is an enzyme that cross-links many proteins, including milk proteins. In this study, the effects of TGase on some physico-chemical properties of milk were studied. TGase-treated milk was not coagulable by rennet, which was due to failure of the primary (enzymic) stage of rennet action rather than the non-enzymic secondary phase. Dissociation of TGase-treated casein micelles by urea or sodium citrate or removal of colloidal calcium phosphate by acidification and dialysis was reduced, presumably due to the formation of cross-links between the caseins. Casein micelles in TGase-treated milks were also resistant to high pressure treatment and to hydrolysis by plasmin. Results of the present study show that milk proteins are fundamentally modified by the action of TGase, which may have applications in the manufacture of functional proteins for use as novel food ingredients.

Effect of pH and calcium concentration on proteolysis in mozzarella cheese.

Low-moisture Mozzarella cheeses (LMMC), varying in calcium content and pH, were made using a starter culture (control; CL) or direct acidification (DA) with lactic acid or lactic acid and glucono-delta-lactone. The pH and calcium concentration significantly affected the type and extent of proteolysis in Mozzarella cheese during the 70-d storage period at 4 degrees C. For cheeses with a similar pH, reducing the calcium-to-casein ratio from -29 to 22 mg/g of protein resulted in marked increases in moisture content and in primary and secondary proteolysis, as indicated by polyacrylamide gel electrophoresis and higher levels of pH 4.6- and 5%-PTA-soluble N. Increasing the pH of DA cheeses of similar moisture content, from approximately 5.5 to 5.9, while maintaining the calcium-to-casein ratio almost constant at approximately 29 mg/g, resulted in a decrease in primary proteolysis but had no effect on secondary proteolysis. Comparison of CL and DA cheeses with a similar composition showed that the CL cheese had higher levels of alpha(s1)-CN degradation, pH 4.6- and 5%-PTA-soluble N. Analysis of pH 4.6-soluble N extracts by reverse-phase HPLC showed that the CL cheese had higher concentrations of compounds with low retention times, suggesting higher concentrations of low molecular mass peptides and free amino acids.

Effect of pH and calcium concentration on some textural and functional properties of mozzarella cheese.

The effects of Ca concentration and pH on the composition, microstructural, and functional properties of Mozzarella cheese were studied. Cheeses were made using a starter culture (control) or by direct acidification of the milk with lactic acid or lactic acid and glucono-delta-lactone. In each of three trials, four cheeses were produced: a control, CL, and three directly-acidified cheeses, DA1, DA2, and DA3. The cheeses were stored at 4 degrees C for 70 d. The Ca content and pH were varied by altering the pH at setting, pitching, and plasticization. The mean pH at 1 d and the Ca content (mg/g of protein) of the various cheeses were: CL, 5.42 and 27.7; DA1, 5.96 and 21.8; DA2, 5.93 and 29.6; DA3, 5.58 and 28.7. For cheeses with a high pH (i.e., approximately 5.9), reducing the Ca content from 29.6 to 21.8 mg/g of protein resulted in a significant decrease in the protein level and increases in the moisture content and mean level of nonexpressible serum (g/g of protein). Reducing the Ca concentration also resulted in a more swollen, hydrated para-casein matrix at 1 d. The decrease in Ca content in the high-pH cheeses coincided with increases in the mean stretchability and flowability of the melted cheese over the 70-d storage period. The fluidity of the melted cheese also increased when the Ca content was reduced, as reflected by a lower elastic shear modulus and a higher value for the phase angle, delta, of the melted cheese, especially after storage for <12 d. The melt time, flowability, and stretchability of the low-Ca, high-pH DA1 cheese at 1 d were similar to those for the CL cheese after storage for > or = 12 d. In contrast, the mean values for flowability and stretchability of the high-pH, high-Ca DA2 cheese over the 70-d period were significantly lower than those of the CL cheese. Reducing the pH of high-Ca cheese (27.7 to 29.6 mg/g of protein) from -5.95 to 5.58 resulted in higher flowability, stretchability, and fluidity of the melted cheese. For cheeses with similar pH and Ca concentration, the method of acidification had little effect on composition, microstructure, flowability, stretchability, and fluidity of the melted cheese.

Effect of transglutaminase on the heat stability of milk: a possible mechanism.

Treatment of milk with transglutaminase (TGase) affects its heat stability, but the manner in which it does so depends on whether or not the milk had been preheated before incubation and on the temperature of preheating. In raw milk, it appears that cross-link formation between the individual caseins is responsible for preventing the dissociation of kappa-casein from the micelles at pH values in the region of minimum stability. In milks preheated before incubation with TGase, denaturation of whey protein may have allowed the formation of cross-links by TGase between denatured whey proteins and the individual caseins which, in combination with cross-linking of the caseins, contributed to greatly improved heat stability at pH > 6.5. It appears from the results of this study that TGase has potential commercial applications as a food-grade additive capable of improving the heat stability of milk.

Solution structures of casein peptides: NMR, FTIR, CD, and molecular modeling studies of alphas1-casein, 1-23.

To determine its potential for interacting with other components of the casein micelle, the N-terminal section of bovine alphas1-casein-B, residues 1-23, was investigated with nuclear magnetic resonance (NMR), Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies, and molecular modeling. NMR data were not consistent with conventional alpha-helical or beta-sheet structures, but changes in N-H proton chemical shifts suggested thermostable structures. Both CD and FTIR predicted a range of secondary structures for the peptide (30-40% turns, 25-30% extended) that were highly stable from 5 degrees C to 25 degrees C. Other conformational elements, such as loops and polyproline II helix, were indicated by FTIR only. Molecular dynamics simulation of the peptide predicted 32% turns and 27% extended, in agreement with FTIR and CD predictions and consistent with NMR data. This information is interpreted in accord with recent spectroscopic evidence regarding the nature of unordered conformations, leading to a possible role of alphas1-casein (1-23) in facilitating casein-casein interactions.

Ethanol-dependent heat-induced dissociation of casein micelles.

The dissociation of casein micelles when heated to approximately 65 degrees C in the presence of ethanol [1:1 mixture (v/v) of milk and 65% (w/w) aqueous ethanol] was investigated using L* values and transmission measurements. Mixtures of milk and ethanol became transparent on heating, which suggests dissociation of casein micelles. Results of experiments using confocal laser scanning microscopy, light scattering (static and dynamic), and dialysis to examine the changes of milk during heating in the presence of ethanol supported the assertion that such treatments result in dissociation of casein micelles, as did studies of model beta-casein micellar systems.

Mechanism for the ethanol-dependent heat-induced dissociation of casein micelles.

An explanation as to how casein micelles dissociate when heated in the presence of ethanol is presented. Dissociation of casein micelles in milk-ethanol mixtures was studied using (1)H NMR, and the effects of addition of CaCl(2), NaCl, or EDTA or alteration of milk pH on this dissociation were studied. It is proposed that at low temperatures, ethanol reduces the solvent quality of milk serum, but above a critical temperature (approximately 30 degrees C in a 35% ethanol solution), ethanol enhances solvent quality and dissociates the casein micelles. Ethanol reduced protein hydrophobicity and increased the pK(a) value of phosphoserine, effects that are likely to be significant in the dissociating effect of ethanol at elevated temperatures.

Microbiological and biochemical characteristics of Canestrato Pugliese cheese made from raw milk, pasteurized milk or by heating the curd in hot whey.

Canestrato Pugliese cheeses were produced from raw ewes' milk (R and R(II) cheeses), pasteurized ewes' milk (P cheese) and by heating the curd in hot whey according to a traditional protocol (T cheese). R(II) differed from R cheese mainly by having been produced from raw milk with a higher number of somatic cells, 950.000 vs. 750.000 ml(-1), respectively. Compared to P and T cheeses, R and R(II) cheeses had a higher concentration (one or two orders of magnitude) of cheese-related bacteria such as adventitious mesophilic lactobacilli, enterococci and staphylococci. At the end of ripening, all cheeses contained less than 1.0 log cfu g(-1) of total and fecal coliforms, and Escherichia coli and Staphylococcus aureus were not detected. As shown by phenotypic identification and RAPD-PCR, R cheese contained the largest number of mesophilic lactobacilli species and the greatest diversity of strains within the Lactobacillus plantarum species. Primary proteolysis did not differ appreciably among the cheeses. On the contrary, both urea-PAGE and the RP-HPLC analyses of the water-soluble N fractions showed the more complex profiles in cheeses produced by raw milks. R and R(II) cheeses had the highest values of water-soluble N/total N (ca. 30%) and the highest concentration of total free amino acids (ca. 40 mg g(-1) which approached or exceeded those reported for Italian cheeses with very high level of proteolysis during ripening. The main differences between R-R(II) and P-T cheeses were the concentrations of aspartic acid, proline, alanine, isoleucine, histidine and lysine. The water-soluble extracts of R and R(II) cheeses contained levels of amino-, imino- and di-peptidase activities, which were about twice those found in P and T cheeses. Cheeses differed slightly in the concentration of total free fatty acids that ranged between 1673 and 1651 mg kg(-1) in R and R(II) cheeses, and 1397 and 1334 mg kg(-1) in P and T cheeses. Butyric, caproic, capric, palmitic, oleic and linoleic acids were found at the highest concentrations.

Short communication: influence of transglutaminase on the heat stability of milk.

Skim milk powders were prepared from control and transglutaminase-treated skimmed milk. The heat stability of reconstituted transglutaminase-treated skimmed milk (9.0% total solids) was markedly increased in the pH region of minimum stability (pH 6.8 to 7.1) compared with control milk, while the heat stability of reconstituted concentrated transglutaminase-treated skimmed milk (22.5% total solids) increased progressively as a function of pH relative to control milk. The effect of transglutaminase treatment on the heat stability of skimmed milk may have commercial applications, but extensive research is necessary to gain a better understanding of the mechanism by which transglutaminase improves heat stability.

Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov., isolated from the surface of a smear-ripened cheese.

Ten isolates each of two different bacterial species isolated from the surface of a smear-ripened cheese were found to exhibit many characteristics of the genus Corynebacterium. The isolates were Gram-positive, catalase-positive, non-spore-forming rods that did not undergo a rod/coccus transformation when grown on complex media. Chemotaxonomic investigation revealed that the strains belonged unambiguously to the genus Corynebacterium. Their cell walls contained arabinose, galactose and short-chain mycolic acids (C22 to C36) and their peptidoglycan contained meso-diaminopimelic acid. The G+C content of the DNA was 51-60 mol%. MK-9 (H2) was the principal menaquinone. The 16S rDNA sequences of four isolates of each bacterium were determined and aligned with those of other members of the coryneform group. Phylogenetic analysis showed that the strains represented two new sublines within the genus Corynebacterium; Corynebacterium variabile and Corynebacterium ammoniagenes were their nearest known phylogenetic neighbours. Corynebacterium variabile and Corynebacterium ammoniagenes showed the highest levels of sequence homology with the isolates; however, DNA-DNA hydridization studies indicated that the Corynebacterium strains isolated from the cheese smear did not belong to either Corynebacterium variabile or Corynebacterium ammoniagenes (26 and 46% chromosomal similarity, respectively). On the basis of the phylogenetic and phenotypic distinctiveness of the unknown isolates, it is proposed that the bacteria be classified as two new Corynebacterium species, for which the names Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov. are proposed. Type strains have been deposited in culture collections as Corynebacterium mooreparkense LMG S-19265T (= NCIMB 30131T) and Corynebacterium casei LMG S-19264T (= NCIMB 30130T).

Effect of beta-lactoglobulin and precipitation of calcium phosphate on the thermal coagulation of milk.

The effect of beta-lactoglobulin and heat-induced precipitation of calcium phosphate on the pH dependence and mechanism of thermal coagulation of milk throughout the pH range 6.3-7.3 was studied using serum protein-free milk and sodium caseinate as models for micellar and non-micellar milk protein systems respectively. It appears that the specific effect of beta-lactoglobulin at the pH of maximum stability may be related to its ability to chelate calcium. The effect of beta-lactoglobulin at the pH of minimum stability does not appear to be directly related to heat-induced dissociation of K-casein or micellar integrity but may be due to its ability to sensitize casein micelles to heat-induced precipitation of calcium phosphate, by increasing micellar hydrophobicity. The extent of heat-induced precipitation of calcium phosphate, as a function of pH, is an inverse reflection of the pH dependence of heat stability. Micellar integrity appears to play a critical role in the heat stability of milk but for reasons not previously appreciated.

Transcatheter intraarterial infusion of rt-PA for acute lower limb ischemia: results and complications.

PURPOSE: To determine the success and complication rates of intraarterial recombinant tissue-type plasminogen activator (rt-PA) infusion for the treatment of acute lower extremity artery and bypass graft occlusions. MATERIALS AND METHODS: The results of 74 limbs in 70 patients (mean age, 66 y) treated with catheter-directed rt-PA infusion for the treatment of acute lower extremity ischemia were retrospectively evaluated. The group included 42 bypass grafts and 32 native arteries. All limbs were viable at presentation. The mean duration of symptoms was 11.9 days. rt-PA was infused for a mean of 27.9 hours for a mean total dose of 38.7 mg. Initial infusion rates of 3-6 mg/h were lowered to a preferred rate of 1.5 mg/h. Thrombolytic success was defined as 95% thrombolysis of an occluded segment with return of antegrade flow. Major bleeding complications were defined as any hemorrhagic event leading to surgery, extended or unexpected hospitalization, transfusion, death, intracranial hemorrhage, or a decrease in hemoglobin of 5 g/dL or in hematocrit of 15%. Thirty-day mortality and amputation rates were calculated. Patient characteristics and infusion parameters were evaluated as to whether they contributed to thrombolytic success or major bleeding events. RESULTS: Thrombolytic success was achieved in 64 limbs (86%). Major bleeding complications occurred in 33 (47%) patients. In 22 of these patients, bleeding occurred at a vascular puncture site, whereas remote bleeding occurred in seven patients. Remote bleeding complications included two retroperitoneal hematomas, two rectus sheath hematomas, one lower gastrointestinal hemorrhage, one episode of hemoptysis, and one dehiscence of a femoral-popliteal bypass graft revision. No parameters were found to be predictive of thrombolytic success, whereas a negative history of smoking, increasing duration of infusion, and a low preprocedural ankle-brachial index (ABI) were found to be associated with major hemorrhagic events. Four patients (6%) underwent amputation and one patient (1%) died, resulting in a 30-day amputation-free survival rate of 93%. CONCLUSION: Catheter-directed rt-PA infusion is effective in achieving thrombolysis. Despite a significant number of bleeding complications, 30-day mortality and amputation rates were favorable. Nonetheless, complication rates related to bleeding were not trivial and further evaluation with use of variable dosing regimens is indicated.

Purification and characterization of an extracellular proline iminopeptidase from Corynebacterium variabilis NCDO 2101.

AIMS: To screen the extracellular proteolytic and lipolytic activities of Corynebacterium variabilis NCDO 2101 and to purify and characterize a proline iminopeptidase enzyme in order to investigate the role of the major component of the smear of bacterial surface-ripened cheeses. METHODS AND RESULTS: Four chromatographic steps were used to purify the enzyme and a three-factor, five-level Central Composite Design was used to study the interactive effects of cheese-related values of pH, NaCl and temperature. The proline iminopeptidase showed some biochemical properties different from the same enzyme purified from lactic acid bacteria and other smear bacteria. It tolerated NaCl concentrations up to 7.5% and was sensitive to low values of pH especially when they were combined with low temperature. CONCLUSION: The proline iminopeptidase of C. variabilis NCDO 2101 may have a role in proteolysis during ripening of smear surface-ripened cheeses. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of the enzymology of smear bacteria in order to improve the ripening of bacterial surface-ripened cheeses.

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.