As we discussed previously, probiotic viability and vitality are critical for ensuring functional probiotics — probiotics that do what they’re supposed to do once consumed (or applied topically). But how, exactly, does one optimize viability and vitality to ensure they’re producing the best performing probiotic strains possible?
There are two major factors that can be manipulated to ensure probiotic strains survive fermentation, concentration, preservation, storage, and passage through the upper gastrointestinal (GI) tract. While they aren’t the only factors that can be optimized, adjusting culture conditions and culture media can yield huge dividends for producing robust, highly active probiotic strains.
Probiotics production: optimizing culture conditions
There are a number of culture conditions that can be adjusted and tuned specifically for your strain(s) to ensure robust growth and metabolic activity. This is critical not just during the fermentation stage of production: the microorganisms must also survive the stressful spray- or freeze-drying process (i.e., concentration) as well as dormancy while on the shelf. Then, they must reassume normal metabolic activity once consumed.
Setting up the right culture conditions can ensure that your probiotic strain is as robust as possible and armed to survive concentration and storage once the fermentation process is complete.
Several different parameters can be optimized:
Fermentation time: During fermentation, bacteria undergo four major stages of growth: lag phase, exponential phase, stationary phase, and death phase. The duration of the fermentation process determines the phase that the majority of bacterial cells are in when the process stops. Most processes are stopped somewhere between the exponential and stationary phases, when the bacteria are robust and cell numbers are at their highest. However, metabolic activity changes throughout these stages, and the desired probiotic characteristics, such as the ability to adhere to intestinal epithelial cells, may not be at their highest during the exponential phase of growth. Therefore, producers must determine on a strain-by-strain and use-case basis the optimum fermentation time.
Fermentation temperature: Several studies have demonstrated that the temperature during fermentation has a significant impact both on total cell numbers and on probiotic resilience and ability to survive freeze-drying. While higher temperatures may lead to faster growth, they don’t always lead to higher total cell numbers and can negatively impact a probiotic strain’s ability to survive post-fermentation processes. And even if fermentation temperature does not negatively impact growth, metabolic activity changes at different temperatures — so fermentation temperature must be selected to support both viability and functionality of each specific strain.
Fermentation pH: Studies have shown that the pH during fermentation can impact the ability of rehydrated probiotic strains to survive low pH, such as that encountered in the human stomach, and high bile salt concentrations, which are found in the small intestine. However, research has also revealed that optimal pH for surviving acid and bile salt stress is non-optimal for surviving the freeze-drying process. Therefore, producers must determine the optimum pH for each strain that achieves the best balance between both survival parameters.
Agitation rate: Agitation during fermentation increases the probability of bacteria (or yeast) being exposed to key nutrients (carbon and nitrogen sources) in the media. However, not all fermentation processes are improved with agitation. Additionally, a faster agitation rate doesn’t always equate to healthier microorganisms. Agitation rate should be optimized for your strain and will likely also vary with pH, temperature, and even the nutrients present in the media.
Culture media is perhaps the most critical part in the fermentation process. Without the right nutrients, your probiotic strains will not grow and won’t be functional.
Optimizing culture media during probiotics production
Probiotic strains, the majority of which are lactic acid bacteria (LAB), have more complex nutritional requirements than typical laboratory strains, such as E. coli. E. coli, for example, has been grown in the nutrient-rich Luria-Bertani broth for decades, and this broth works well for all common laboratory strains used.
In contrast, LAB have more complex nutritional requirements, with riboflavin and acetate being two compounds necessary for growth for several species. Certain additives, such as cysteine and pantothenic acid can further boost growth for a number of species, including L. casei. Additionally, different LAB species, though closely related, can vary widely in their nutritional requirements. For example, while riboflavin is necessary for growth of several species, others can synthesize it and thus do not require it in the culture medium.
Regardless of their varying nutritional requirements, all LAB have one nutritional need in common: nitrogen. Nitrogen is a building block for ribonucleic acids, amino acids, and vitamins required for growth.
Nitrogen sources can be inorganic, such as ammonium salts, or organic, such as amino acids, proteins, or urea. Due to the high concentration of nitrogen in soil and plants, agriculture and food production by-products are a common source of nitrogen for industrial fermentation.
Yeast-based nutrients have also been added as a nitrogen source in bacterial growth media for decades due to its high vitamin, mineral, and nitrogen content. It has proven critical for influencing LAB viability and vitality. Yeast-derived free amino acids, peptides, nucleotides, vitamins, and micronutrients (i.e., yeast-based nutrients) can also be added to the culture medium independently or in ratios tailored to individual bacterial strains. In contrast to other nitrogen sources, yeast-based nutrients are animal-free, natural, non-GMO, allergen-free and cleaner.
Choosing your nutrients
There are many different combinations of nutrients that can be provided to your probiotic strains to optimize both viability and vitality. It is important to consider that the nutrients that optimize growth may not be the best nutrients for enabling probiotic strain(s) to survive post-fermentation production processes and passage through the human GI tract.
The key is choosing the right nutrients to maximize both, rather than maximizing one at the expense of the other.
A trustworthy, expert partner can be a critical tool for selecting the best nutrients to optimize survival during and post-production. Procelys has decades of experience producing high-quality yeast-based nutrients for probiotic strains, and the scientific expertise to help you select the right nutrients for your probiotic production needs. Contact us today to find out more about how we can support your probiotic production.
