Jeffery R. Broadbent

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.

Comparative High-Density Microarray Analysis of Gene Expression during Growth of Lactobacillus helveticus in Milk versus Rich Culture Medium

ABSTRACT 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 Bonferronis 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.

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.

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

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

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.

Use of Exopolysaccharide-Producing Cultures to Improve the Functionality of Low Fat Cheese

Abstract Lactic acid bacteria may produce exopolysaccharide (EPS) that is tightly associated with the bacterial cell wall (capsular EPS) or liberated into the growth medium (ropy EPS). Because EPS have viscosity enhancing and stabilizing properties, exopolysaccharide-producing (EPS + ) starter cultures are commonly used to enhance water binding and viscosity in yogurt and fermented milks. Previous work has shown that low fat Mozzarella cheese manufactured with an EPS + starter pair, Streptococcus thermophilus MR-1C and Lactobacillus delbrueckii subsp. bulgaricus MR-1R, contained significantly more moisture and had better melt properties than cheese made with a control starter pair. Genetic studies demonstrated that this effect was due to the MR-1C capsular EPS, and industrial cheese trials confirmed that MR-1C can effect a 1.5% moisture increase in part-skim Mozzarella. Because EPS accumulation in cheese whey may retard whey protein concentration and drying efficiency, the effect of capsular and ropy S. thermophilus starter bacteria on Mozzarella cheese and whey was also compared. Moisture and melt properties were improved in cheeses made with either EPS + S. thermophilus , but 5× concentrated whey from the ropy S. thermophilus was significantly more viscous than concentrated wheys from capsule-producing or non-EPS-producing S. thermophilus . These data indicate that encapsulated, but not ropy, EPS + S. thermophilus can be used to increase moisture content and improve melt in Mozzarella cheese, without deleteriously affecting whey viscosity.

Perspectives on the contribution of lactic acid bacteria to cheese flavor development

It has been known since the 1960s that lactic acid bacteria are essential for the development of cheese flavor. In the ensuing 50 years significant research has been directed at understanding the microbiology, genetics and biochemistry of this process. This review briefly covers the current status of cheese flavor development and then provides our vision for approaches which will enhance our understanding of this process. The long-term goal of this area of research is to enable technology (i.e. cultures and enzymes) that results in consistent rapid development of cheese variety-specific characteristic flavors.

Identification of Endopeptidase Genes from the Genomic Sequence of Lactobacillus helveticus CNRZ32 and the Role of These Genes in Hydrolysis of Model Bitter Peptides

ABSTRACT 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 DH5α derivatives expressing CNRZ32 endopeptidases PepE, PepE2, PepF, PepO, PepO2, and PepO3 to hydrolyze the model bitter peptides, β-casein (β-CN) (f193-209) and αS1-casein (αS1-CN) (f1-9), under cheese-ripening conditions (pH 5.1, 4% NaCl, and 10°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 αS1-CN (f1-9) and β-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 β-CN (f193-209) and αS1-CN (f1-9) under cheese-ripening conditions in single-peptide reactions, in a defined peptide mix, and in Cheddar cheese serum. Peptides αS1-CN (f1-9), αS1-CN (f1-13), and αS1-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 β-CN (f193-209) and αS1-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 strains ability to reduce bitterness in cheese.

Genetic diversity in proteolytic enzymes and amino acid metabolism among Lactobacillus helveticus strains1

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.

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.