Lactobacillus casei expressing methylglyoxal synthase causes browning and heterocyclic amine formation in Parmesan cheese extract.

Undesired browning of Parmesan cheese can occur during the latter period of ripening and cold storage despite the relative absence of reducing sugars and high temperatures typically associated with Maillard browning. Highly reactive α-dicarbonyls such as methylglyoxal (MG) are products and accelerants of Maillard browning chemistry and can result from the microbial metabolism of sugars and AA by lactic acid bacteria. We demonstrate the effects of microbially produced MG in a model Parmesan cheese extract using a strain of Lactobacillus casei 12A engineered for inducible overexpression of MG synthase (mgsA) from Thermoanaerobacterium thermosaccharolyticum HG-8. Maximum induction of plasmid-born mgsA led to 1.6 mM MG formation in Parmesan cheese extract and its distinct discoloration. The accumulation of heterocyclic amines including ß-carboline derivatives arising from mgsA expression were determined by mass spectrometry. Potential MG-contributing reaction mechanisms for the formation of heterocyclic amines are proposed. These findings implicate nonstarter lactic acid bacteria may cause browning and influence nutritional aspects of Parmesan by enzymatic conversion of triosephosphates to MG. Moreover, these findings indicate that the microbial production of MG can lead to the formation of late-stage Maillard reaction products such as melanoidin and ß-carbolines, effectively circumventing the thermal requirement of the early- and intermediate- stage Maillard reaction. Therefore, the identification and control of offending microbiota may prevent late-stage browning of Parmesan. The gene mgsA may serve as a genetic biomarker for cheeses with a propensity to undergo MG-mediated browning.

Correction to: Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species.

In the published article, the co-author Abdelmoneim Abdalla's affiliation has been published incompletely. The additional affiliation is given below.

Lactobacillus demonstrate thiol-independent metabolism of methylglyoxal: Implications toward browning prevention in Parmesan cheese.

Endogenous production of α-dicarbonyls by lactic acid bacteria can influence the quality and consistency of fermented foods and beverages. Methylglyoxal (MG) in Parmesan cheese can contribute toward undesired browning during low temperature ripening and storage conditions, leading to the economic depreciation of affected cheeses. We demonstrate the effects of exogenously added MG on browning and volatile formation using a Parmesan cheese extract (PCE). To determine the influence of Lactobacillus on α-dicarbonyls, strains were screened for their ability to modulate concentrations of MG, glyoxal, and diacetyl in PCE. It was found that a major metabolic pathway of MG in Lactobacillus is a thiol-independent reduction, whereby MG is partially or fully reduced to acetol and 1,2-propanediol, respectively. The majority of lactobacilli grown in PCE accumulated the intermediate acetol, whereas Lactobacillus brevis 367 formed exclusively 1,2-propanediol and Lactobacillus fermentum 14931 formed both metabolites. In addition, we determined the inherent tolerance to bacteriostatic concentrations of MG among lactobacilli grown in rich media. It was found that L. brevis 367 reduces MG exclusively to 1,2-propanediol, which correlates to both its ability to significantly decrease MG concentrations in PCE, as well as its significantly higher tolerance to MG, in comparison to other lactobacilli screened. These findings have broader implications toward lactobacilli as a viable solution for reducing MG-mediated browning of Parmesan cheese.

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

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

Application of ARISA to assess the influence of salt content and cation type on microbiological diversity of Cheddar cheese.

UNLABELLED: The structure and dynamics of microbial populations play a significant role during cheese manufacture and ripening. Therefore, fast and accurate methods for identification and characterization of the microbial populations are of fundamental importance to the cheese industry. In this study, we investigate the application of the automated ribosomal intergenic spacer analysis (ARISA) for the assessment of the microbial dynamics in cheeses differing in salt cation level and type. We developed a database of the observed and theoretical length of the 16S-23S intergenic spacer of common lactic acid bacteria (LAB) found in cheese and used the database to describe the structure and dynamics of microbial populations during ripening. Salt content and cation concentration did not significantly influence the overall bacteria structure, except that lower salt levels resulted in enhanced starter survival. Presence of nonstarter LAB was detected by ARISA and denaturing gradient gel electrophoresis (DGGE) after 3 months for all the cheeses analysed. ARISA used as fingerprinting method, proved to be a rapid and inexpensive technique for the discrimination of LAB in cheese and demonstrated higher resolution and performance in comparison with DGGE. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial communities play important roles during cheese making and ripening, hence rapid inexpensive methods to characterize this microbiota are of great interest to both academic and industrial scientists. The application of automated ribosomal intergenic spacer analysis (ARISA) was used to examine the microbial ecology of Cheddar cheese differing in salt level and type. ARISA is well suited to the analysis of the microbial ecology of cheese during ripening. Additionally, the results confirm that salt concentration influences starter culture survival in the cheese matrix, while significant differences were not observed in the nonstarter lactic acid bacteria.

Influence of polysorbate 80 and cyclopropane fatty acid synthase activity on lactic acid production by Lactobacillus casei ATCC 334 at low pH.

Lactic acid is an important industrial chemical commonly produced through microbial fermentation. The efficiency of acid extraction is increased at or below the acid's pKa (pH 3.86), so there is interest in factors that allow for a reduced fermentation pH. We explored the role of cyclopropane synthase (Cfa) and polysorbate (Tween) 80 on acid production and membrane lipid composition in Lactobacillus casei ATCC 334 at low pH. Cells from wild-type and an ATCC 334 cfa knockout mutant were incubated in APT broth medium containing 3 % glucose plus 0.02 or 0.2 % Tween 80. The cultures were allowed to acidify the medium until it reached a target pH (4.5, 4.0, or 3.8), and then the pH was maintained by automatic addition of NH4OH. Cells were collected at the midpoint of the fermentation for membrane lipid analysis, and media samples were analyzed for lactic and acetic acids when acid production had ceased. There were no significant differences in the quantity of lactic acid produced at different pH values by wild-type or mutant cells grown in APT, but the rate of acid production was reduced as pH declined. APT supplementation with 0.2 % Tween 80 significantly increased the amount of lactic acid produced by wild-type cells at pH 3.8, and the rate of acid production was modestly improved. This effect was not observed with the cfa mutant, which indicated Cfa activity and Tween 80 supplementation were each involved in the significant increase in lactic acid yield observed with wild-type L. casei at pH 3.8.

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.

Optimal growth of Lactobacillus casei in a Cheddar cheese ripening model system requires exogenous fatty acids.

Flavor development in ripening Cheddar cheese depends on complex microbial and biochemical processes that are difficult to study in natural cheese. Thus, our group has developed Cheddar cheese extract (CCE) as a model system to study these processes. In previous work, we found that CCE supported growth of Lactobacillus casei, one of the most prominent nonstarter lactic acid bacteria (NSLAB) species found in ripening Cheddar cheese, to a final cell density of 10(8) cfu/mL at 37°C. However, when similar growth experiments were performed at 8°C in CCE derived from 4-mo-old cheese (4mCCE), the final cell densities obtained were only about 10(6) cfu/mL, which is at the lower end of the range of the NSLAB population expected in ripening Cheddar cheese. Here, we report that addition of Tween 80 to CCE resulted in a significant increase in the final cell density of L. casei during growth at 8°C and produced concomitant changes in cytoplasmic membrane fatty acid (CMFA) composition. Although the effect was not as dramatic, addition of milk fat or a monoacylglycerol (MAG) mixture based on the MAG profile of milk fat to 4mCCE also led to an increased final cell density of L. casei in CCE at 8°C and changes in CMFA composition. These observations suggest that optimal growth of L. casei in CCE at low temperature requires supplementation with a source of fatty acids (FA). We hypothesize that L. casei incorporates environmental FA into its CMFA, thereby reducing its energy requirement for growth. The exogenous FA may then be modified or supplemented with FA from de novo synthesis to arrive at a CMFA composition that yields the functionality (i.e., viscosity) required for growth in specific conditions. Additional studies utilizing the CCE model to investigate microbial contributions to cheese ripening should be conducted in CCE supplemented with 1% milk fat.

Growth of Lactobacillus paracasei ATCC 334 in a cheese model system: a biochemical approach.

Growth of Lactobacillus paracasei ATCC 334, in a cheese-ripening model system based upon a medium prepared from ripening Cheddar cheese extract (CCE) was evaluated. Lactobacillus paracasei ATCC 334 grows in CCE made from cheese ripened for 2 (2mCCE), 6 (6mCCE), and 8 (8mCCE) mo, to final cell densities of 5.9×10(8), 1.2×10(8), and 2.1×10(7)cfu/mL, respectively. Biochemical analysis and mass balance equations were used to determine substrate consumption patterns and products formed in 2mCCE. The products formed included formate, acetate, and D-lactate. These data allowed us to identify the pathways likely used and to initiate metabolic flux analysis. The production of volatiles during growth of Lb. paracasei ATCC 334 in 8mCCE was monitored to evaluate the metabolic pathways utilized by Lb. paracasei during the later stages of ripening Cheddar cheese. The 2 volatiles detected at high levels were ethanol and acetate. The remaining detected volatiles are present in significantly lower amounts and likely result from amino acid, pyruvate, and acetyl-coenzyme A metabolism. Carbon balance of galactose, lactose, citrate, and phosphoserine/phosphoserine-containing peptides in terms of D-lactate, acetate, and formate are in agreement with the amounts of substrates observed in 2mCCE; however, this was not the case for 6mCCE and 8mCCE, suggesting that additional energy sources are utilized during growth of Lb. paracasei ATCC 334 in these CCE. This study provides valuable information on the biochemistry and physiology of Lb. paracasei ATCC 334 in ripening cheese.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains.

Lactobacillus helveticus CNRZ 32 is recognized for its ability to decrease bitterness and accelerate flavor development in cheese, and has also been shown to release bioactive peptides in milk. Similar capabilities have been documented in other strains of Lb. helveticus, but the ability of different strains to affect these characteristics can vary widely. Because these attributes are associated with enzymes involved in proteolysis or AA catabolism, we performed comparative genome hybridizations to a CNRZ 32 microarray to explore the distribution of genes encoding such enzymes across a bank of 38 Lb. helveticus strains, including 2 archival samples of CNRZ 32. Genes for peptidases and AA metabolism were highly conserved across the species, whereas those for cell envelope-associated proteinases varied widely. Some of the genetic differences that were detected may help explain the variability that has been noted among Lb. helveticus strains in regard to their functionality in cheese and fermented milk.

Intrinsic and inducible resistance to hydrogen peroxide in Bifidobacterium species.

Interest in, and use of, bifidobacteria as a probiotic delivered in functional foods has increased dramatically in recent years. As a result of their anaerobic nature, oxidative stress can pose a major challenge to maintaining viability of bifidobacteria during functional food storage. To better understand the oxidative stress response in two industrially important bifidobacteria species, we examined the response of three strains of B. longum and three strains of B. animalis subsp. lactis to hydrogen peroxide (H2O2). Each strain was exposed to a range of H2O2 concentrations (0-10 mM) to evaluate and compare intrinsic resistance to H2O2. Next, strains were tested for the presence of an inducible oxidative stress response by exposure to a sublethal H2O2 concentration for 20 or 60 min followed by challenge at a lethal H2O2 concentration. Results showed B. longum subsp. infantis ATCC 15697 had the highest level of intrinsic H2O2 resistance of all strains tested and B. animalis subsp. lactis BL-04 had the highest resistance among B. lactis strains. Inducible H2O2 resistance was detected in four strains, B. longum NCC2705, B. longum D2957, B. lactis RH-1, and B. lactis BL-04. Other strains showed either no difference or increased sensitivity to H2O2 after induction treatments. These data indicate that intrinsic and inducible resistance to hydrogen peroxide is strain specific in B. longum and B. lactis and suggest that for some strains, sublethal H2O2 treatments might help increase cell resistance to oxidative damage during production and storage of probiotic-containing foods.

Screening of Lactobacillus casei strains for their ability to bind aflatoxin B1.

It has been proposed that the consumption of lactic acid bacteria capable of binding or degrading foodborne carcinogens would reduce human exposure to these deleterious compounds. In the present study, the ability of eight strains of Lactobacillus casei to bind aflatoxin B1 in aqueous solution was investigated. Additionally, the effect of addition of bile salts to the growth medium on aflatoxin B1 binding was assessed. The eight strains tested were obtained from different ecological niches (cheese, corn silage, human feces, fermented beverage). The strains exhibited different degrees of aflatoxin binding; the strain with the highest AFB1 binding was L. casei L30, which bound 49.2% of the available aflatoxin (4.6 microg/mL). In general, the human isolates bound the most aflatoxin B1 and the cheese isolates the least. Stability of the bacterial-aflatoxin complex was assessed by repeated washings. Binding was to a limited degree (0.6-9.2% release) reversible; the L. casei 7R1-aflatoxin B1 complex exhibited the greatest stability. L. casei L30, a human isolate, was the strain least sensitive to the inhibitory effects of bile salts. Exposure of the bacterial cells to bile significant increased aflatoxin B1 binding and the differences between the strains was reduced.

Search for protein adhesin gene in Bifidobacterium longum genome using surface phage display technology.

A fragment of the nucleotide sequence encoding polypeptide binding to HT-29 epithelial cells was cloned from VMKB44 Bifidobacterium longum genome library using surface phage display technology. Sequencing of this polypeptide consisting of 26 amino acid residues showed that it is an extracellular fragment of a large BL0155 transmembrane protein belonging to the ABC transport protein superfamily. The genes encoding homologues of this protein were detected in genomes of not only bifidobacteria of different species, but also in many other enteric commencals and pathogens.

Lactobacillus casei metabolic potential to utilize citrate as an energy source in ripening cheese: a bioinformatics approach.

AIMS: To identify potential pathways for citrate catabolism by Lactobacillus casei under conditions similar to ripening cheese. METHODS AND RESULTS: A putative citric acid cycle (PCAC) for Lact. casei was generated utilizing the genome sequence, and metabolic flux analyses. Although it was possible to construct a unique PCAC for Lact. casei, its full functionality was unknown. Therefore, the Lact. casei PCAC was evaluated utilizing end-product analyses of citric acid catabolism during growth in modified chemically defined media (mCDM), and Cheddar cheese extract (CCE). Results suggest that under energy source excess and limitation in mCDM this micro-organism produces mainly L-lactic acid and acetic acid, respectively. Both organic acids were produced in CCE. Additional end products include D-lactic acid, acetoin, formic acid, ethanol, and diacetyl. Production of succinic acid, malic acid, and butanendiol was not observed. CONCLUSIONS: Under conditions similar to those present in ripening cheese, citric acid is converted to acetic acid, L/D-lactic acid, acetoin, diacetyl, ethanol, and formic acid. The PCAC suggests that conversion of the citric acid-derived pyruvic acid into acetic acid, instead of lactic acid, may yield two ATPs per molecule of citric acid. Functionality of the PCAC reductive route was not observed. SIGNIFICANCE AND IMPACT OF THE STUDY: This research describes a unique PCAC for Lact. casei. Additionally, it describes the citric acid catabolism end product by this nonstarter lactic acid bacteria during growth, and under conditions similar to those present in ripening cheese. It provides insights on pathways preferably utilized to derive energy in the presence of limiting carbohydrates by this micro-organism.

Construction and evaluation of food-grade vectors for Lactococcus lactis using aspartate aminotransferase and alpha-galactosidase as selectable markers.

AIMS: We report development of two food-grade cloning vectors for Lactococcus lactis, which utilize either a lactococcal aspartate aminotransferase gene (aspC), or Bifidobacterium longumalpha-galactosidase gene (aglL) as selectable markers. METHODS AND RESULTS: The theta-replicon of lactococcal plasmid, pW563, was combined with aspC and a multiple cloning site. When electroporated into L. lactis JLS400 (AspC-), the resulting vector, pSUW611 (3.9 kbp), restores ability of the mutant to grow in milk thus allowing for selection of the transformants. The vector is stable during 100 generations of nonselective growth (0.2% loss per generation). The second vector, pSUW711 (5.1 kbp), was constructed by exchanging aspC with aglL under the control of usp45 promoter. Lactococcus lactis transformed with pSUW711 produced distinctive colonies within 48-72 h on melibiose-containing plates. Expression of two Lactobacillus helveticus peptidases was attempted using these new vehicles. Introduction of pepN on pSUW611 and pSUW711 into L. lactis led to a sixfold, or 27-fold increase in aminopeptidase activity, respectively. However, no changes in endopeptidase activity were recorded upon transformation with pSUW611 carrying pepO2 under control of three different promoters. Attempts were also made to construct high copy variants of pSUW711. CONCLUSIONS: The aspC and aglL can be employed as food-grade genetic markers for L. lactis. The vectors, pSUW611 and pSUW711, were successfully used to express Lact. helveticus PepN in L. lactis. SIGNIFICANCE AND IMPACT OF THE STUDY: Two novel food-grade vectors were developed which provide simple and convenient selection and maintenance in L. lactis.

Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange.

AIMS: To determine whether conversion of lactocepin substrate binding regions by gene replacement can alter lactocepin specificity in Lactococcus lactis starter bacteria without affecting other important strain properties. METHODS AND RESULTS: We utilized two-step gene replacement to convert substrate-binding determinants in the L. lactis prtP genes encoding group h (bitter) lactocepin in two industrial strains into the corresponding group b (nonbitter) variant. Analysis of lactocepin activity toward alpha(s1)-casein (f 1-23) by reversed-phase high-pressure liquid chromatography demonstrated enzyme specificity among isogenic derivatives had been altered in a manner that was consistent with predicted amino acid substitutions in substrate binding regions. Milk acidification properties of some mutants were not statistically different (P > 0.05) from wild-type parent strains, and strain propensity for autolysis was also not significantly (P > 0.05) changed. CONCLUSIONS: Conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of L. lactis without significantly affecting other important strain properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Methodology outlined in this study can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect, and prtP derivatives developed by this approach should be suitable for commercial application.

Succinate production and citrate catabolism by Cheddar cheese nonstarter lactobacilli.

AIMS: To identify strains of Cheddar cheese nonstarter lactobacilli that synthesize succinate from common precursors and characterize the biochemical pathways utilized. METHODS AND RESULTS: Whole cell incubations of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus zeae and Lactobacillus rhamnosus, were used to identify strains that accumulated succinate from citrate, l-lactate, aspartic acid or isocitrate. In vivo 13C-nuclear magnetic resonance spectroscopy (13C-NMR) identified the biochemical pathway involved at pH 7.0, and under conditions more representative of the cheese ripening environment (pH 5.1/4% NaCl/13 degrees C). Enzyme assays on cell-free extracts were used to support the pathway suggested by 13C-NMR. CONCLUSIONS: The Lact. plantarum strains studied synthesize succinate from citrate by the reductive tricarboxylic acid (TCA) cycle at either pH 7.0 or pH 5.1/4% NaCl/13 degrees C. Lactobacillus casei, Lact. zeae and Lact. rhamnosus strains lack one or more enzymatic activities present in this pathway, and do not accumulate succinate from any of the four precursors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of Lact. plantarum strains to milk during cheese manufacture may increase the accumulation of the flavour enhancer succinate.

Analysis of promoter sequences from Lactobacillus helveticus CNRZ32 and their activity in other lactic acid bacteria.

AIMS: To clone and analyse seven putative promoter fragments (pepC, pepN, pepX, pepO, pepE, pepO2, hsp17) from Lactobacillus helveticus CNRZ32 for their expression in Lact. helveticus CNRZ32, Lact. casei ATCC334 and Lactococcus lactis MG1363. METHODS AND RESULTS: Promoter fragments were fused to the promoter-less beta-glucuronidase (gusA) gene on pNZ272(RBS-) (ATG-). The resulting constructs were evaluated for their ability to drive the expression of active GusA with 0.5 mmol l(-1) 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide. All promoters except P(pepN)::gusA were active in the examined strains. Northern hybridization was performed to examine the promoter strength. Sequence analysis of these promoters identified well conserved putative ribosomal binding and putative -10 hexamers sites. CONCLUSIONS: Seven promoter fragments from Lact. helveticus CNRZ32 were recognized in the lactic acid bacteria, Lact. casei ATCC334 and L. lactis MG1363, as well as in Escherichia coli. P(pepN)::gusA could not be maintained in the strains examined because of toxicity associated with heterologous protein over-expression driven by P(pepN). SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that desirable levels of heterologous food-grade protein production in GRAS organisms can be obtained with the application of natural promoter fragments from closely related organisms.

Accumulation of short n-chain ethyl esters by esterases of lactic acid bacteria under conditions simulating ripening Parmesan cheese.

EstA from Lactobacillus helveticus CNRZ32 (Lbh-EstA), EstB, and EstC from Lactobacillus casei LILA, and EstA from Lactococcus lactis MG1363 (Lcl-EstA) were evaluated for their ability to accumulate esters in a model system simulating Parmesan cheese ripening conditions (10 degrees C, 2 to 3% NaCl, pH 5.4 to 5.5, aw = 0.850 to 0.925) using Capalase K from kid goat as a positive control. All of the LAB esterases and Capalase K mediated the accumulation of esters in the model system in an enzyme specific manner, which was influenced by a, and selectivity for fatty acid chain-length. In general, enzyme mediated accumulation of ethyl esters was higher at aw values of 0.850 and 0.900 than at aw of 0.925, demonstrating that aw is a critical parameter influencing ester accumulation. The substrate selectivity of esterases, aw, and enzyme type may be important factors in the development of fruity flavors, as evidenced by results in this model system simulating Parmesan cheese ripening conditions.

Nucleotide sequencing, purification, and biochemical properties of an arylesterase from Lactobacillus casei LILA.

An esterase gene, designated estB, was isolated from a genomic library of Lactobacillus casei LILA. Nucleotide sequencing of the estB gene revealed a 954-bp open reading frame encoding a putative peptide of 35.7 kDa. The deduced amino acid sequence of EstB contained the characteristic GXSXG active-site serine motifidentified in most lipases and esterases. An EstB fusion protein containing a C-terminal 6-histidine tag was constructed and purified to electrophoretic homogeneity by affinity chromatography. The native molecular weight of EstB was 216.5 +/- 2.5 kDa, while the subunit molecular weight was 36.7 +/- 1.0 kDa. Optimum pH, temperature, and NaCl concentration for EstB were determined to be pH 7.0,50 to 55 degrees C, and 15% NaCl, respectively. EstB had significant activity under conditions simulating those of ripening cheese (pH 5.1, 10 degrees C, and 4% NaCl). Kinetic constants (KM and Vmax) were determined for EstB action on a variety of ethyl esters and ester compounds consisting of substituted phenyl alcohols and short n-chain fatty acids. For comparison purposes, EstA from Lb. helveticus CNRZ32 was purified to electrophoretic homogeneity and its substrate selectivity determined in a similar fashion. Different substrate selectivities were observed for EstB and EstA.