Lactobacillus casei

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A Microbial Biorealm page on the genus Lactobacillus casei

Lactobacillus casei ATCC 334 from Jeff Broadbent, Utah State University

Classification

Higher order taxa

Bacteria; Firmicutes; Bacilli; Lactobacillales; Lactobacillaceae; Lactobacillus

Species

NCBI: Taxonomy

Lactobacillus casei

Strains

Listed are some of the many strains/isolates of Lactobacillus casei: (1)

HUMAN origin: L3, L6, L9, L14, L19, L25, L30, CRF28, Shirota

CHEESE origin: ATCC 334, ASCC 428, ASCC 477, ASCC 1087, ASCC 1088, ASCC 1123, DPC 3971, DPC 3968, DPC 4249, DPC 4748

PLANT origin: 12A, 32G, BI0231, USDA-P, KH-1

Description and significance

Lactobacillus casei is one of the many species of bacteria belonging in the genus Lactobacillus. It is a mesophilic bacteria that is gram positive, rod shaped, nonsporing, nonmotile, anaerobic, and contains no cytochromes (5). L. casei can be found in various environments such as raw and fermented dairy products, intestinal tracts and reproductive systems of humans and animals, and fresh and fermented plant products (1). The optimum pH for L. casei is 5.5. The lactic acid produced by L. casei through fermentation is very important since it can be used to make cheeses and yogurts, reduce cholesterol levels, enhance immune response, control diarrhea, alleviate lactose intolerance, inhibit intestinal pathogens, and serve as probiotics (8). Probiotics are viable microorganisms that promote or support a beneficial balance of microbes to live in the gastrointestinal tract (5).

There are many strains/isolates of L.casei from different origins and geographical locations. That is why molecular typing of L.casei is crucial to understanding the evolutionary adaptation of this species to different ecological niches (1). Another reason for having L. casei's genome sequenced is to determine the phylogenetic relationships between various groups of bacteria in Lactobacillus. L. casei, L. paracasei, L. rhamnosus, and L. zeae form a closely related taxonomic group within Lactobacillus (3). Sometimes the classification L. casei is loosely applied to strains of any of these species by commercial companies. By having the genome sequenced, species boundaries could be drawn and names can be attached to those species (3).

Genome structure

NCBI: Genome

The genome of Lactobacillus casei strain ATCC 334 is composed of one circular chromosome and one plasmid. The chromosome has 2.9 million base pairs and the plasmid has 0.029 million base pairs. The chromosome encodes for 2,751 proteins and the sequencing was completed at the US DOE Joint Genome Institute and The Lactic Acid Bacteria Genome Consortium and Fidelity Systems, Inc. Currently, the genome of the plasmid of Lactobacillus casei is being sequenced.

One of L. casei's qualities is its ability to live in various diverse ecological niches. Research through comparative genomic analyses has suggested that extensive gene loss and gene acquisitions during the evolution of lactobacilli, presumably via bacteriophage or conjugation-mediated horizontal gene transfers have facilitated L. casei's adaptation to diverse ecological niches (1). The study used 40 different strains of L. casei showing that there is a high degree of recombination and phylogenetic diversity among the species.

Another feature in the genome of L.casei is the csp-A gene. This gene codes for a cold shock protein Csp A (66 amino acid residues) which allows the bacteria to adapt to low temperatures (11). (see Ecology)

Cell structure and metabolism

L. casei is a facultatively anaerobic organism that gets its energy through fermentation. Most L. casei strains can ferment galactose, glucose, fructose, mannose, mannitol, N-acetylglucosamine, and tagatose (1). The ability to ferment lactose is less common in strains isolated from plant materials than in those from cheese and human gastrointestinal tracts (1). The conditions of fermentation such as temperature, pH, the type of growth media, oxygen, and some neutralizers also play a role in the growth activity of L. casei (4).

The most important compound that L. casei produces is lactic acid. It is obtained by fermenting glucose and lactate formation. Lactic acid is a hydroxy acid that can be produced chemically from acetaldehyde and hydrogen cyanide or by microbial fermentation (2). It is used for numerous industrial processes such as chemical and biological production of organic acids, the use as a flavoring in food, the manufacturing of cosmetics, and the production of biodegradable plastics (2). (for more uses see Biotechnology)

Ecology

Lactobacillus casei has the ability to adapt to a variety of ecological niches. One of these niches is the gastrointestinal tract. L. casei functions as a probiotic in the gastrointestinal tract. Probiotics are originally defined as microorganisms promoting the growth of other microorganisms (5). The characteristics of a successful probiotic are acid and bile tolerance, antimicrobial activity against intestinal pathogens, and ability to adhere and colonize the intestinal tract (8). In order for the probiotics to carry out their functions, the probiotic live cells must not be lower than 10^6-10^7 cfu/g (9). The strains of L. casei that live in the intestines are sensitive to the intestinal conditions by having high bile salt concentrations and have the permeabilization and release of intracellular lactase to produce lactic acid (5).

L. casei is very important in regulating the immune system of the gastrointestinal tract. L. casei will bind to the luminal surface of gastrointestinal cells and stimulate gut-associated lymphoid tissue (6). This will strengthen the innate immune response and give local and systemic immunity to the body. To fight off the pathogens that may invade the immune system, L. casei can compete for nutrients or adhesion site against the pathogens. They can also inhibit the growth of pathogenic bacteria by a pH reduction through the production of organic acids such as acetic, propionic, or lactic acid, or by producing hydrogen peroxide (7). Furthermore, L. casei can secrete bacteriocins, antimicrobial peptides of cationic, amphiphilic molecules, to get rid of the pathogens in the body.

Another interesting characteristic of L. casei is its ability to adapt to colder temperatures, cold shock response. Research has shown that cold shock can cause a sudden growth stop or significantly reduced growth rate by decreasing membrane fluidity, and arrest or decrease of the synthesis of most housekeeping proteins (11). The cold shock would turn on the csp-A gene to make cold shock proteins (CSP A) to help the cell adjust to its colder environment. The research has also shown that CSP is needed not only for cold shock response, but for optimal growth in normal, unstressed cells (11).

Pathology

L. casei is generally considered nonpathogenic and safe. However, cases of sepsis, meningitis, and infections localized in organs have been reported (10).

Application to Biotechnology

Lactobacillus casei produces lactic acid which is used in various applications in biotechnology since it has numerous beneficial effects such as an increase in immune system response, a decreased risk for bladder cancer, and reduced cholesterol levels. Most of the biotechnology applications are related to the food industry.

Waste banana are bananas that cannot be sold on the international market since they might be undersized, have a spotty or marked peels, or had suffered mechanical damage. Instead of just throwing the bananas away, these waste bananas can be added with lactic acid which will increase its value and allow the farmers to diversify their harvest and receive higher income (2).

Fermented cabbage juice is another application to biotechnology. Cabbage is a cruciferous vegetable that is rich in minerals, vitamin C, dietary fibers, and phytochemicals (14). The cabbage juice added with lactic acid can be a healthy beverage for vegetarians and lactose-allergic customers.

Both of these applications, waste banana fermentation and fermented cabbage juice were not successful in using L. casei. In order for these biotechnology applications to work, the right substrate or food must be chosen for the lactic acid bacteria to grow in. For the waste banana study, there was not enough of the nutrients that L. casei needed for growth and production of lactic acid. For the fermented cabbage juice, L. casei could not survive over time in the low pH and high acidity of the cabbage juice environment.

Chocolate bars with lactic acid is one application that has proven successful. L. casei could survive in the chocolate bars and actually did not change the taste of chocolate. This healthy chocolate bar would be great for consumers, especially children, since it not only tastes good, it is also good for you too. (see Current Research)

Overall, for these applications for biotechnology to work for food products, the pH, availability of oxygen, water content, the presence of inhibitors and competing microorganisms, storage temperature, fermentation time, etc. need to be considered (14).

Current Research

The effect of milk fermented by yogurt cultures plus Lactobacillus casei DN-114001 on the immune response of subjects under academic examination stress

Psychological stress such as taking an exam can cause a decrease in the immune system response and increase the susceptibility to infection and disease. One way to counteract this suppression in immune system response would be to ingest lactic acid bacteria that are supposed to increase the immune system response. This idea was investigated by giving fermented milk to students attending a university in Madrid, Spain. The control group drank 200 mL semi-skimmed milk each day, while the experimental group had two 100 mL portions of Actimel (fermented milk with L. casei cultures) (6). This experiment was done over a period of 9 weeks, where 3 of those weeks were academic examination periods. The study showed that there was no decline of the immune system cells (lymphocytes, white blood cells, etc.) indicating that fermented milk with L. casei was able to stop the decrease in immune system response even during stress.

Dark chocolates supplemented with Lactobacillus strains

The use of lactic acid bacteria such as L. casei in foods would contribute to enhanced beneficial impact on human health due to its numerous health benefits. Putting the lactic acid bacteria into a dark chocolate bar would allow consumers to be healthy and enjoy chocolate. This would become a novel approach of manufacturing non-dairy products (9). This new and improved dark chocolate bar would have fewer calories and won't cause cavities so it can be consumed by diabetics. This study experimented with producing the chocolate bar and storing the chocolate bar to make sure that the lactic acid bacteria was still alive and functional over a period of 12 months. Then the bars were evaluated on sensory attributes (shape, color, texture, taste, etc.) Results show that for the chocolate bar to have its optimal lactic acid activity would be to store the bars in the refrigerator (around 4º Celsius). Also, the chocolate bars must not be exposed to oxygen while it is being made or it will kill the lactic acid bacteria. The study shows that lactic acid bacteria can be incorporated into chocolate bars without changing the taste of chocolate.

Kinetics analysis of growth and lactic acid production in pH-controlled batch cultures of Lactobacillus casei KH-1 using yeast extract/corn steep liquor/glucose medium

For the L. casei to grow and ferment lactic acid, the proper conditions for fermentation must be met such as temperature, pH, and type of growth medium. To produce a batch of L. casei in the laboratory would require yeast extract. Yeast extract is used for lactic acid fermentations but it costs too much. So in this article, a yeast/extract/corn steep liquor/glucose medium was used to grow L. casei. This growth medium is cheap and easy to harvest, but its effects on L. casei are unknown. Results show that the yeast extract/corn steep liquor/glucose medium is only good for production of lactic acid but not growth of the L. casei bacteria (4).

References

1. Cai, H., Rodriguez, B.T., Zhang, W., Broadbent, J.R., and Steele, J.L. “Genotypic and phenotypic characterization of Lactobacillus casei strains isolated from different ecological niches suggests frequent recombination and niche specificity”. Microbiology. 2007. Volume 153. p. 2655-2665.

2. Chan-Blanco,Y., Bonilla-Leiva, A.R., and Velazquez, A.C. “Using banana to generate lactic acid through batch process fermentation”. Applied Microbiology Biotechnology. 2003. Volume 63. p. 147-152.

3. Desai, A.R., Shah, N.P., and Powell, I.B. “Discrimination of Dairy Industry Isolates of the Lactobacillus casei group”. Journal of Dairy Science. 2006. Volume 89. p. 3345-3351.

4. Ha, M.Y., Kim, S.W., Lee, Y.W., Kim, M.Y., and Kim, S.J. "Kinetics analysis of growth and lactic acid production in pH-controlled batch cultures of Lactobacillus casei KH-1 using yeast extract/corn steep liquor/glucose medium. Journal of Bioscience and Bioengineering. 2003. Volume 96. p. 134-140.

5. Holzapfel, W.H., Haberer, P., Geisen, R., Bjorkroth, J., and Schillinger, U. “Taxonomy and important features of probiotic microorganisms in food and nutrition”. The American Journal of Clinical Nutrition. 2001. Volume 73. p. 365S-373S.

6. Marcos, A., Warnberg, J., Nova, E., Gomez, S., Alvarez, A., Alvarez, R., Mateos, J.A., and Cobo, J.M. “The effect of milk fermented by yogurt cultures plus Lactobacillus casei DN-114001 on the immune response of subjects under academic examination stress”. European Journal of Nutrition. 2004. Volume 43. p. 381-389.

7. Millette, M., Luquet, F.M., and Lacroix, M. "In vitro growth of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei- fermented milk". Letters in Applied Microbiology. 2007. Volume 44. p. 314-319.

8. Mishra, V. and Prasad, D.N. “Application of in vitro methods for selection of Lactobacillus casei strains as potential probiotics”. International Journal of Food Microbiology. 2005. Volume 103. p. 109-115.

9. Nebesny, E., Zyzelewicz, D., Motyl, I., and Libudzisz, Z. "Dark chocolates supplemented with Lactobacillus strains". European Food Resource Technology. 2007. Volume 225. p. 33-42.

10. Salvatore, S., Hauser, B., Devreker, T., Vierira, M.C., Luini, C., Arrigo, S., Nespoli, L., and Vandeplas, Y. “Probiotics and zinc in acute infectious gastroenteritis in children: are they effective?”. Nutrition. 2007. Volume 23. p. 498-506.

11. Sauvageot, N., Beaufils, S., Maze, A., Deutscher, J., and Hartke, A. “Cloning and characterization of a gene encoding a cold-shock protein in Lactobacillus casei”. FEMS Microbiology Letters. 2006. Volume 254. p. 55-62.

12. Takeda, K. and Okumura, K. “Effects of a fermented milk drink containing Lactobacillus casei strain Shirota on the human NK-cell activity”. The Journal of Nutrition. 2007. Volume 137. p. 791S-793S.

13. Ventura, M., Canchaya, C., Bernini, V., Altermann, E., Barrangou, R., McGrath, S., Claesson, M.J., Li, Y., Leahy, S., Walker, C.D., Zink, R., Neviani, E., Steele, J., Broadbent, J., Klaenhammer, T.R., Fitzgerald, G.F., O’Toole, P.W., and van Sinderen, D. “Comparative genomics and transcriptional analysis of prophages identified in the genomes of Lactobacillus gasseri, Lactobacillus salivarius, and Lactobacillus casei”. Applied and Environmental Microbiology. 2006. Volume 72. p. 3130-3146.

14. Yoon, K.Y., Woodams, E.E., and Hang, Y.D. “Production of probiotic cabbage juice by lactic acid bacteria”. Bioresource Technology. 2006. Volume 97. p. 1427-1430.

Edited by Alison Wong, student of Rachel Larsen

Edited by KLB