By Mike Montemarano, Associate Editor04.08.24
From strain discovery to new product development, manufacturing of probiotics is a complex process. Quality controls and best practices at several stops along the R&D and commercialization pipelines aim to deliver consumers best-in-class dietary supplements that provide measurable health benefits.
Probiotical’s in-house research laboratory has a suite of discovery and development tools, including strain sequence profiling, bioinformatic probing, and pilot strain production capabilities, Almada said. The company’s pilot fermentation experiments also enable it “to model and predict scalability and stability, and to align with a strong pre-clinical science package,” he added.
Strain discovery begins with research on a non-pathogenic species usually isolated from human, animal, or plant sources. Many strains are also selected through third-party research on species used in other areas of the food industry, said Nena Dockery, scientific affairs manager at Stratum Nutrition.
There are many literature reviews to investigate, and researchers to collaborate with, to identify strains with sought-after health benefits, said Jordi Riera, chief business officer at Kaneka Probiotics. “Then the data is organized and prioritized based on factors such as health benefits, stability, formulation, and demand.”
ADM’s library of more than 500 microbial strains goes through a pre-clinical screening program, which the company’s team of scientists use to complete DNA sequencing and understand the genes that are present in the different microorganisms. “From there, the strains go through pre-clinical models to characterize them further before moving to human clinical trials,” said Vaughn DuBow, global director of marketing at ADM.
After identification of potential benefits, strains need to be tested to ensure they have sufficient baseline resistance to gastric acid and bile salts, adherence and interaction with intestinal mucosa, and some interaction with immune factors or antagonistic activity toward pathogenic microbes, said Dockery.
“Advances in culturomics and metagenomics have enabled probiotics developers to accurately identify and distinguish strains from tissue samples taken from both healthy and diseased human subjects,” said Dockery. “And, because of the great diversity in human gut, oral cavity, and skin microbiomes, this opens the door for a dramatic increase in the source for probiotic strains.”
Dive Deeper: Microbiome Modulation: Emerging Probiotic Benefits
“Production of commercial probiotics must be at the strain level, not just the species level. And this has led to the realization that strain-specific sensitivities, such as nutrient requirements and ideal pH and temperature conditions, which impact production efficiency, are also present,” Dockery said.
Therefore, manufacturers have been challenged to identify those strain-dependent sensitivities during production, she added, from cultivation to drying, and to develop strategies for overcoming them while maintaining sustainable production.
“The biggest developments in probiotic production stem from the need by manufacturers to have dependable production methods that produce precise bacterial strains that are robust and stable throughout a shelf-life that extends beyond a few months,” Dockery said. “Novel technologies have been developed to improve cell counts of desired bacterial strains during fermentation, enabling better production efficiencies by increasing bacterial load and decreasing fermentation time.”
Almada pointed to two distinctive areas of improvement: cryoprotectants and culture optimization. “What was once limited to [substrates of] skim milk powder and vitamin C, and often undeclared animal and allergen-rich derivatives, has evolved into what can be implemented as a systematic and sustainable program of producing probiotic strains,” he said.
Advancements in protecting fermented strains from harsh freeze-drying conditions and validating best-in-class, clean-label growth/fermentation media are areas “often overlooked,” he added.
Keep Reading: Prescription Probiotics and Microbiome Modulation
Certain strains have higher natural resistance or tolerance to the harsh conditions of the GI tract, said Riera, and Kaneka has prioritized selecting these strains in order to work with delivery formats beyond capsules.
Tests to evaluate the performance of an encapsulation technology within the digestive tract continue to gain sophistication. Many technologies have been tested using SHIME (Simulator of the Human Intestinal Microbial Ecosystem) for greater insight on the timeline of a product’s digestion.
Lonza’s Capsugel DRcaps designed-release capsules are a vegetarian solution that targets release “approximately 45 minutes after a standard gelatin-based capsule,” is taken said Zain Saiyed, PhD, FACN, director of research and development, dosage form solutions, at Lonza.
“These capsules are robust enough to help protect dietary supplement ingredients from degradation in the low pH environment of the stomach,” he noted.
For another option that can combine probiotics with otherwise incompatible ingredients, “capsule-in-capsule technology offers endless customization and innovation potential,” said Saiyed. In the case of probiotics, live bacteria can be incorporated into a smaller capsule that is inserted into a larger liquid-filled capsule containing other complementary ingredients.
“A study investigating the release behaviors of several different Capsugel Duocap capsule-in-capsule combinations revealed that a mix of Lonza’s designed release capsules, DRcaps capsule in a DRcaps capsule, exhibited the potential for the slowest possible release profile, indicating they can be used to effectively target delivery to the colon.”
Riera noted that organic, oily suspensions “can enhance the survival of probiotics in the GI tract, and also offer the potential for combining fat-soluble vitamins with probiotic strains, which opens up new avenues for synergistic health benefits.”
Probiotical developed and patented Microbac, a technique of encasing microbial cells within a vegetable lipid matrix, said Almada. “We undertook comparator clinical trials and showed a five-fold greater delivery and viability in the stool relative to uncoated cells.”
To prove stability, Riera said key parameters include CFUs of bacteria across a range of conditions (including different climate zones), as well as how stable bacterial strains are in various steps of the production process, like in cold chain and in the context of varied water activity levels.
“Compatibility studies with different combinations are important to analyze, Riera said, as well as combinations of our proprietary probiotic strains with vitamins, minerals, fibers, and other active ingredients.”
As with many other ingredients, manufacturers often include overages of certain bacteria to ensure efficacious dosages through shelf life. But that’s not a one-size-fits-all solution, said Riera, as each strain will have a unique stability profile.
The standard way of determining the quantity of live viable cells in a supplement is through plate counting of bacterial colonies, but this technique has limitations and can’t always be used for heterogeneous blends of strains. Flow cytometry, which is suitable for such combinations can also distinguish between live, damaged, and dead cells.
“Other methods, such as fluorescence in situ hybridization (FISH) and molecular techniques based on the detection of nucleic acid sequences are beginning to gain attention for the ability to differentiate individual microbes, at least to the species level,” noted Dockery.
Pointing to recently-published comparisons of various counting methods for probiotic strains, Almada said: “We have pioneered flow cytometry, and the associated testing values of active fluorescent units (AFU) and total fluorescent units (TFU) to validate the presence of viable cells and ensure the adequacy of input addition in the monitoring of manufacturing and shelf-stability over time, respectively.”
“Probiotical developed AFU-based models to determine the predictive stability of probiotic compositions, which can be achieved within a few months of serial evaluations,” he said. “This can accelerate the path to market and contract the launch cycle.”
“The introduction of novel probiotic species, such as strict anaerobes or postbiotics, presents additional challenges and necessitates a consensus in the industry on the most appropriate counting methodology,” he said. “These new species may have unique requirements that current methodologies are not equipped to accurately assess.”
Probiotics are live microorganisms interacting with the gut microbiome, and as such, their behavior and stability can’t be fully accounted for through traditional chemical analysis, Riera added.
The introduction of novel fermentation media will also present opportunities. “The bulk of research in alternative growth mediums for probiotic bacteria has come within the context of the food industry,” said Dockery, “leading to evidence that there can be a symbiotic relationship between select plant components and probiotics, which can interact favorably with the gut commensal bacteria. This has led to the development and introduction to the market of several non-dairy probiotic beverages, frozen desserts, and vegan milk replacements.”
Microbial strains don’t always “play well together,” since they might have overlapping functions or may have antagonistic relationships. “Traditionally, manufacturers have combined individual probiotic strains together to produce multi-strain formulations without much thought regarding the impact that the strains might have on each other … The practice has continued because methods for differentiating the strains by cell count after production are still limited,” Dockery said.
New strategies, however, involve the use of immobilization and carrier molecules to enable efficient and efficacious production of multi-strain probiotics, she added.
It can be challenging to articulate the full scope of mechanisms at play in multi-strain formulations, noted Almada, offering Probiotical’s four-strain Bifizen ingredient as an example.
“Our rational, in vitro investigations pointed to a variety of biological activities: controlling a dysbiotic state and intestinal permeability, exerting a positive role on inflammation and oxidative stress, and even a strain that is an overproducer of GABA. We then subjected this quartet of microbes to three distinct, controlled clinical investigations, which demonstrated effects upon behavior, mood, sleep indices, and even circulating serotonin. Although we have not yet demonstrated an unambiguous role of the gut as the mediator of these behavioral and mood changes, we feel confident that our rational approach yielded a compelling and ever-expanding evidence base.”
Probiotics can’t simply be combined with any ingredient, Almada noted. “Many natural products, especially of botanical origin, possess mild bacteriostatic/antimicrobial activity.”
ADM’s spore-forming probiotic, DE111, can retain functionality through challenging formulation environments that may otherwise damage conventional probiotics, according to DuBow. “On top of that, DE111 is backed by studies showing that it may support digestive health and may help support healthy immune function.”
While spore-forming probiotics resolve many challenges, they may create others, Dockery noted. “These tough bacterial species can be a cleaning challenge for a manufacturing facility since they are so resistant. Their applications for human health are also more limited than strains that are derived from human sources.”
About the Author: Mike Montemarano has been the Associate Editor of Nutraceuticals World since 2020. He can be reached at mmontemarano@rodmanmedia.com.
Probiotic Strain Discovery
The pace of probiotics research has been “meteoric,” said Anthony Almada, MSc, president and chief science officer at IMAGINutrition and scientific expert at Probiotical. New strain discovery requires deep scientific expertise.Probiotical’s in-house research laboratory has a suite of discovery and development tools, including strain sequence profiling, bioinformatic probing, and pilot strain production capabilities, Almada said. The company’s pilot fermentation experiments also enable it “to model and predict scalability and stability, and to align with a strong pre-clinical science package,” he added.
Strain discovery begins with research on a non-pathogenic species usually isolated from human, animal, or plant sources. Many strains are also selected through third-party research on species used in other areas of the food industry, said Nena Dockery, scientific affairs manager at Stratum Nutrition.
There are many literature reviews to investigate, and researchers to collaborate with, to identify strains with sought-after health benefits, said Jordi Riera, chief business officer at Kaneka Probiotics. “Then the data is organized and prioritized based on factors such as health benefits, stability, formulation, and demand.”
ADM’s library of more than 500 microbial strains goes through a pre-clinical screening program, which the company’s team of scientists use to complete DNA sequencing and understand the genes that are present in the different microorganisms. “From there, the strains go through pre-clinical models to characterize them further before moving to human clinical trials,” said Vaughn DuBow, global director of marketing at ADM.
After identification of potential benefits, strains need to be tested to ensure they have sufficient baseline resistance to gastric acid and bile salts, adherence and interaction with intestinal mucosa, and some interaction with immune factors or antagonistic activity toward pathogenic microbes, said Dockery.
“Advances in culturomics and metagenomics have enabled probiotics developers to accurately identify and distinguish strains from tissue samples taken from both healthy and diseased human subjects,” said Dockery. “And, because of the great diversity in human gut, oral cavity, and skin microbiomes, this opens the door for a dramatic increase in the source for probiotic strains.”
Dive Deeper: Microbiome Modulation: Emerging Probiotic Benefits
Probiotic Cultivation & Production
Several technologies are improving best cultivation-practices. For example, Dockery noted a recent discovery that exposure to specific radiofrequency electromagnetic fields improved growth rates of several bacterial species during production.“Production of commercial probiotics must be at the strain level, not just the species level. And this has led to the realization that strain-specific sensitivities, such as nutrient requirements and ideal pH and temperature conditions, which impact production efficiency, are also present,” Dockery said.
Therefore, manufacturers have been challenged to identify those strain-dependent sensitivities during production, she added, from cultivation to drying, and to develop strategies for overcoming them while maintaining sustainable production.
“The biggest developments in probiotic production stem from the need by manufacturers to have dependable production methods that produce precise bacterial strains that are robust and stable throughout a shelf-life that extends beyond a few months,” Dockery said. “Novel technologies have been developed to improve cell counts of desired bacterial strains during fermentation, enabling better production efficiencies by increasing bacterial load and decreasing fermentation time.”
Almada pointed to two distinctive areas of improvement: cryoprotectants and culture optimization. “What was once limited to [substrates of] skim milk powder and vitamin C, and often undeclared animal and allergen-rich derivatives, has evolved into what can be implemented as a systematic and sustainable program of producing probiotic strains,” he said.
Advancements in protecting fermented strains from harsh freeze-drying conditions and validating best-in-class, clean-label growth/fermentation media are areas “often overlooked,” he added.
Keep Reading: Prescription Probiotics and Microbiome Modulation
Probiotic Encapsulation and Ensuring Stability
Encapsulation technology has seen “tremendous growth and fine-tuning in recent years,” Dockery noted. “Great strides have been made in improving technical parameters and using additives to protect the viability of probiotic strains, and target their delivery to the intended location within the GI tract.”Certain strains have higher natural resistance or tolerance to the harsh conditions of the GI tract, said Riera, and Kaneka has prioritized selecting these strains in order to work with delivery formats beyond capsules.
Tests to evaluate the performance of an encapsulation technology within the digestive tract continue to gain sophistication. Many technologies have been tested using SHIME (Simulator of the Human Intestinal Microbial Ecosystem) for greater insight on the timeline of a product’s digestion.
Lonza’s Capsugel DRcaps designed-release capsules are a vegetarian solution that targets release “approximately 45 minutes after a standard gelatin-based capsule,” is taken said Zain Saiyed, PhD, FACN, director of research and development, dosage form solutions, at Lonza.
“These capsules are robust enough to help protect dietary supplement ingredients from degradation in the low pH environment of the stomach,” he noted.
For another option that can combine probiotics with otherwise incompatible ingredients, “capsule-in-capsule technology offers endless customization and innovation potential,” said Saiyed. In the case of probiotics, live bacteria can be incorporated into a smaller capsule that is inserted into a larger liquid-filled capsule containing other complementary ingredients.
“A study investigating the release behaviors of several different Capsugel Duocap capsule-in-capsule combinations revealed that a mix of Lonza’s designed release capsules, DRcaps capsule in a DRcaps capsule, exhibited the potential for the slowest possible release profile, indicating they can be used to effectively target delivery to the colon.”
Riera noted that organic, oily suspensions “can enhance the survival of probiotics in the GI tract, and also offer the potential for combining fat-soluble vitamins with probiotic strains, which opens up new avenues for synergistic health benefits.”
Probiotical developed and patented Microbac, a technique of encasing microbial cells within a vegetable lipid matrix, said Almada. “We undertook comparator clinical trials and showed a five-fold greater delivery and viability in the stool relative to uncoated cells.”
To prove stability, Riera said key parameters include CFUs of bacteria across a range of conditions (including different climate zones), as well as how stable bacterial strains are in various steps of the production process, like in cold chain and in the context of varied water activity levels.
“Compatibility studies with different combinations are important to analyze, Riera said, as well as combinations of our proprietary probiotic strains with vitamins, minerals, fibers, and other active ingredients.”
As with many other ingredients, manufacturers often include overages of certain bacteria to ensure efficacious dosages through shelf life. But that’s not a one-size-fits-all solution, said Riera, as each strain will have a unique stability profile.
The standard way of determining the quantity of live viable cells in a supplement is through plate counting of bacterial colonies, but this technique has limitations and can’t always be used for heterogeneous blends of strains. Flow cytometry, which is suitable for such combinations can also distinguish between live, damaged, and dead cells.
“Other methods, such as fluorescence in situ hybridization (FISH) and molecular techniques based on the detection of nucleic acid sequences are beginning to gain attention for the ability to differentiate individual microbes, at least to the species level,” noted Dockery.
Pointing to recently-published comparisons of various counting methods for probiotic strains, Almada said: “We have pioneered flow cytometry, and the associated testing values of active fluorescent units (AFU) and total fluorescent units (TFU) to validate the presence of viable cells and ensure the adequacy of input addition in the monitoring of manufacturing and shelf-stability over time, respectively.”
“Probiotical developed AFU-based models to determine the predictive stability of probiotic compositions, which can be achieved within a few months of serial evaluations,” he said. “This can accelerate the path to market and contract the launch cycle.”
Novel Probiotic Species and New Technologies
As companies develop new formulations, certain testing challenges will become more prevalent, according to Riera.“The introduction of novel probiotic species, such as strict anaerobes or postbiotics, presents additional challenges and necessitates a consensus in the industry on the most appropriate counting methodology,” he said. “These new species may have unique requirements that current methodologies are not equipped to accurately assess.”
Probiotics are live microorganisms interacting with the gut microbiome, and as such, their behavior and stability can’t be fully accounted for through traditional chemical analysis, Riera added.
The introduction of novel fermentation media will also present opportunities. “The bulk of research in alternative growth mediums for probiotic bacteria has come within the context of the food industry,” said Dockery, “leading to evidence that there can be a symbiotic relationship between select plant components and probiotics, which can interact favorably with the gut commensal bacteria. This has led to the development and introduction to the market of several non-dairy probiotic beverages, frozen desserts, and vegan milk replacements.”
Microbial strains don’t always “play well together,” since they might have overlapping functions or may have antagonistic relationships. “Traditionally, manufacturers have combined individual probiotic strains together to produce multi-strain formulations without much thought regarding the impact that the strains might have on each other … The practice has continued because methods for differentiating the strains by cell count after production are still limited,” Dockery said.
New strategies, however, involve the use of immobilization and carrier molecules to enable efficient and efficacious production of multi-strain probiotics, she added.
It can be challenging to articulate the full scope of mechanisms at play in multi-strain formulations, noted Almada, offering Probiotical’s four-strain Bifizen ingredient as an example.
“Our rational, in vitro investigations pointed to a variety of biological activities: controlling a dysbiotic state and intestinal permeability, exerting a positive role on inflammation and oxidative stress, and even a strain that is an overproducer of GABA. We then subjected this quartet of microbes to three distinct, controlled clinical investigations, which demonstrated effects upon behavior, mood, sleep indices, and even circulating serotonin. Although we have not yet demonstrated an unambiguous role of the gut as the mediator of these behavioral and mood changes, we feel confident that our rational approach yielded a compelling and ever-expanding evidence base.”
Probiotics can’t simply be combined with any ingredient, Almada noted. “Many natural products, especially of botanical origin, possess mild bacteriostatic/antimicrobial activity.”
Probiotic in Foods and Beverages
Spore-forming probiotics, which can survive harsh manufacturing conditions, have been added to sodas, drink powders, bars, cookies, chewable pet supplements, and more.ADM’s spore-forming probiotic, DE111, can retain functionality through challenging formulation environments that may otherwise damage conventional probiotics, according to DuBow. “On top of that, DE111 is backed by studies showing that it may support digestive health and may help support healthy immune function.”
While spore-forming probiotics resolve many challenges, they may create others, Dockery noted. “These tough bacterial species can be a cleaning challenge for a manufacturing facility since they are so resistant. Their applications for human health are also more limited than strains that are derived from human sources.”
About the Author: Mike Montemarano has been the Associate Editor of Nutraceuticals World since 2020. He can be reached at mmontemarano@rodmanmedia.com.
