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.

Genome Sequences of Two Leuconostoc pseudomesenteroides Strains Isolated from Danish Dairy Starter Cultures.

The lactic acid bacterium Leuconostoc pseudomesenteroides can be found in mesophilic cheese starters, where it produces aromatic compounds from, e.g., citrate. Here, we present the draft genome sequences of two L. pseudomesenteroides strains isolated from traditional Danish cheese starters.

Genome Sequence of Leuconostoc mesenteroides subsp. cremoris Strain T26, Isolated from Mesophilic Undefined Cheese Starter.

Leuconostoc is the main group of heterofermentative bacteria found in mesophilic dairy starters. They grow in close symbiosis with the Lactococcus population and are able to degrade citrate. Here we present a draft genome sequence of Leuconostoc mesenteroides subsp. cremoris strain T26.

Effect of sodium, potassium, magnesium, and calcium salt cations on pH, proteolysis, organic acids, and microbial populations during storage of full-fat Cheddar cheese.

Sodium reduction in cheese can assist in reducing overall dietary Na intake, yet saltiness is an important aspect of cheese flavor. Our objective was to evaluate the effect of partial substitution of Na with K on survival of lactic acid bacteria (LAB) and nonstarter LAB (NSLAB), pH, organic acid production, and extent of proteolysis as water-soluble nitrogen (WSN) and protein profiles using urea-PAGE, in Cheddar cheese during 9mo of storage. Seven Cheddar cheeses with molar salt contents equivalent to 1.7% salt but with different ratios of Na, K, Ca, and Mg cations were manufactured as well as a low-salt cheese with 0.7% salt. The 1.7% salt cheeses had a mean composition of 352g of moisture/kg, 259g of protein/kg and 50% fat-on-dry-basis, and 17.5g of salt/kg (measured as Cl(-)). After salting, a faster initial decrease in cheese pH occurred with low salt or K substitution and it remained lower throughout storage. No difference in intact casein levels or percentage WSN levels between the various cheeses was observed, with the percentage WSN increasing from 5% at d 1 to 25% at 9mo. A greater decrease in intact αs1-casein than ß-casein was detected, and the ratio of αs1-casein (f121-199) to αs1-casein could be used as an index of ripening. Typical changes in bacteria microflora occurred during storage, with lactococci decreasing gradually and NSLAB increasing. Lowering the Na content, even with K replacement, extended the crossover time when NSLAB became dominant. The crossover time was 4.5mo for the control cheese and was delayed to 5.2, 6.0, 6.1, and 6.2mo for cheeses with 10, 25, 50, and 75% K substitution. Including 10% Mg or Ca, along with 40% K, further increased crossover time, whereas the longest crossover time (7.3mo) was for low-salt cheese. By 9mo, NSLAB levels in all cheeses had increased from initial levels of ≤10(2) to approximately 10(6)cfu/g. Lactococci remained at 10(6) cfu/g in the low-salt cheese even after 9mo of storage. The propionic acid concentration in the cheese increased when NSLAB numbers were high. Few other trends in organic acid concentration were observed as a function of Na content.

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.

Survival of microencapsulated probiotic Lactobacillus paracasei LBC-1e during manufacture of Mozzarella cheese and simulated gastric digestion.

An erythromycin-resistant strain of probiotic Lactobacillus paracasei ssp. paracasei LBC-1 (LBC-1e) was added to part-skim Mozzarella cheese in alginate-microencapsulated or free form at a level of 10(8) and 10(7)cfu/g, respectively. Survival of LBC-1e and total lactic acid bacteria (LAB) was investigated through the pasta filata process of cheese making (in which the cheese curd was heated to 55 °C and stretched in 70 °C-hot brine), followed by storage at 4 °C for 6 wk and simulated gastric and intestinal digestion. This included incubation in 0.1 M and 0.01 M HCl, 0.9 M H(3)PO(4), and a simulated intestinal juice consisting of pancreatin and bile salts in a pH 7.4 phosphate buffer. Some reductions were observed in both free and encapsulated LBC-1e during heating and stretching, with encapsulated LBC-1e surviving slightly better. Changes in total LAB losses during heating and stretching did not reach statistical significance. During storage, a decrease was observed in total LAB, but no statistically significant decrease was observed in LBC-1e. Survival during gastric digestion in HCl was dependent on the extent of neutralization of HCl by the cheese, with more survival in the weaker acid, in which pH increased to 4.4 after cheese addition. The alginate microcapsules did not provide any protection against the HCl. It is interesting that survival of the encapsulated LBC-1e was greater during incubation in H(3)PO(4) than in the HCl gastric juices. Proper selection of simulated gastric digestion media is important for predicting the delivery of probiotic bacteria into the human intestinal tract. Neither free nor encapsulated LBC-1e was affected by incubation in the pancreatin-bile solution. Based on the level of probiotic bacteria in cheese needed to provide a health benefit and its survival during simulated gastric digestion, as determined in this study, it should theoretically be possible to lower the amount that needs to be ingested in cheese by up to a factor of 10(3) compared with other fermented dairy foods or when consumed as supplements.

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.

Remifentanil patient-controlled analgesia for labour: a complete audit cycle.

Remifentanil patient-controlled analgesia (PCA) was introduced to a small maternity unit where an extensive epidural service was difficult to provide. This was a new service and the New Zealand College of Midwives had serious doubts about the efficacy and safety of remifentanil, so auditing its use was important. In a two-stage audit, clinical notes of 244 consecutive remifentanil users were studied between January 2008 and November 2009. We developed a questionnaire to assess the parturients' satisfaction with remifentanil PCA and designed a proforma to evaluate it against four standards of best practice. During the two audit periods, timely commencement of PCA was achieved in 65% and 82% of cases, respectively. A 70% compliance rate with monitoring standards fell to 10% after the withdrawal of supervision by an acute pain team, but improved to 91% following implementation of regular midwifery training sessions and a redesigned partogram and prescription flowchart. Ninety-four percent of women rated remifentanil PCA as excellent, very good or good. Maternal side-effects were nausea, pruritus and drowsiness. A comparison of Apgar scores of consecutive neonates born by normal vaginal delivery to women receiving no analgesia, with those born to women using remifentanil PCA, demonstrated no difference. As a result of our audit, remifentanil PCA is now viewed by our midwives as an effective and safe method when accompanied by 1:1 care and appropriate monitoring. With our input other maternity units have introduced it, especially where epidural service provision is limited, and for patients in whom epidural analgesia is contraindicated.

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.

Efficacy of washing meat surfaces with 2% levulinic, acetic, or lactic acid for pathogen decontamination and residual growth inhibition.

We compared spray washing at 55.4 °C with 2% levulinic acid to that with lactic or acetic acid for decontamination of pathogenic bacteria inoculated onto meat surfaces, and their residual protection against later growth of pathogenic bacteria. The model systems included Escherichia coli O157:H7 on beef plate, Salmonella on chicken skin and pork belly, and Listeria monocytogenes on turkey roll. In the decontamination studies, acid washes lowered recoverable numbers of pathogens by 0.6 to 1 log/cm(2) as compared to no-wash controls, and only lactic acid lowered the number of pathogens recovered as compared to the water wash. Washing with levulinic acid at 68.3 or 76.7 °C did not result in additional decontamination of E. coli. Acetic acid prevented residual growth of E. coli and L. monocytogenes, and it reduced numbers of Salmonella on chicken skin to below recoverable levels. Overall, levulinic acid did not provide as effective decontamination as lactic acid nor residual protection as acetic acid.

Protein-protein interactions among the components of the biosynthetic machinery responsible for exopolysaccharide production in Streptococcus thermophilus MR-1C.

AIM: This study identified protein-protein interactions among the biosynthetic machinery responsible for exopolysaccharide (EPS) production in Streptococcus thermophilus MR-1C. METHODS AND RESULTS: Protein-protein interactions were investigated using the yeast two-hybrid system. A strong protein-protein interaction was detected between the transmembrane activation protein Wzd and the protein tyrosine kinase Wze. Weaker protein-protein interactions were detected between two duplicate Wze proteins and between Wze and the phosphotyrosine phosphatase Wzh. Protein-protein interactions involving a Wzd/Wze fusion protein and Wzd and Wze may indicate that these proteins form multi-protein complexes. All combinations of the Wzh, Wzd, Wze, Wzg (regulation), CpsE (glycosyl-1-phosphate transferase), CpsS (polymerization), CpsL (unknown), CpsW (regulation) and CpsU (membrane translocation) were analysed for protein-protein interactions but no additional interactions were discovered using the yeast two-hybrid system. CONCLUSIONS: Interactions among the phosphotyrosine phosphatase, tyrosine kinase, and transmembrane activation protein are important in the regulation of capsule biosynthesis in Strep. thermophilus MR-1C. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides some valuable insight into the organization and interactions between the many proteins involved in EPS production. A better understanding of this process may facilitate the genetic manipulation of capsule production to impart desirable properties to dairy starter cultures.

External concentration of organic acid anions and pH: key independent variables for studying how organic acids inhibit growth of bacteria in mildly acidic foods.

Although the mechanisms by which organic acids inhibit growth of bacteria in mildly acidic foods are not fully understood, it is clear that intracellular accumulation of anions is a primary contributor to inhibition of bacterial growth. We hypothesize that intracellular accumulation of anions is driven by 2 factors, external anion concentration and external acidity. This hypothesis follows from basic chemistry principles that heretofore have not been fully applied to studies in the field, and it has led us to develop a novel approach for predicting internal anion concentration by controlling the external concentration of anions and pH. This approach overcomes critical flaws in contemporary experimental design that invariably target concentration of either protonated acid or total acid in the growth media thereby leaving anion concentration to vary depending on the pK(a) of the acids involved. Failure to control external concentration of anions has undoubtedly confounded results, and it has likely led to misleading conclusions regarding the antimicrobial action of organic acids. In summary, we advocate an approach for directing internal anion levels by controlling external concentration of anions and pH because it presents an additional opportunity to study the mechanisms by which organic acids inhibit bacterial growth. Knowledge gained from such studies would have important application in the control of important foodborne pathogens such as Listeria monocytogenes, and may also facilitate efforts to promote the survival in foods or beverages of desirable probiotic bacteria.

Comparative evaluation of yogurt and low-fat cheddar cheese as delivery media for probiotic Lactobacillus casei.

This study used Lactobacillus casei 334e, an erythromycin-resistant derivative of ATCC 334, as a model to evaluate viability and acid resistance of probiotic L. casei in low-fat Cheddar cheese and yogurt. Cheese and yogurt were made by standard methods and the probiotic L. casei adjunct was added at approximately 10(7) CFU/g with the starter cultures. Low-fat cheese and yogurt samples were stored at 8 and 2 degrees C, respectively, and numbers of the L. casei adjunct were periodically determined by plating on MRS agar that contained 5 microg/mL of erythromycin. L. casei 334e counts in cheese and yogurt remained at 10(7) CFU/g over 3 mo and 3 wk, respectively, indicating good survival in both products. Acid challenge studies in 8.7 mM phosphoric acid (pH 2) at 37 degrees C showed numbers of L. casei 334e in yogurt dropped from 10(7) CFU/g to less than 10(1) CFU/g after 30 min, while counts in cheese samples dropped from 10(7) CFU/g to about 10(5) after 30 min, and remained near 10(4) CFU/g after 120 min. As a whole, these data showed that low-fat Cheddar cheese is a viable delivery food for probiotic L. casei because it allowed for good survival during storage and helped protect cells against the very low pH that will be encountered during stomach transit.

Control of Listeria monocytogenes in ready-to-eat meats containing sodium levulinate, sodium lactate, or a combination of sodium lactate and sodium diacetate.

This study investigated the use of sodium levulinate to prevent outgrowth of Listeria monocytogenes in refrigerated ready-to-eat (RTE) meat products. Turkey breast roll and bologna were formulated to contain 1%, 2%, or 3% (w/w) sodium levulinate, 2% sodium lactate, a 2% combination of sodium lactate and sodium diacetate (1.875% sodium lactate and 0.125% sodium diacetate), or no antimicrobial (control). Samples of the RTE products were sliced, inoculated with 10(2) to 10(3) CFU/cm(2) of a 5-strain cocktail of L. monocytogenes, vacuum packaged, and stored at refrigeration temperature for 0 to 12 wk. Counts reached 10(8) CFU/cm(2) on control turkey roll product after 8 wk, and over 10(7) CFU/cm(2) on control bologna after 12 wk. Addition of 2% or more sodium levulinate to turkey roll and 1% or more sodium levulinate to bologna completely prevented growth of L. monocytogenes during 12 wk of refrigerated storage. A consumer taste panel with pathogen-free samples found no differences in the overall liking among the preparations of turkey roll or among preparations of bologna. These results show that sodium levulinate is at least as effective at inhibiting outgrowth of L. monocytogenes in RTE meat products as the current industry standards of lactate or lactate and diacetate, and levulinate addition does not alter the overall liking of the RTE meat products.

Enhanced nutty flavor formation in cheddar cheese made with a malty Lactococcus lactis adjunct culture.

Nutty flavor in Cheddar cheese is desirable, and recent research demonstrated that 2- and 3-methyl butanal and 2-methyl propanal were primary sources of nutty flavors in Cheddar. Because malty strains of Lac-tococcus lactis (formerly Streptococcus lactis var. malti-genes) are characterized by the efficient production of these and other Strecker aldehydes during growth, this study investigated the influence of a malty L. lactis adjunct culture on nutty flavor development in Cheddar cheese. Cheeses made with different adjunct levels (0, 10(4) cfu/mL, and 10(5) cfu/mL) were ripened at 5 or 13 degrees C and analyzed after 1 wk, 4 mo, and 8 mo by a combination of instrumental and sensory methods to characterize nutty flavor development. Cheeses ripened at 13 degrees C developed aged flavors (brothy, sulfur, and nutty flavors) more rapidly than cheeses held at 5 degrees C. Additionally, cheeses made with the adjunct culture showed more rapid and more intense nutty flavor development than control cheeses. Cheeses that had higher intensities of nutty flavors also had a higher concentration of 2/3-methyl butanal and 2-methyl propanal compared with control cheeses, which again confirmed that these compounds are a source of nutty flavor in Cheddar cheese. Results from this study provide a simple methodology for cheese manufacturers to obtain consistent nutty flavor in Cheddar cheese.

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.