Microbiologist Duane Sowards analyzes probiotic samples for enumeration at the Eurofins Microbiology Laboratory Center of Excellence for Probiotic Testing. Photo courtesy of Eurofins Scientific
Microbiologist Duane Sowards analyzes probiotic samples for enumeration at the Eurofins Microbiology Laboratory Center of Excellence for Probiotic Testing. Photo courtesy of Eurofins Scientific
Defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host, probiotics are being used in numerous dietary supplements and food and beverage products. Research on the potential health benefits of probiotics has focused on such health conditions as pediatric acute infectious diarrhea, antibiotic-associated diarrhea, irritable bowel syndrome, hypercholesterolemia, obesity, and others. Many strains of bacteria have been shown to have a health benefit, but the benefit depends on the specific strain and dose as well as the number of live cells at time of consumption. The microorganisms most often used in probiotic products are Lactobacillus, Bifidobacterium, Saccharomyces, Streptococcus, Enterococcus, Escherichia, and Bacillus. Since probiotics don’t colonize the gastrointestinal tract, they must be consumed daily.
Lactic acid bacteria (LAB), such as Lactobacillus and Bifidobacterium, don’t survive processing that requires heating to a high temperature, so they are commonly used in refrigerated or frozen products (primarily yogurt). They are also used in shelf-stable food products such as dry cereals if very dry, low– water activity conditions are maintained. On the other hand, spore-forming microorganisms such as Bacillus can survive heat processing and therefore can be used in a variety of processed food products.
A probiotic intended to be used as an ingredient in a food product must either be approved by the U.S. Food and Drug Administration as a food additive or be generally recognized as safe. While a food containing probiotics can’t make a health claim (i.e., that it treats, cures, or prevents a disease), it can make a structure/function claim (i.e., that it maintains normal functioning of the body) if appropriate scientific substantiation is available. It can also state that it includes probiotics or live and active cultures.
DuPont Nutrition & Biosciences is the major manufacturer of LAB probiotics, which it markets under the HOWARU trade name. Vanessa Bailey, global marketing leader, DuPont, said that the food and beverage category has historically been a few steps behind dietary supplements in terms of probiotic strain variety and potency. Consumers are becoming increasingly aware of the health benefits of probiotics, and this has created demand for new formats besides capsules. DuPont has increased its efforts to understand how different probiotic strains survive in different food formats, especially those that align with current food trends and consumption habits such as increased snacking and eating on the go. The end goal is to deliver a product that consistently provides a clinically studied dose of probiotics in a delicious and convenient format. Bailey expects to see more and more foods and beverages with new probiotic strains and health benefits hitting shelves as knowledge about strain stability and new formats increases.
The challenge is to find ways to ensure that an efficacious dose of probiotic strains is delivered all the way to the end of the shelf life. Bailey said that HOWARU is one of the largest portfolios of clinically studied strains as it contains more than 600 published clinical studies providing more options for potential health benefits and formats. In addition to digestive health benefits, DuPont has clinical results to also support immune health, women’s health, and weight management. Bailey added that dietary supplement brands are already selling probiotic products with health claims that go beyond digestive health and she expects that food isn’t far behind.
Kellogg’s Special K Berries & Peaches with Probiotics cereal says it contains live and active probiotic cultures.
Kellogg’s Special K Berries & Peaches with Probiotics cereal says it contains live and active probiotic cultures.
In July 2019, DuPont opened a new state-of-the-art probiotics fermentation unit, said to be the largest fermenter in the world dedicated to probiotics production. DuPont’s range of probiotic strains can all potentially be used in foods and beverages, Bailey said, but certain strains have shown better stability in food and beverage formats. The company works closely with its customers to understand what their end goal is in terms of benefits, claims, and formats and can make recommendations on how to formulate products and handle the strains in their production facility. Most of the probiotics are provided in a powdered concentrate form, but in some cases frozen pellets or concentrate preblended with an excipient may be more convenient.
Kerry ProActive Health is the major manufacturer of spore-forming probiotics. Don Cox, research and development director, Kerry, said that Ganeden BC30 (the trade name for the spore-forming strain Bacillus coagulans GBI- 30 6086) was selected through an extensive probiotic screening process, and more than 25 published papers have demonstrated its positive effects on digestive health. He said that other companies also offer spore-forming probiotics but that Kerry is the only company with as much published research. A study published in Food Research International in September 2019 showed that Ganeden BC30 has beneficial effects on gastrointestinal symptoms and upper respiratory tract infection symptoms in children. Cox said that while there is already a large body of evidence for the digestive and immune health benefits of the probiotic, this research is the first to focus on children and shows the potential of Ganeden BC30 as a functional ingredient in products for children.
Kerry’s probiotic is manufactured in a proprietary process that starts by growing the cells that create spores. The spores survive most food processing conditions, including shear, hightemperature, and high-pressure processes, enabling manufacturers to integrate probiotics into foods and beverages that do not necessarily require refrigeration or freezing. The spores’ shell protects them from stomach acid and bile and allows them to survive transit through the gastrointestinal tract and be recovered as vegetative cells.
Kerry provides the probiotic to food and beverage manufacturers as a freeze-dried powder blended with additives (such as inulin or milk powder) that serve as food for the bacteria. The company works with its customers to determine where the probiotic should be added in the food and beverage manufacturing process and requires each customer to submit a finishedproduct sample to Kerry to confirm that the correct count of probiotics is present. Kerry developed a Food Chemicals Codex monograph describing the method for enumerating B. coagulans GBI-30 6086.
Cox said that label claims such as “supports immune health,” “supports digestive health,” and “supports protein utilization” can be made on product labels, provided that the probiotic is live when consumed, has been shown to be efficacious by research studies, and provides a specified number of live cells per day. For example, to make a simple claim that the product contains a probiotic, the product should meet the definition of a probiotic. If the label identifies the probiotic by brand name (e.g., Ganeden BC30) or strain (Bacillus coagulans GBI-30 6086), the product must provide 500 million cfu/day for a probiotic claim or 1 billion cfu/day to say it supports digestive health. Cox said that meeting the growing consumer demand for probiotic products will require a wide range of stable product offerings. The more products there are, he said, the more likely consumers will routinely consume the efficacious amount of probiotic.
Andrzej A. Benkowski, technical leader at Eurofins Microbiology Laboratories, answered questions about probiotics testing.
Q: What are the most recent developments regarding the formulation and analysis of foods containing probiotics?
A: Formats are becoming more and more complex, leading to challenges when testing for enumeration and/or identification. Incorporation of probiotic strains with other active ingredients, addition of blends of strains, and use of microencapsulated strains are examples of the complexities that enumeration laboratories face when testing for probiotic potency. Guidance documents have been, and are being, developed by multiple trade associations in an attempt to get everyone on the same page for the best ways to manufacture and test probiotic products. Genotypic methods for identifying multiple strains in a blend differ from, and are often more involved than, those used to identify a single-strain product.
Dannon’s Activia yogurt says it provides billions of live and active probiotics in every cup.
Dannon’s Activia yogurt says it provides billions of live and active probiotics in every cup.
Q: What approach does Eurofins take with regard to probiotic testing?
A: There are two main concerns: ensuring that products are safe and confirming that they provide the expected dose of probiotic microorganisms. However, this is complicated by the need for speed and the risks associated with using inappropriate methods. Ensuring safety by detecting pathogens in probiotic preparations, whether by rapid pathogen detection platforms or by traditional cultural methods, can be challenging. United States Pharmacopeia methods are often appropriate for probiotics because method suitability is demonstrated for each new formulation, but these are traditional cultural methods and not particularly fast. Eurofins has validated methodologies to detect pathogens in probiotics using rapid techniques, which is particularly important for functional foods when shorter shelf lives require faster turnaround times, and has published and presented extensively on probiotic methods.
Confirming the expected dose involves enumeration. There are numerous traditional enumeration methodologies because each probiotic producer has methods optimized for their particular strains, and this introduces a lot of complexity for a testing laboratory. An extra degree of precision is required to help mitigate some of the intrinsic variance associated with the methods. In the past several years, flow cytometry has been making some inroads into enumeration of probiotics because it provides a faster result than traditional plating methods and can indicate the proportion of live, injured, and dead cells in a probiotic preparation. Using an inappropriate method for probiotic enumeration, such as a quantitative microbial contaminant test, can lead to inaccuracies in the counts, resulting in production issues and delays downstream.
Q: Does your approach for testing spore-forming probiotics differ from those for testing vegetative probiotics?
A: Yes, although there are many similarities. With regard to cultural enumeration methods, the principle is consistent whether testing a spore or a vegetative cell: add a sample to a diluent and homogenize, serially dilute, plate, grow, and estimate a result based on the colony counts. Spore-enumeration methods typically incorporate a heat-shock step that will kill off any vegetative cells present. Since probiotic spores can survive food production processes, a wider variety of matrices are being tested for spore enumerations, potentially requiring additional method scrutiny to ensure recovery. Highly concentrated probiotic spore preparations also require additional aseptic preventive measures to minimize the risk of crosscontamination due to their survivability. This is especially important when a laboratory is testing for both probiotic potency and the absence of microbial contaminants. Spatially separating these tests is critical to minimize risk, and Eurofins has a dedicated probiotic laboratory for that purpose.
Q: What methods and instruments is Eurofins using for enumeration and identification of probiotics?
A: Our workhorse methods continue to be cultural enumerations. The suite of methods continues to expand as companies tailor methods to their portfolio of functional food products. We use flow cytometry for enumeration and have expanded its capability to spores and spore blends. We have also been using polymerase chain reaction and whole genome sequencing for identification testing of strains and blends, respectively. Eurofins also has extensive capability in genomic testing, including microbiome services and expertise in method development and validation.
Q: The Eurofins website says that it offers clinical trial support. Does that include clinical studies with foods that contain probiotics?
A: We offer enumeration support for the probiotic product consumed or applied during the course of a clinical trial by confirming stability/efficacy over time. A structure/function claim on a probiotic label is usually established on an efficacious concentration of a particular strain or strains. It is important to maintain stability of the strains through the shelf life of the food to confirm efficacy and substantiate the claim.
Q: What are the major challenges ahead with regard to foods and beverages that contain probiotics?
A: The sheer number of cultural enumeration methods out there is one of the major challenges for testing laboratories as methods become more and more customized to the matrices being tested. For example, we may have four or five different variations of the same method that are tailored to and validated for a particular food or beverage matrix containing the same strain of probiotic. I expect this trend to continue as more and more formats are being utilized in conjunction with new strains being found, so-called next-generation probiotics that will add even more complexities to the operation. Regulatory harmonization when it comes to label claims is and will continue to be a challenge when selling probiotic foods and beverages in the international market. Improving method precision is an ongoing focus of our continuous improvement program. There is also an educational aspect to the challenges industry faces as more and more companies enter the growing probiotic marketplace. I can’t stress enough the importance of understanding the unique properties of these living microscopic organisms compared to traditional dietary ingredients, how to keep them alive and viable over time, and how to test them and make sure that the label reflects the composition of the product.
