Problem Based Learning Flashcards
Which are the most common bacteria present in probiotics?
Lactobacillus (phylum: Firmicutes) and Bifidobacterium (phylum: Actinobacteria) are the most common bacteria prevalent in probiotics,
Example species like L. acidophilus, B. bifidum
What are the most common phylum of bacteria in the gut microbiome?
Firmicutes and bacteroidetes
Describe phylum, class, order, family, genus, species and strain for Lactobacillus Plantarum 299V
Firmicutes
Bacilli
Lactobacilales
Lactobacillacea
How do environmental factors impact probiotics - what is the most optimal environment?
The optimal environment for GROWTH of probiotic bacteria:
temperature - usually around 37 °C
pH - slightly acidic environment pH 4-6
water activity - higher water activity, above 0.90
oxygen - some need low-levels of oxygen and some are anaerobic
The optimal environment for STORAGE of probiotic bacteria:
temperature - lower temperatures, often refrigerated at 4°C to reduce metabolic activity and prolong shelf life
pH - maintain stability
water activity - low water activity, below 0.30, achieved through freeze-drying (lyophilization)/spray-drying, inhibits reactions and microbial growth (bacterial growth inhibited when water activity is between 0-0.6
oxygen - minimal during storage (many probiotics are sensitive to oxidation), might be even packed in vacuum
How do you ensure a stable/viable/good starting culture?
Selecting robust bacterial strains and using high quality growth media, maintain sterile conditions. Using proper storage/handling procedures to prevent contamination and loss of viability.
What is an appropriate dose of these common probiotics?
The appropriate dose varies, but generally ranges from 1-10 billion CFU per day for most probiotic supplements. Common dose is 10^9 CFU/day. (CFU= colony forming units). And approx. 10^6 CFU/grams of probiotic food products.
What is an appropriate process chain for producing the common probiotics?
strain selection
culture maintenance
scale-up
*fermentation
harvesting
formulation
*stabilization
*packaging
storage
Which steps could be harming our products and should be extra controlled?
*fermentation
*stabilisation
*packaging
*critical points where viability can be compromised due to stress on the bacteria
Potentially problematic steps:
Inoculation: Do we have the correct starting concentration? Are they alive?
Centrifugation: Do we have particles that lyse bacteria during centrifugation in the media?
Freeze drying: Can our strain handle freeze drying?
Exiting factory:Last chance to easily find a bad batch
Transport: Are the products stored at an acceptable temperature? Integrity of the primary packaging.
What should be tested in the process controls?
regular sampling
testing for viability
pH monitoring
temperature checks
moisture checks
contamination checks
Oxygen consumption (P02)
Glucose consumption
What methods could be used for characterization and to identify the probiotics used?
To identify and characterize specific strains of probiotics:
molecular techniques (PCR, DNA sequencing)
culture-based methods (selective media)
sanger sequencing
BLAST
Gram-staining methods
Viability and quantification:
qPCR
ATP measuring
flow cytometry
microscopic analysis
What legislation exists regarding probiotic products?
Legislation varies by region and usually includes guidelines, safety and GMPs. EFSA in Europe (European food safety authority) provides guidelines, FDA in the US (food and drug administration) regulates probiotics as dietary supplements.
What can we learn from faecal samples, and what analysis methods can we use?
Gut microbiota composition, 16S sequencing to identify different microbial species both what species and the abundance.
Identification of potential biomarkers.
Culturing - looking at live bacteria
Gene based methods; PCR or qPCR if we want to quantify.
Inflammatory markers can be found by doing ELISA and PCR.
What information about the microbiota do we “miss” when we do fecal sampling? What additional samples do we need to complete the info?
Miss the upper GI tract, we can do a saliva sample to test the microbiota in the mouth. Hard to get representative samples from the entire GI tract. Due to spatial differences the microbiota can differ. Depending on the time you take the sample it can affect the microbiota composition.
Blood samples can give information of microbiota in the blood.
If the microbes are in low abundance or are rare they can easily be missed.
Biopsy can maybe be relevant for the tissue in the cells in the mucosa, or ultrasound or in vivo methods, not looking at the microbes but gives information of how the intestines are doing.
What are the requirements for an in vivo study to be representable?
Includes a statistically significant amount of population to be representative for the group on which we do the experiment. Clearly described and easily repeatable - reproducible.
Aim for as high variety in the group as possible within the investigated group. Diversity within the group or limiting the group. Also needs a control group. Double-blind placebo is good.
If we use an animal model we need to choose wisely if it is going to be applicable for humans. A standardized procedure, and good written records. If something is an outlier, then it can be found.
Clear instructions for the participants so it’s possible to be repeated. Easy instructions to follow.
What is an allergic response?
An immune response to a harmless allergen
