India’s probiotic controversy: Industry questions methodology used by researchers who criticised label claims
The study, involving researchers from Apollo Children’s Hospital in Chennai, Madras Medical Mission, and NITTE University, was published on the journal Cureus last month.
It claimed that there was “very poor correlation” between the declared contents on the pack and lab values in viable cell count colonies, the genus and species strain identification, and contaminants were detected.
The paper has since attracted significant attention from the industry in India, especially for the test protocol used.
“I understand the analytical method used in this article had limitations to ensure the right identification of organism and counts.
“Most leading products reported have validated label claims backed with clinical trials and are also produced by good companies that have strong GMP standards too,” Amit Srivastava, founder of idea-to-commercialisation consultancy Nutrify India said.
The study has used the sample and culture technique to count colony forming units (CFU) of the probiotics.
Depending on the contents declared on the sachet, one gram of the probiotic powder was first inoculated onto 5ml of liquid broth, yeast extract peptone broth, and thioglycolate broth
Serial tenfold dilutions up to 106 were made and plated on the de Man, Rogosa and Sharpe (MRS) agar or tryptic soy agar.
After incubation for 24 to 48 hours at 37-degree Celsius, the colonies were counted.
Twenty probiotics products which are the most prescribed in the country and had a minimum shelf life of 12 months were randomly selected for the study.
Of which, eight products contained single strain probiotics, while 12 were multiple strains.
The International Probiotics Association (IPA) technical and scientific committees, in response to queries from NutraIngredients-Asia, said while some products did seem to contain undeclared organisms, there were also flaws with the methodologies used in the study.
“Some products do indeed seem to contain undeclared organisms even at high levels. This is unacceptable and not in line with the IPA recommendations,” the committees said.
On the methodological flaws, first, the committees said the researchers have used one standard culture method for all organisms (check) when assessing the bacteria count, which it said, was an oversimplification.
“Different organisms may require different conditions.
“This may explain some deviations from the numbers reported on the label. However, the researchers did not explain major differences or even the absence of detectable viable organisms in products.
“This is not in line with IPA recommendations,” the committees said.
Second, a 48-hour incubation may not be enough time to allow for all cells to form colony forming units on the plate. This is especially the case when using a general method not tailored to the strain(s) present.
Third, the committees said that heat shocking the initial dilution for spore preparations may have the potential to yield lower recovery than heat shocking the final dilutions to be plated.
“Most probiotic spore methods heat shock at 70 to 75 degrees Celsius for 30 minutes, as opposed to the 80 degrees for 10 minutes used in the study,” the committees said.
In the study, the researchers had the first suspension of spore-forming bacteria incubated at 80 degree Celsius in a water bath for 10 minutes, before further dilution to kill vegetative cells and allow germination of spores.
The number of probiotic cells was then determined by spread plating 100 μL of each dilution on respective selective media, according to the label-claimed organisms.
The committees added that it was difficult to repeat and confirm the study done by the researchers.
This is because the researchers did not explicitly describe the various methods used for lactic acid bacteria, spore, and yeast and link them to the data produced.
“The correlation [between the method and data produced] is opaque, which makes the study difficult to repeat and confirm,” said the committees.
The committees also pointed out issues with the researchers’ choice of media.
On the use of MRS agar, the committees said the adding of L-cysteine HCL has shown to aid in the recovery of strict anaerobes, in turn, producing more accurate counts.
Whereas the use of Tryptic Soy Agar (TSA) is an acceptable media for spore enumerations, the committees said there were better growth media.
An example is the glycerol yeast extract agar (GYEA) which is used in the probiotic Food Chemicals Codex (FCC) monograph.
FCC monographs set ingredient-specific quality standards to guide food manufacturers.
The IPA committees also pointed out that there was a wrong citation used in the paper.
In the paper, the researchers said they have adopted methodology described by Aureli et al – which is stated as citation number 21 – to determine the number of culturable probiotic cells. The methods are used to analyse probiotic food supplements in Italy.
However, the actual citation made reference to an article written by Bizzini A et al instead.
“Seeing that this is supposed to be the reference of the cultural methodology used to obtain the quantities in table 4 [of the paper], this is a red flag that should have been caught during the editing/review process.
“Even if it was pointing to the correct reference, I would ask the authors why they chose the methods employed in a similar paper published in Italy, versus the interlaboratory validated, internationally recognised ISO methods,” the committees said.
The committees said while the researchers did mention that they have used ISO methods where available, the ISO that they eventually used was a general document (ISO 7218), and not the ISO probiotic method documents.
On the other hand, the committees said there was misnaming of probiotics, which could be attributed to the use of outdated taxanomy.
Examples include Lactobacillus sporogenes and Streptococcus fecalis, said the committees.
Lastly, the committees explained that the dosage claims made on the product labels were often times formulated from the strain manufacturer’s certificate of analysis (COA) using in-house methods.
As such, the committee said it would expect some discrepancies between the results of the study and the product labels.
“The paper has highlighted the need for enumeration methods that can be standardised and are thus not culture dependent, and this is something IPA has been promoting for years.
“This study highlights that IPA is on the right track with our initiative to develop standardised methods for the industry and to rally behind and follow collectively, in order to minimise these types of findings in the future,” the committee said.
Composition and Laboratory correlation of commercial probiotics in India
Authors: Dhanasekhar Kesavelu Sr, et al