Galactose-positive adjunct cultures prevent gas formation by Paucilactobacillus wasatchensis WDC04 in a model gas production test.

Gas production by obligatory heterofermentative lactic acid bacteria such as Paucilactobacillus wasatchensis is a sporadic problem in Cheddar cheese and results in undesired slits and cracks in the cheese. Growth of Pa. wasatchensis is not rapid, which makes investigations of gas production difficult to consistently execute. A primary objective of this study was to develop a model gas production test that could be used to investigate the effect of galactose and ribose utilization on gas production by Pa. wasatchensis and determine whether galactose-fermenting adjunct cultures could prevent gas formation. Paucilactobacillus wasatchensis WDC04 was inoculated at 101 to 106 cfu/mL into carbohydrate-restricted MRS broth containing different ribose and galactose levels and incubated for up to 21 d at 23°C. Gas production in the broth was detected using a Durham tube inverted on a 6-cm-long capillary tube; cells were enumerated at 4, 8, and 12 d; and residual galactose was also measured. Gas production was sporadic except for when 105 cfu/mL of Pa. wasatchensis WDC04 was inoculated into broth containing 0.3% ribose and 0.7% galactose. In those tubes, gas production was consistently observed after 8-d incubation, by which time galactose levels had decreased to 0.15%. Co-inoculation of Pa. wasatchensis WDC04 with as few as 103 cfu/mL of a lactose-negative galactose-positive adjunct culture (Pediococcus acidilactici 23F, Lacticaseibacillus paracasei UW4, or Lactobacillus helveticus 7995) resulted in galactose depletion by d 4 and no observable gas production by d 12. With less galactose available to the slower-growing Pa. wasatchensis WDC04, its growth was limited to 108 cfu/mL when any of the adjunct cultures was co-inoculated, compared with 109 cfu/mL when grown on its own. We concluded that galactose-fermenting adjunct cultures have potential for preventing unwanted gas production in cheese by competition for resources and especially by removing the 6-carbon galactose before it can be utilized for energy by an obligatory heterofermentative lactobacilli such as Pa. wasatchensis and produce carbon dioxide.

Characterization of microbial communities in ethanol biorefineries.

Bacterial contamination of corn-based ethanol biorefineries can reduce their efficiency and hence increase their carbon footprint. To enhance our understanding of these bacterial contaminants, we temporally sampled four biorefineries in the Midwestern USA that suffered from chronic contamination and characterized their microbiomes using both 16S rRNA sequencing and shotgun metagenomics. These microbiotas were determined to be relatively simple, with 13 operational taxonomic units (OTUs) accounting for 90% of the bacterial population. They were dominated by Firmicutes (89%), with Lactobacillus comprising 80% of the OTUs from this phylum. Shotgun metagenomics confirmed our 16S rRNA data and allowed us to characterize bacterial succession at the species level, with the results of this analysis being that Lb. helveticus was the dominant contaminant in this fermentation. Taken together, these results provide insights into the microbiome of ethanol biorefineries and identifies a species likely to be commonly responsible for chronic contamination of these facilities.

Hot topic: Geographical distribution and strain diversity of Lactobacillus wasatchensis isolated from cheese with unwanted gas formation.

Lactobacillus wasatchensis, an obligate heterofermentative nonstarter lactic acid bacteria (NSLAB) implicated in causing gas defects in aged cheeses, was originally isolated from an aged Cheddar produced in Logan, Utah. To determine the geographical distribution of this organism, we isolated slow-growing NSLAB from cheeses collected in different regions of the United States, Australia, New Zealand, and Ireland. Seven of the cheeses showed significant gas defects and 12 did not. Nonstarter lactic acid bacteria were isolated from these cheeses on de Man, Rogosa, and Sharpe medium supplemented with ribose, a preferred substrate for Lb. wasatchensis. Identification was confirmed with 16S rRNA gene sequencing and the API50CH (bioMérieux, Marcy l'Etoile, France) carbohydrate panel. Isolates were also compared with one another by using repetitive element sequence-based PCR (rep-PCR). Lactobacillus wasatchensis was isolated only from cheeses demonstrating late-gas development and was found in samples from 6 of the 7 cheeses. This supports laboratory evidence that this organism is a causative agent of late gas production defects. The rep-PCR analysis produced distinct genetic fingerprints for isolates from each cheese, indicating that Lb. wasatchensis is found in several regions across the United States and is not a local phenomenon.

Lactobacillus wasatchensis sp. nov., a non-starter lactic acid bacteria isolated from aged Cheddar cheese.

A Gram-stain positive, rod-shaped, non-spore-forming strain (WDC04T), which may be associated with late gas production in cheese, was isolated from aged Cheddar cheese following incubation on MRS agar (pH 5.2) at 6 °C for 35 days. Strain WDC04T had 97 % 16S rRNA gene sequence similarity with Lactobacillus hokkaidonensis DSM 26202T, Lactobacillus oligofermentans 533, 'Lactobacillus danicus' 9M3, Lactobacillus suebicus CCUG 32233T and Lactobacillus vaccinostercus DSM 20634T. API 50 CH carbohydrate fermentation panels indicated strain WDC04T could only utilize one of the 50 substrates tested, ribose, although it does slowly utilize galactose. In the API ZYM system, strain WDC04T was positive for leucine arylamidase, valine arylamidase, cysteine arylamidase (weakly), naphthol-AS-BI-phosphohydrolase and ß-galactosidase activities. Total genomic DNA was sequenced from strain WDC04T using a whole-genome shotgun strategy on a 454 GS Titanium pyrosequencer. The sequence was assembled into a 1.90 Mbp draft genome consisting of 105 contigs with preliminary genome annotation performed using the RAST algorithm (rast.nmpdr.org). Genome analysis confirmed the pentose phosphate pathway for ribose metabolism as well as galactose, N-acetylglucosamine, and glycerol fermentation pathways. Genomic analysis places strain WDC04T in the obligately heterofermentative group of lactobacilli and metabolic results confirm this conclusion. The result of genome sequencing, along with 16S rRNA gene sequence analysis, indicates WDC04T represents a novel species of the genus Lactobacillus, for which the name Lactobacillus wasatchensis sp. nov. is proposed. The type strain is WDC04T ( = DSM 29958T = LMG 28678T).

Growth and gas formation by Lactobacillus wasatchensis, a novel obligatory heterofermentative nonstarter lactic acid bacterium, in Cheddar-style cheese made using a Streptococcus thermophilus starter.

A novel slow-growing, obligatory heterofermentative, nonstarter lactic acid bacterium (NSLAB), Lactobacillus wasatchensis WDC04, was studied for growth and gas production in Cheddar-style cheese made using Streptococcus thermophilus as the starter culture. Cheesemaking trials were conducted using S. thermophilus alone or in combination with Lb. wasatchensis deliberately added to cheese milk at a level of ~10(4) cfu/mL. Resulting cheeses were ripened at 6 or 12°C. At d 1, starter streptococcal numbers were similar in both cheeses (~10(9) cfu/g) and fast-growing NSLAB lactobacilli counts were below detectable levels (<10(2) cfu/g). As expected, Lactobacillus wasatchensis counts were 3×10(5) cfu/g in cheeses inoculated with this bacterium and below enumeration limits in the control cheese. Starter streptococci decreased over time at both storage temperatures but declined more rapidly at 12°C, especially in cheese also containing Lb. wasatchensis. Populations of fast-growing NSLAB and the slow-growing Lb. wasatchensis reached 5×10(7) and 2×10(8) cfu/g, respectively, after 16 wk of storage at 12°C. Growth of NSLAB coincided with a reduction in galactose concentration in the cheese from 0.6 to 0.1%. Levels of galactose at 6°C had similar decrease. Gas formation and textural defects were only observed in cheese with added Lb. wasatchensis ripened at 12°C. Use of S. thermophilus as starter culture resulted in galactose accumulation that Lb. wasatchensis can use to produce CO2, which contributes to late gas blowing in Cheddar-style cheeses, especially when the cheese is ripened at elevated temperature.

Late blowing of Cheddar cheese induced by accelerated ripening and ribose and galactose supplementation in presence of a novel obligatory heterofermentative nonstarter Lactobacillus wasatchensis.

Lactobacillus wasatchensis sp. nov. has been studied for growth and gas formation in a control Cheddar cheese and in cheese supplemented with 0.5% ribose, 0.5% galactose, or 0.25% ribose plus 0.25% galactose using regular and accelerated cheese ripening temperatures of 6 and 12°C, respectively. Milk was inoculated with (1) Lactococcus lactis starter culture, or (2) Lc. lactis starter culture plus Lb. wasatchensis (10(4) cfu/mL). In the control cheese with no added Lb. wasatchensis, starter numbers decreased from 10(7) initially to ~10(4) cfu/g over 23 wk of ripening at 6°C. When the cheese was ripened at 12°C, or if Lb. wasatchensis was added, the final starter counts were 1 log lower. In contrast, nonstarter lactic acid bacteria in the cheese increased from <10(2) cfu/g at press to 10(6) to 10(7) cfu/g after 23 wk, with higher numbers being observed with ripening at 12°C. In cheese with no added Lb. wasatchensis, levels of Lb. wasatchensis were initially below the enumeration threshold but counts of up to 10(3) cfu/g were detected after 23 wk. When the cheese was inoculated with Lb. wasatchensis, it could be enumerated throughout ripening, with final levels at 23 wk being dependent on whether ribose had been added to the cheese curd. With added ribose (with or without added galactose), Lb. wasatchensis grew to 10(7) to 10(8) cfu/g after 23 wk, whereas without added ribose it was 1 log lower. In all cheeses with added Lb. wasatchensis, greater gas formation was observed at 12°C, with most gas production occurring after ~16 wk. Very little gas production was detected in cheese without added Lb. wasatchensis ripened at 12°C or in cheese with added Lb. wasatchensis ripened at 6°C. Adding a combination of ribose and galactose caused more gas formation, putatively because of the ability of Lb. wasatchensis to co-utilize both sugars and grow to high numbers, and then produce gas from galactose as ribose levels were depleted. Even without sugar supplementation, gas was observed in cheese with added Lb. wasatchensis after 16 wk. We also observed that Lb. wasatchensis could grow to high cell densities when grown in carbohydrate-restricted broth containing lactococcal cell lysate. This suggests that during cheese ripening, lysis of starter bacteria provides sufficient substrates (such as ribose) to allow growth of Lb. wasatchensis and, if fermentable hexose is available, the cheese will become gassy. We conclude that Lb. wasatchensis is a previously undetected contributor to late gas formation in Cheddar cheese and the defect is more pronounced when elevated ripening temperatures are used.

Growth and gas production of a novel obligatory heterofermentative Cheddar cheese nonstarter lactobacilli species on ribose and galactose.

An obligatory heterofermentative lactic acid bacterium, Lactobacillus wasatchii sp. nov., isolated from gassy Cheddar cheese was studied for growth, gas formation, salt tolerance, and survival against pasteurization treatments at 63°C and 72°C. Initially, Lb. wasatchii was thought to use only ribose as a sugar source and we were interested in whether it could also utilize galactose. We conducted experiments to determine the rate and extent of growth and gas production in carbohydrate-restricted (CR) de Man, Rogosa, and Sharpe (MRS) medium under anaerobic conditions with various combinations of ribose and galactose at 12, 23, and 37°C, with 23°C being the optimum growth temperature of Lb. wasatchii among the 3 temperatures studied. When Lb. wasatchii was grown on ribose (0.1, 0.5, and 1%), maximum specific growth rates (µmax) within each temperature were similar. When galactose was the only sugar, compared with ribose, µmax was 2 to 4 times lower. At all temperatures, the highest final cell densities (optical density at 640 nm) of Lb. wasatchii were achieved in CR-MRS plus 1% ribose, 0.5% ribose and 0.5% galactose, or 1% ribose and 1% galactose. Similar µmax values and final cell densities were achieved when 50% of the ribose in CR-MRS was substituted with galactose. Such enhanced utilization of galactose in the presence of ribose to support bacterial growth has not previously been reported. It appears that Lb. wasatchii co-metabolizes ribose and galactose, utilizing ribose for energy and galactose for other functions such as cell wall biosynthesis. Co-utilization of both sugars could be an adaptation mechanism of Lb. wasatchii to the cheese environment to efficiently ferment available sugars for maximizing metabolism and growth. As expected, gas formation by the heterofermenter was observed only when galactose was present in the medium. Growth experiments with MRS plus 1.5% ribose at pH 5.2 or 6.5 with 0, 1, 2, 3, 4, or 5% NaCl revealed that Lb. wasatchii is able to grow under salt and pH conditions typical of Cheddar cheese (4 to 5% salt-in-moisture, pH ~5.2). Finally, we found that Lb. wasatchii cannot survive low-temperature, long-time pasteurization but survives high-temperature, short-time (HTST) laboratory pasteurization, under which a 4.5 log reduction occurred. The ability of Lb. wasatchii to survive HTST pasteurization and grow under cheese ripening conditions implies that the presence of this nonstarter lactic acid bacterium can be a serious contributor to gas formation and textural defects in Cheddar cheese.

The Streptococcus thermophilus protein Wzh functions as a phosphotyrosine phosphatase.

Amino acid residues that are important for metal binding and catalysis in Gram-positive phosphotyrosine phosphatases were identified in the Wzh protein of Streptococcus thermophilus MR-1C by using sequence comparisons. A His-tagged fusion Wzh protein was purified from Escherichia coli cultures and tested for phosphatase activity against synthetic phosphotyrosine and phosphoserine-threonine peptides. Purified Wzh released 2316.5 ± 138.7 pmol PO4·min(-1)·µg(-1) from phosphotyrosine peptide-1 and 2345.7 ± 135.2 pmol PO4·min(-1)·µg(-1) from phosphotyrosine peptide-2. The presence of the phosphotyrosine phosphatase inhibitor sodium vanadate decreased purified Wzh activity by 45%-50% at 1 mmol·L(-1), 74%-84% at 5 mmol·L(-1), and by at least 88% at 10 mmol·L(-1). Purified Wzh had no detectable activity against the phosphoserine-threonine peptide. These results clearly establish that S. thermophilus MR-1C Wzh functions as a phosphotyrosine phosphatase that could function to remove phosphate groups from proteins involved in exopolysaccharide biosynthesis, including the protein tyrosine kinase Wze and priming glycosyltransferase.

Complete genome sequence of Lactobacillus helveticus R0052, a commercial probiotic strain.

Lactobacillus helveticus R0052 is a commercially available strain that is widely used in probiotic preparations. The genome sequence consisted of 2,129,425 bases. Comparative analysis showed that it was unique among L. helveticus strains in that it contained genes encoding mucus-binding proteins similar to those found in Lactobacillus acidophilus.

Intraspecific and interspecific interactions among proteins regulating exopolysaccharide synthesis in Streptococcus thermophilus, Streptococcus iniae, and Lactococcus lactis subsp. cremoris and the assessment of potential lateral gene transfer.

Using the yeast two-hybrid system, intraspecific protein interactions were detected in Streptococcus iniae and Lactococcus lactis subsp. cremoris between the transmembrane activation protein (CpsC and EpsA, respectively) and the protein tyrosine kinase (CpsD and EpsB, respectively), between two protein tyrosine kinases, and between the protein tyrosine kinase and the phosphotyrosine phosphatase (CpsB and EpsC, respectively). For each of these intraspecific interactions, interspecific interactions were also detected when one protein was from S. iniae and the other was from Streptococcus thermophilus . Interactions were also observed between two protein tyrosine kinases when one protein was from either of the Streptococcus species and the other from L. lactis subsp. cremoris. The results and sequence comparisons performed in this study support the conclusion that interactions among the components of the tyrosine kinase - phosphatase regulatory system are conserved in the order Lactobacillales and that interspecific genetic exchanges of the genes that encode these proteins have the potential to form functional recombinants. A better understanding of intraspecific and interspecific protein interactions involved in regulating exopolysaccharide biosynthesis may facilitate construction of improved strains for industrial uses as well as identification of factors needed to form functional regulatory complexes in naturally occurring recombinants.

Comparison of the complete genome sequences of Bifidobacterium animalis subsp. lactis DSM 10140 and Bl-04.

Bifidobacteria are important members of the human gut flora, especially in infants. Comparative genomic analysis of two Bifidobacterium animalis subsp. lactis strains revealed evolution by internal deletion of consecutive spacer-repeat units within a novel clustered regularly interspaced short palindromic repeat locus, which represented the largest differential content between the two genomes. Additionally, 47 single nucleotide polymorphisms were identified, consisting primarily of nonsynonymous mutations, indicating positive selection and/or recent divergence. A particular nonsynonymous mutation in a putative glucose transporter was linked to a negative phenotypic effect on the ability of the variant to catabolize glucose, consistent with a modification in the predicted protein transmembrane topology. Comparative genome sequence analysis of three Bifidobacterium species provided a core genome set of 1,117 orthologs complemented by a pan-genome of 2,445 genes. The genome sequences of the intestinal bacterium B. animalis subsp. lactis provide insights into rapid genome evolution and the genetic basis for adaptation to the human gut environment, notably with regard to catabolism of dietary carbohydrates, resistance to bile and acid, and interaction with the intestinal epithelium. The high degree of genome conservation observed between the two strains in terms of size, organization, and sequence is indicative of a genomically monomorphic subspecies and explains the inability to differentiate the strains by standard techniques such as pulsed-field gel electrophoresis.

Role of cystathionine beta-lyase in catabolism of amino acids to sulfur volatiles by genetic variants of Lactobacillus helveticus CNRZ 32.

Catabolism of sulfur-containing amino acids plays an important role in the development of cheese flavor. During ripening, cystathionine beta-lyase (CBL) is believed to contribute to the formation of volatile sulfur compounds (VSCs) such as methanethiol and dimethyl disulfide. However, the role of CBL in the generation of VSCs from the catabolism of specific sulfur-containing amino acids is not well characterized. The objective of this study was to investigate the role of CBL in VSC formation by Lactobacillus helveticus CNRZ 32 using genetic variants of L. helveticus CNRZ 32 including the CBL-null mutant, complementation of the CBL-null mutant, and the CBL overexpression mutant. The formation of VSCs from methionine, cystathionine, and cysteine was determined in a model system using gas chromatography-mass spectrometry with solid-phase microextraction. With methionine as a substrate, CBL overexpression resulted in higher VSC production than that of wild-type L. helveticus CNRZ 32 or the CBL-null mutant. However, there were no differences in VSC production between the wild type and the CBL-null mutant. With cystathionine, methanethiol production was detected from the CBL overexpression variant and complementation of the CBL-null mutant, implying that CBL may be involved in the conversion of cystathionine to methanethiol. With cysteine, no differences in VSC formation were observed between the wild type and genetic variants, indicating that CBL does not contribute to the conversion of cysteine.

Comparative high-density microarray analysis of gene expression during growth of Lactobacillus helveticus in milk versus rich culture medium.

Lactobacillus helveticus CNRZ32 is used by the dairy industry to modulate cheese flavor. The compilation of a draft genome sequence for this strain allowed us to identify and completely sequence 168 genes potentially important for the growth of this organism in milk or for cheese flavor development. The primary aim of this study was to investigate the expression of these genes during growth in milk and MRS medium by using microarrays. Oligonucleotide probes against each of the completely sequenced genes were compiled on maskless photolithography-based DNA microarrays. Additionally, the entire draft genome sequence was used to produce tiled microarrays in which noninterrupted sequence contigs were covered by consecutive 24-mer probes and associated mismatch probe sets. Total RNA isolated from cells grown in skim milk or in MRS to mid-log phase was used as a template to synthesize cDNA, followed by Cy3 labeling and hybridization. An analysis of data from annotated gene probes identified 42 genes that were upregulated during the growth of CNRZ32 in milk (P < 0.05), and 25 of these genes showed upregulation after applying Bonferroni's adjustment. The tiled microarrays identified numerous additional genes that were upregulated in milk versus MRS. Collectively, array data showed the growth of CNRZ32 in milk-induced genes encoding cell-envelope proteinases, oligopeptide transporters, and endopeptidases as well as enzymes for lactose and cysteine pathways, de novo synthesis, and/or salvage pathways for purines and pyrimidines and other functions. Genes for a hypothetical phosphoserine utilization pathway were also differentially expressed. Preliminary experiments indicate that cheese-derived, phosphoserine-containing peptides increase growth rates of CNRZ32 in a chemically defined medium. These results suggest that phosphoserine is used as an energy source during the growth of L. helveticus CNRZ32.

Identification of endopeptidase genes from the genomic sequence of Lactobacillus helveticus CNRZ32 and the role of these genes in hydrolysis of model bitter peptides.

Genes encoding three putative endopeptidases were identified from a draft-quality genome sequence of Lactobacillus helveticus CNRZ32 and designated pepO3, pepF, and pepE2. The ability of cell extracts from Escherichia coli DH5alpha derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, beta-casein (beta-CN) (f193-209) and alpha(S1)-casein (alpha(S1)-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10 degrees C) was examined. CNRZ32 PepO3 was determined to be a functional paralog of PepO2 and hydrolyzed both peptides, while PepE and PepF had unique specificities towards alpha(S1)-CN (f1-9) and beta-CN (f193-209), respectively. CNRZ32 PepE2 and PepO did not hydrolyze either peptide under these conditions. To demonstrate the utility of these peptidases in cheese, PepE, PepO2, and PepO3 were expressed in Lactococcus lactis, a common cheese starter, using a high-copy vector pTRKH2 and under the control of the pepO3 promoter. Cell extracts of L. lactis derivatives expressing these peptidases were used to hydrolyze beta-CN (f193-209) and alpha(S1)-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides alpha(S1)-CN (f1-9), alpha(S1)-CN (f1-13), and alpha(S1)-CN (f1-16) were identified from Cheddar cheese serum and included in the defined peptide mix. Our results demonstrate that in all systems examined, PepO2 and PepO3 had the highest activity with beta-CN (f193-209) and alpha(S1)-CN (f1-9). Cheese-derived peptides were observed to affect the activity of some of the enzymes examined, underscoring the importance of incorporating such peptides in model systems. These data indicate that L. helveticus CNRZ32 endopeptidases PepO2 and PepO3 are likely to play a key role in this strain's ability to reduce bitterness in cheese.

Overexpression of Lactobacillus casei D-hydroxyisocaproic acid dehydrogenase in cheddar cheese.

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 alpha-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 alpha-keto acids to more-stable alpha-hydroxy acids via the action of alpha-keto acid dehydrogenases such as d-hydroxyisocaproic acid dehydrogenase. To further characterize the role of this enzyme in cheese flavor, the Lactobacillus casei d-hydroxyisocaproic acid dehydrogenase gene was cloned into the high-copy-number vector pTRKH2 and transformed into L. casei ATCC 334. Enzyme assays confirmed that alpha-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 alpha-keto acids, but sensory work indicated that this effect retarded cheese flavor development.

Influence of coagulant level on proteolysis and functionality of mozzarella cheeses made using direct acidification.

Nonfat (0% fat), reduced-fat (11% fat), and control (19% fat) mozzarella cheeses were made using direct acidification to test the influence of three levels (0.25X, 1X, and 4X) of coagulant concentration on proteolysis, meltability and rheological properties of cheeses during 60 d of storage at 5 degrees C. Changes in meltability, level of intact alpha(s1)-casein and beta-casein (by capillary electrophoresis), 12.5% TCA-soluble nitrogen, and complex modulus were measured. There were differences in rate of proteolysis and functional properties as a function of fat content of the cheese, but some of these differences could be attributed to differences in moisture contents of the cheeses. As fat level decreased, the percent moisture-in-nonfat-substance of the cheeses also decreased. Cheeses with the lower fat contents (and consequently the lowest moisture-in-nonfat-substance content) had slower rates of proteolysis. Fat content influenced the complex modulus of the cheese, with the biggest effect occurring when fat content was reduced from 11 to 0%. Coagulant level had only a small effect on initial modulus. Cheeses became softer during storage, and the decrease in modulus was influenced by the level of coagulant. At 0.25X, there was very little decrease in modulus after 60 d, while at 1X and 4X coagulant levels the softening of the cheese was more evident. The influence of coagulant level and fat content on cheese melting was similar to their effects on complex modulus. In general, higher fat contents promoted more melting and so did higher coagulant levels. Melting increased during storage although very little change was observed in the nonfat cheese.

Identification and characterization of Lactobacillus helveticus PepO2, an endopeptidase with post-proline specificity.

A post-proline endopeptidase (PepO2) was detected in cell extracts from a genomic library of Lactobacillus helveticus CNRZ32 by using the synthetic substrate N-acetyl-beta-casein-(f203-209)-rho-nitroanilide in a coupled reaction with aminopeptidase N. Isolates with activity for this substrate contained plasmids with visually indistinguishable restriction profiles. Nucleotide sequence analysis revealed a 1,947-bp open reading frame, designated pepO2, encoding a putative 71.4-kDa protein. Analysis of the predicted peptide sequence revealed that L. helveticus PepO2 contained the zinc-dependent metalloprotease motif HEXXH and exhibited levels of amino acid sequence similarity of 72, 61, 59, and 53% to L. helveticus PepO, Lactococcus lactis PepO2, L. lactis PepO, and Lactobacillus rhamnosus PepO, respectively. Northern hybridization results indicated that the transcript containing pepO2 was monocistronic. Despite the high degrees of amino acid similarity to PepO proteins from other lactic acid bacteria, the specificity of the L. helveticus PepO2 for post-proline bonds distinguishes it from other PepO-type endopeptidases characterized to date. The specificity for post-proline bonds also suggests that this enzyme may play a central role in the hydrolysis of casein-derived bitter peptides, such as beta-casein(f193-209).

Hydrolysis of casein-derived peptides alpha(S1)-casein(f1-9) and beta-casein(f193-209) by Lactobacillus helveticus peptidase deletion mutants indicates the presence of a previously undetected endopeptidase.

Peptides derived from hydrolysis of alpha(S1)-casein(f1-9) [alpha(S1)-CN(f1-9)] and beta-CN(f193-209) with cell extracts of Lactobacillus helveticus CNRZ32 and single-peptidase mutants (Delta pepC, Delta pepE, Delta pepN, Delta pepO, and Delta pepX) were isolated by using reverse-phase high-performance liquid chromatography and were characterized by mass spectrometry. The peptides identified suggest that there was activity of an endopeptidase, distinct from previously identified endopeptidases (PepE and PepO), with specificity for peptide bonds C terminal to Pro residues. Identification of hydrolysis products derived from a carboxyl-blocked form of beta-CN(f193-209) confirmed that the peptides were derived from the activity of an endopeptidase.

Contribution of Lactococcus lactis cell envelope proteinase specificity to peptide accumulation and bitterness in reduced-fat Cheddar cheese.

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 alpha(S1)-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.