Lactobacillus Casei

Swiss-type cheeses. Although some strains of propionibacteria can ferment lactose, none is available during the time at which these bacteria are given the opportunity to grow (i.e., several weeks after the primary lactose fermentation is complete). Instead, lactate is the only energy source available. Lactate fermentation occurs via the propionate pathway, which yields two moles of propionate, and one each of acetate and CO2 per three moles of lactate fermented. The cell nets one mole of ATP per lactate. In cheese, the actual amount of end-products varies as a result of condensation reactions, co-metabolism with amino acids, or strain variation. The propionic acid pathway is quite complex and requires several metal-containing enzymes and vitamin co-factors (Chapter 5).

Metabolism of molds

As noted above, the metabolic activities of Penicillium, Aspergillus, and other fungi are quite unlike those of bacteria and yeasts. The latter have a mostly fermentative metabolism, growing on simple sugars and producing just a few different end-products. In contrast, fungal metabolism is characterized by secretion of numerous proteolytic, amylolytic, and lipolytic enzymes. These enzymatic end-products then serve as substrates for further metabolism.

Often, the metabolic pathways used by fungi result in an array of unique products. For example, when P. roqueforti, the blue mold organism, grows in cheese, substantial proteo-lysis occurs through elaboration of several extracellular proteinases, endopeptidases, and exopeptidases. The resulting amino acids are subsequently metabolized via deaminases and decarboxylases releasing amines, ammonia, and other possible flavor compounds.

More end-products that are characteristic of blue cheese flavors are generated, however, from lipid metabolism. About 20% of triglycerides in milk are initially hydrolyzed by P roqueforti-produced lipases, releasing free fatty acids, including short chain, volatile fatty acids, such as butyric and caproic acids. Metab olism of free fatty acids via p-oxidation pathways then yields a variety of methylketones, compounds that are responsible for the characteristic flavor and aroma of blue cheese. Similarly, growth of the Brie cheese mold, P. camemberti, results in a similar sequence of metabolic events. Proteinases and lipases diffuse through the cheese (since the mold grows only at the surface), generating amino and free fatty acids. Subsequent metabolism of the amino acids results in formation of ammonia, methanethiol, and other sulfur compounds, presumably derived from sulfur-containing amino acids. Lipolysis of triglycerides and fatty acid metabolism by P. camemberti are just as important in Brie-type cheeses as in blue-veined cheese, and methylketones are abundant. In the cheese environment, both P.roque-forti and P. camemberti can use lactic acid as a carbon source, which causes the pH to rise, often to near-neutral levels.

Metabolic Engineering

As described earlier in this chapter as well as throughout this text, the key to a successful fermentation is control. The difference between fermentation and spoilage really boils down to controlled growth and metabolism versus uncontrolled growth and metabolism. Therefore, one way to improve a given fermentation process would be to impose either greater control on that process or, alternatively, simply engineer the preferred metabolic route directly into the organism of interest.The latter strategy, referred to as metabolic engineering, provides for a more precise and consistent metabolic result. For example, if increased di-acetyl production by a lactic acid bacterium is the goal, rather than manipulate substrate or oxygen levels, the pathway can be genetically manipulated, such that carbon flow is directed to diacetyl rather than lactate (e.g., by inactivating lactate dehydrogenase). The availability of sequenced genomes, along with computational tools, now makes it possible to screen those genomes, not just for a particular enzyme, but for entire pathways or clusters of

Box 2—5. Fermentation Microbiology: From Pasteur's Ferment to Functional Genomics

In 1999, the American Society of Microbiology published a chronology of "Microbiology's fifty most significant events during the past 125 years" (ASM News, 1999).The list started with Pasteur's discovery in 1861 that yeast produced more ethanol during anaerobic fermentative growth compared to growth under aerobic, respiring conditions (the aptly called "Pasteur ef-fect").The list ended with the report in 1995 that described, for the first time, the complete genome sequence of a bacterium (Fleischmann et al., 1995).

The latter event marked, perhaps, the end of one era and the start of another. From 1995 to the present (June, 2005), more than 230 microbial genomes have been sequenced (and published), and another 370 are nearly finished (according to the National Center for Biotechnology Information,www.ncbi.nlm.nih.gov). Sequencing genomes has evolved from an expensive, multi-year, labor-intensive endeavor to one that is affordable, fast, and highly automated.At the same time, the development of bioinformatics and various computational tools used to analyze and compare genome information has also advanced at an equally rapid rate.

Genomes of Food Fermentation Organisms

Among organisms involved in food fermentations, the genome of the common yeast, Saccha-romyces cerevisiae, was published in 1996 (Goffeau et al., 1996).Although the sequence strain was a lab strain, and was quite different from the typical bakers' or brewers' yeasts used in industry, the sequence nonetheless provided valuable information on yeast biology and genetics. Several years later, the genome sequence of the lactic acid bacterium, Lactococcus lactis subsp. lactis, was reported (Bolotin et al., 2001). In the past several years, groups in the United States, Europe, and New Zealand have completed sequencing projects for more than 30 other lactic acid and related bacteria (Table 1). A major collaborative effort was also begun in 2002, when the Lactic Acid Bacteria Genome Consortium (in collaboration with the U.S. Department of Energy's Joint Genome Institute) was organized (Klaenhammer et al., 2002). The genomes of eleven commercially important bacteria were sequenced as part of this project (Figure 1; Makarova et al., 2006).

Genome Sequences are Only the Beginning

The actual output of a genome sequencing project is really just the linear order of nucleotides arranged as a continuous circle.It is not until the nucleotide sequences (i.e.,strings of Gs, Cs,As, and Ts) are analyzed computationally, that open reading frames (orf) and putative genes can be inferred or predicted. Comparison of those orfs (or the translated proteins) to genome data bases provides a basis for assigning probable functions to those genes.The latter process, called genome annotation, usually results in about 70% of the orfs having assigned functions, although as databases grow, so does the likelihood a given orf will have a homolog (i.e., find a match to a previously assigned protein).

The genome may also contain regions encoding for insertion sequence elements,prophages, and tRNA and rRNA. Of course, predicted function is just that—it is based on the statistical probability that a gene codes for a particular protein.To confirm an actual biological function for a gene requires some form of biochemical demonstration (e.g., creating loss-of-function mutations having the expected phenotype).Therefore, genome sequencing and annotation are really only the first steps toward understanding how pathways are constructed, how regulatory networks function, and how these bacteria ultimately behave during food fermentations. Functional and comparative genomics not only addresses these questions, but also provides insight into their phylogeny and evolution.

Box 2—5. Fermentation Microbiology: From Pasteur's Ferment to Functional Genomics (Continued)

Table 1. Sequenced genomes of lactic acid bacteria1.

Organism Strain(s)

Lactobacillus acidophilus NCFM Lactobacillus brevis ATCC 367

Lactobacillus delbrueckii subsp. bulgaricus ATCC 11842,DN-100107,ATCC BAA-365

Lactobacillus caseiATCC 334, BL23

Lactobacillus gasseri ATCC 33323

Lactobacillus helveticus CNRZ 32, DPC 4571

Lactobacillus johnsonii NCC 533

Lactobacillus plantarum WCRS1

Lactobacillus reuteri 100-23, DSM 20016T

Lactobacillus rhamnosus HN001

Lactobacillus sakei 23K

Lactococcus lactis subsp. cremoris MG 1363, SK11

Lactococcus lactis subsp. lactis IL 1403

Leuconostoc mesenteroides ATCC 8293

Leuconostoc citreum KM20

Oenococcus oeni PSU-1

Pediococcus pentosaceus ATCC 25745

Streptococcus thermophilus LMG 18311, CNRZ 1066, LMD-9

Adapted from Klaenhammer et al., 2002

What have the genomes revealed?

The genomes of the lactic acid bacteria are generally small, with several less than 2 Mb.Accord-ingly, the genomes reflect the rather specialized metabolic capabilities of these bacteria and the specific environmental niches in which they live (Makarova et al., 2006). For example, most species contain genes encoding for only a few of the carbon-utilization pathways (i.e., homo-and heterofermentation; pyruvate dissimulation; and pentose, citrate, and malate fermentation). These bacteria are auxotrophic for several amino acids, and, therefore, contain many genes encoding for protein and peptide catabolism. In fact, given their overall dependence on obtaining nutrients from the environment, it is not surprising that the genomes are replete (between 13% and 18%) with transport system genes (Lorca et al., 2006).

Genome evolution analyses have revealed that the loss of biosynthetic genes over time, and the corresponding acquisition of catabolic genes, are consistent with the view that the lactic acid bacteria have recently adapted from nutritionally-poor, plant-type habitats to nutritionally-complex food environments.This suggestion is supported by several observations. First, some of the lactic acid bacteria contain a large number of plasmids and transposons that accelerate horizontal gene transfer (genetic exchange between related organisms) and that promote adaptation to new environments. Second, there are a large number of non-functional pseudogenes (containing mutations or truncation) within the genomes that likely represent "gene decay" or loss of genes that are no longer needed (Makarova et al.,2006). For example, 10% of the genome of the nutritionally-fastidious Streptococcus thermophilus are classified as pseudogenes, with many encoding specifically for energy metabolism and transport functions (Bolotin et al., 2004). As lactic acid bacteria have evolved, therefore, significant genome reduction, with modest genome expansion, has apparently become the norm (Makarova et al., 2006).

References

ASM News. Microbiology's fifty most significant events during the past 125 years. Poster supplement. 65.

Box 2—5. Fermentation Microbiology: From Pasteur's Ferment to Functional Genomics (Continued)

Bolotin,A., P.Wincker, S. Mauger, O.Jaillon, K. Malarme, J.Weissenbach, S.D. Ehrlich, and A. Sorokin. 2001.The complete genome sequence of the lactic acid bacterium Lactococcus lactis ssp. lactis IL1403. Genome Res. 11:731-753.

Fleischmann, R.D., M.D.Adams, et al., (38 other authors). 1995.Whole-genome random sequencing and assembly of Haemophilus influenza Rd. Science 269:496-512. Goffeau,A., B. G. Barrell, H. Bussey, R.W. Davis, B. Dujon, H. Feldmann, F. Galibert, J. D. Hoheisel, C.Jacq, M. Johnston, E.J. Louis, H.W. Mewes,Y. Murakami, P. Philippsen, H.Tettelin, and S. G. Oliver. 1996. Life with 6000 genes. Science 274:546-567. Klaenhammer, T., E. Altermann, et al., (35 other authors). 2002. Discovering lactic acid bacteria by genomics.Antonie van Leeuwenhoek 82:29-58. Lorca, G.L., R.D. Barabote,V. Zlotopolski, C.Tran, B.Winnen, R.H. Hvorup,A.J. Stonestrom, E. Nguyen, L.-W. Huang, D. Kempton, and M.H. Saier. 2006.Transport capabilities of eleven Gram-positive bacteria: comparative genomic analyses. In press. Makarova, K.,Y.Wolf, et al., (and 43 other authors) 2006. Comparative genomics of the lactic acid bacteria. 2006. Proc. Nat.Acad. Sci. In Press.

Lactobacillus Garicus

Figure 1. Electron micrographs of lactic acid and related bacteria: A, Lactobacillus delbrueckkii subsp. bul-garicus; B, Lactobacillus brevis; C, Pediococcus pentosaceus; D, Lactobacillus casei; E, Lactococcus lactis; F, Brevibacterium linens; G, Lactobacillus helveticus; H, Streptococcus thermophilus; and I, Bifidobacterium longum. Scale bars are 3.0 ^m, unless otherwise indicated. Not shown: Lactobacillus gasseri and Oenococcus oeni. Photos courtesy of J. Broadbent and B. McManus, Utah State University, and D. O'Sullivan, University of Minnesota, and with permission from the American Society for Microbiology (ASM News, March, 2005, p. 121-129)

Figure 1. Electron micrographs of lactic acid and related bacteria: A, Lactobacillus delbrueckkii subsp. bul-garicus; B, Lactobacillus brevis; C, Pediococcus pentosaceus; D, Lactobacillus casei; E, Lactococcus lactis; F, Brevibacterium linens; G, Lactobacillus helveticus; H, Streptococcus thermophilus; and I, Bifidobacterium longum. Scale bars are 3.0 ^m, unless otherwise indicated. Not shown: Lactobacillus gasseri and Oenococcus oeni. Photos courtesy of J. Broadbent and B. McManus, Utah State University, and D. O'Sullivan, University of Minnesota, and with permission from the American Society for Microbiology (ASM News, March, 2005, p. 121-129)

pathways (Box 2-5). Moreover, a variety of molecular tools now exist that can be used to inactivate some pathways and activate others.

Bibliography

Axelsson, L. 2004. Lactic acid bacteria: classification and physiology, pp. 1-66. In Salminen, S.,A. von Write, and A. Ouwehand. Lactic Acid Bacteria Microbiological and Functional Aspects Third Edition Marcel Dekker, Inc. New York, New York. Bullerman, L.B. 1993. Biology and health aspects of molds in foods and the environment. J. Korean Soc. Food Nutr. 22:359-366. Dellaglio, F., L.M.T. Dicks, and S.Torriani. 1992.The genus Leuconostoc. In B.J.B. Wood and W.H. Holzapfel, ed.The Lactic Acid Bacteria,Volume 2, The genera of lactic acid bacteria. Blackie Academic and Professional pp 235-268. de Vos, W.M., I. Boerrigter, R.J. van Rooyen, B. Reiche, and W. Hengstenberg. 1990. Characterization of the lactose-specific enzymes of the phospho-transferase system in Lactococcus lactis. J. Biol. Chem. 265:22554-22560. Gancedo, J.M. 1998. Yeast carbon catabolite repression. Microbiol. Mol. Biol. Rev. 62:334-361. Garrity, G.M., J. Bell, and T.G. Lilburn. 2004. Taxo-nomic Outline of the Prokaryotes. Bergey's Man ual of Systematic Bacteriology, Second Edition. Release 5.0, Springer-Verlag. DOI: 10.1007/ bergeysonline.

Hemme, D., C. Foucaud-Scheunemann. 2004. Leuconostoc, characteristics, use in dairy technology and prospects in functional foods. Int. Dairy J. 14:467-494.

Hutkins, R.W. 2001. Metabolism of starter cultures, p. 207. In E.H. Marth and J.L. Steele (ed.), Applied Dairy Microbiology. Marcel Dekker, Inc., New York, NY.

Makarova, K., Y.Wolf, et al., (and 43 other authors) 2006. Comparative genomics of the lactic acid bacteria. 2006. Proc. Nat.Acad. Sci. In Press.

Moore, D. and L. Novak Frazer. 2002. Essential Fungal Genetics. Springer-Verlag. New York, NY

Naumov, G.I., S.A.James, E.S. Naumova, E.J. Louis, and I.N. Roberts. 2000.Three new species in the Sac-charomyces sensu stricto complex: Saccharo-myces cariocanus, Saccharomyces kudriavze-vii and Saccharomyces mikatae. Int. J. Syst. Evol. Microbiol. 50:1931-1942.

Pitt, J.I. and A.D. Hocking. 1999. Fungi and Food Spoilage.Aspen Publishers, Inc. Gaithersburg, MD.

Samson, R.A., E.S. Hoekstra, J.C. Frisvad and O. Filtenborg. 2000. Introduction to Food and Airborne Fungi. Sixth Ed. Centraalbureau voor Schimmelcultures. Utrecht,The Netherlands.

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  • Merja
    Can ferment lactobacillus casei?
    5 years ago

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