Lactobacillus casei: Expression of genes during the Stationary Phase

Cheese,-Milk,-Yogurt--Probiotic-Food

 This website is based on  Wang’s 2012 article Gene expression profile of probiotic Lactobacillus casei Zhang during the late stage of milk fermentation.

Secrets Behind Long-Healthy Lives:

  • Many people want to live a long-healthy lives, but what does it take to stay fit and healthy?
  • Amazingly, in 1900s, Noble Prize-winning Eli Metchnikoff hypothesized long, healthy lives of Bulgarian peasants were due to their consumption of fermented milk products.  These fermented products such as yogurt are referred to as probiotics, which contain beneficial bacteria that protect the “gut” from the harmful bacteria.
  • What are health benefits of lacto-fermented food (yogurt, fermented milk and cheese)? Various health benefits from probiotic consumption: Improves intestinal health. Inhibit pathogens, such as E.Coli or Clostridium perfingens. Restores mucous lining of the “gut”. Helps in the relief of anxiety and depression (enzymes play a huge role in gut-serotonin production!). Stimulates the immune system. Reduces symptoms of lactose intolerance- breaks down sugars, starches, and lactose for easier digestion.  Most importantly, it keeps your digestive system fit and active!probiotics_en
  • What is a probiotic?
    • Probiotics literally means ‘for life’, are micro-organisms such as bacteria that have health benefits in humans.
    • Various strains of Lactobacillus bacteria are widely used as probiotic.

     

  • The genus Lactobacillus
    • Various different strains- focus in on Lactobacillus casei
    • Gram-positive, non-spore forming, aerotolerant anaerobe
    • Generally regarded as safe and provide health benefits as probiotic
    • Normally found in fermented food (dairy products- yogurt, fermented milk) and naturally present in the small and large intestine

     

    Fig1: Lactobacillus casei- One of the most clinically studied probiotics in the world. Image from yakultusa.com

    Fig1: Lactobacillus casei- One of the most clinically studied probiotics in the world.
    Image from yakultusa.com

  • Lactobacillus casei
    • Lactic acid bacteria (LAB)
    • Found in raw and fermented dairy and plant materials
    • Also found in the gastrointestinal tracts of humans and animals (high tolerance to simulated gastric, intestine juices and bile salts)
    • Does not directly use lactose as a carbon source

  • What is fermentation? Fermentation is the process where, in the absence of oxygen, a glucose molecule or other carbohydrate sources are converted into cellular energy and the metabolite lactate.Fermentation
  • Bacteria Growth Curve

bacterial growth curve

    • Logarithmic (exponential phase) is marked by rapid growth
    • Stationary phase is where is limited or no growth due to depletion of essential nutrient
    • Several studies show that bacteria from logarithmic phase are more susceptible to environmental stresses compared with bacteria from stationary phase

 

  • Goal of the study: To identify genes whose expression patterns are altered in the stationary phase in comparison to the late logarithmic phase.

Results:

  • Growth and fermentation characteristics of L.casei Zhang strain in milk incubated at 37◦C.
growth of L.casei Zhang in milk

Fig 2: Growth and Fermentation Characteristic in L.casei. The bacteria start to grow at the time of 3h and grow from the logarithmic phase into the stationary phase at the time of ~16h. During fermentation, there was gradual decrease in pH in the fermented milk which could be explained by sugar in milk being converted into lactic acid by L.casei cells.

  • Expression profiles of L.casei Zhang during growth in milk
    • During stationary phase, where there is depletion of nutrient (glucose), there are certain genes that are up-regulated while others are down-regulated.
    • In the stationary phase, there are 87 differentially expressed genes in the stationary phase compared to the exponential phase.  Of the 87 genes, 61 of them are up-regulated and 26 are down-regulated.
    • Several of the up-regulated genes are involved in carbon metabolism and energy production.  These up-regulated genes encode for transcription, inorganic ion transport, chaperones, and proteins.
    • Genes that are down-regulated are those that encode for nucleotide transport and metabolism, inorganic ion transport and metabolism, and energy production and conversion.

Discussion:

  • Milk-based products are often used as a vehicle for delivering probiotics.  L.casei Zhang is a lactic acid bacterium (LAB) that is used as probiotics in China.  The physiological state (the logarithmic or the stationary growth phase) of the probiotic organisms is a contributing factor that influences the ability of the probiotic to survive in the product and become active in the human gastrointestinal (GI) tract.  In industrial fermentation, to ensure high cell numbers, probiotic and starter strains are harvested in the late exponential or the stationary growth phase. 
  • Depletion of glucose creates stress and this result in activation and repression of various genes during the stationary phase.  There are different types of genes that are expressed in the stationary phase compared to the exponential phase of cultivation.  During depletion of glucose in the stationary phase, L.casei Zhang uses alternative carbon sources such as sucrose, glycerolipid, glyoxylate, and mannose, which is indicated by overexpression of these genes.  In addition to switching from the use of glucose to alternative carbon sources, L.casei Zhang also alters its pyruvate metabolism.  Elevated expression of genes that encode for pyruvate dehydrogenase indicates that there is greater production of energy from glucose, which is used to sustain the cell growth under scarce glucose conditions.
  • While many genes are up-regulated during the stationary phase, certain genes are down-regulated such as genes within the citrate fermentation pathway.  These genes are repressed to facilitate lactate outflux thereby enhancing cells resistance to the inhibitory effect of lactate accumulation. 
  • A good approach in understanding the way L.casei Zhang grows and survives in the late stage of milk fermentation, is to know the effects of glucose depletion on the transcriptome.  Studying transcriptome of L.casei Zhang gives an excellent overview of the changes that occur inside a stressed bacterium: up-regulation of genes encoding amino acid transport and metabolism, carbohydrate transport and metabolism, lipid transport and metabolism, inorganic ion transport and metabolism.  
  • Understanding which genes are up-regulated and down-regulated during the stationary phase can influence the ability of the probiotic to survive in the product during storage and it can also have economical benefit in industrial applications.

Criticism about the Paper:

  • Wang, et al. nicely differentiates changes in gene regulation between logarithmic and stationary phases.  It would have been better if the paper elaborated on the benefits of having certain genes upregulated under stress (glucose depletion).  The paper doesn’t tie the results into how it would benefit human or yogurt production.

“Life is half delicious yogurt, half crap, and your job is to keep the plastic spoon in the yogurt.” -Scott Adams

References:

Parvez. S., et al. (2006). Probiotics and their fermented food products are beneficial for health. Journal of Applied Microbiology. 100, 1171-1185. Link

Rallu, F., et al. (1996). Lactococcus lactis and stress. Antonie Van Leewenhoek. 70, 243-251. Link

Wang, J., et al. (2012). Gene expression profile of probiotic Lactobacillus casei Zhang during the late stage of milk fermentation. Food Control. 25, 321-327. Link

 

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