Comparing the Effect of Different Peptones in Media Formulations on Probiotic Bacterial Growth

Peptones are proteins that have been partially digested by an enzyme, and are used  in bacteriological media formulations to provide a source of nitrogen and carbon in the form of peptides and amino acids.

The source of protein, the enzyme(s) used to digest the protein, as well as the method of hydrolysis used in production may differ among peptones. Companies that produce peptones must meet consumer needs and provide peptones to fit varying demands, such as being animal or animal by-product free, kosher, non-GMO, or free of certain allergens.

Along with the variation in composition of these peptones comes the question of performance: What effect does using these different peptones have on bacterial growth?

Methods:

  • Cultures were grown in liquid media (see media table) using the different peptones shown in Table 1.
  • Amount of peptone used was based on the percent total nitrogen in the peptone.
  • Culture growth rates were monitored by measuring the Absorbance (OD600) of a sample using a Cary300 spectrophotometer with expanded sample carriage.
  • All bacterial cultures were propagated at 37°C in a ~5% CO2 atmosphere or in micro-aerophilic conditions using an anaerobe GasPakTM (BD, New Jersey).

Table 1. Peptones Used in this Study

Peptone % TN AN/TN
Soy A 7.6 0.41
Soy B 9.2 0.21
Soy C 9.2 0.25
Soy D 10.2 0.24
Pea 12.4 0.40

Probiotics:

  • Many types of bacteria in the gut are necessary for our health “live micro-organisms, which when consumed in adequate amounts, confer a health effect on the host” – WHO/FDA
  • Can help balance the gut microbiome; Imbalances in the gut microbiome have been shown to cause disease
  • Of interest to the food industry as additives to promote overall health
  • Examples are:
    – Bifidobacterium infantis
    – Lactobacillus acidophilus
    – Lactobacillus rhamnosus
    – Lactococcus lactis
    – Streptococcus salivarius

Media Recipes:

Lactobacillus sp.

MRS Base:

(2X Solution, 500 mL)
8 g Beef Extract
4 g Yeast Extract
20 g Glucose
1 mL Polysorbate 80
2 g K2HPO4
5 g NaAcetate – 3H2O
2 g Ammonium citrate
0.2 g MgSO4 – 7 H2O
0.05 g MnSO4 – 4 H2O
-Autoclave, then mix 1:1 w/ 0.2 % w/v TN peptone

B. infantis

Reduced Media:

(For 100 mL media)
50 mL Peptone solution
5 mL 10% glucose
20 mL 5x M9 salts
5 mL 10 mg/mL cysteine solution
20 mL sterile H2O
-All components sterilized separately.

Conclusions:

  • Different bacterial species exhibit differing growth behavior based on the nutritional profiles available.
  • L. acidophilus displays growth rate dependence on the yeast extract as well as peptone source.
  • Substituting YE for peptone in MRS media limits growth rate and overall yield of L. acidophilus

Future Work:

  • Work with other strains of probiotics
  • Work with new species of bacteria
  • Metagenomic expression study

References:

BD Bionutrients Technical Manual. 3rd ed. N.p .: Beckton, Dickenson, 2006.
Butel, M. J. (2014). Probiotics, gut microbiota and health. Médecine Et Maladies Infectieuses, 44(1), 1-8.

Acknowledgements:

  • Donato Research Lab
  • UST Chemistry Department
  • Nu-tek Bioscience

Jennifer FitzGerald, Gemma Shaffer, Hailley Cound, Maddy Riemenschneider and Thomas C. Marsh, Ph. D, Chemistry Department, University of Saint Thomas, St. Paul, MN