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Opportunity for research grants to help understand evidence linking live dietary microbes and health

For thousands of years, cultures across the globe have been consuming fermented foods, many of which contain diverse and numerous live microbes. Yet scientists are still puzzling over whether a greater intake of live microbes results in measurably better health. As part of long-term efforts to understand evidence for the health benefits of live dietary microbes and identify research gaps, ILSI North America is presenting a grant opportunity for researchers to help assess current scientific evidence for these links.

Researchers are invited to submit grant proposals, which should include the research approach along with anticipated challenges, resources, timeline, and key deliverables. The ILSI North America Gut Microbiome Committee also requests the inclusion of a suggested publication plan for the work. Budgets in the range of $100-150K will be considered. The deadline to submit the proposal is October 30, 2020 at 11:59PM EST. See here for more details.

ISAPP is supporting long-term efforts in this topic area. Its latest effort is the publication of a review paper (in press) on the links between dietary live microbes and health, called Should there be a recommended daily intake of microbes? The paper is authored by ISAPP board members Prof. Maria Marco, Prof. Colin Hill, Prof. Bob Hutkins, Prof. Dan Tancredi, Prof. Dan Merenstein, and Dr. Mary Ellen Sanders along with well-known nutrition researcher, Prof. Joanne Slavin.

ILSI North America is a non-profit scientific organization whose mission is to advance food safety and nutrition science for the benefit of public health. The organization engages academic, government, and industry experts by conducting­ research projects, workshops, seminars, and publications.

 

How do probiotics stay alive until they are consumed?

By Prof. Gabriel Vinderola PhD,  Associate Professor of Microbiology at the Faculty of Chemical Engineering from the National University of Litoral and Principal Researcher from CONICET at Dairy Products Institute (CONICET-UNL), Santa Fe, Argentina

See the Spanish version of this blog post here.

As a professor, most of my days are spent with people from the academic and scientific world. But through some outreach activities, I am also fortunate to interact with many people who are not scientists by training, but have curious, scientific minds. One question I am often asked is, “Is it really possible for probiotics to still be alive when they are dried and in a capsule?” The answer is yes. Let me provide some basic background on probiotics and explain my response.

The idea of consuming live microbes to promote health is not new. Back in 1907, Élie Metchnikoff, a disciple of Louis Pasteur, the father of microbiology, associated the intake of fermented milks containing live lactobacilli, with a prolonged and healthy life in Bulgarian peasants (see here). This idea was later captured by the concept of probiotics: live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (Hill et al. 2014). Four simple and pragmatic criteria allow one to conclude if specific strains of microorganisms qualify as a probiotic for use in foods and dietary supplements. Probiotic strains must be (i) sufficiently characterized; (ii) safe for the intended use; (iii) supported by at least one human clinical trial showing they are effective; and (iv) alive in the product at an efficacious dose throughout shelf life (Binda et al. 2020). Being alive at the moment of consumption is one of the key characteristics of probiotics.

Life is the condition that distinguishes animals and plants from inorganic matter. Life includes the capacity for growth, for reproduction and for metabolic activity. In order to sustain life, certain environmental conditions must be met, but these differ for different organisms. For microbes, the availability of water and nutrients, adequate temperature and pH (acidity), and the absence of growth inhibitors are essential conditions. However, it is possible to manipulate certain conditions to bring about a state where growth may be put in “stand-by mode”, yet the microbe remains alive. We cannot imagine ourselves in a condition where life is preserved even without any metabolic activity, but for microbes it is possible. Probiotics can be in foods (yoghurts, fermented milks, fruit juices, cereal bars) or in food supplements (capsules, compressed pills) in a “hibernation” state, characterized by no growth, no reproduction and no metabolic activity, waiting for the proper conditions to come back to full metabolic life. This occurs when the microbes reach the gut, which has proper temperature, nutrient availability, lack of inhibitors, adequate acidity and water. Thus, in case of microbes, there is an uncoupling of life and metabolic activity. Even without having any metabolic activity, they can still be alive, but in a dormant state.

Open a food supplement containing probiotics and you will probably find a white dry powder. This is what the microbes may look like in their dormant state, due to a technological process called freeze-drying or lyophilization. Freeze-drying is a two-stage process where cells are first quickly frozen at very low temperatures (-40 to -70°C, or less, using liquid nitrogen for example). Then, frozen water is removed by a gentle process of evaporation at low pressure and temperature, called sublimation. This process removes most of the water from around and inside the cells, leaving the microbes in a dormant state. Water activity is scientists’ way of measuring water availability for the microbes. This technological measure ranges from 0 (no water) to 1 (pure water). A water activity close to 0 impairs growth. In food supplements, freeze-drying leaves water activities less to 0.2, ensuring that no metabolic activity will take place during the shelf life of the product.

Bifidobacteria cells (circled in red) freeze-dried in a probiotic powder. This is a scanning electron microscopy image amplified 10,000 times. Cells are embedded in dry polydextrose.

So yes, probiotics in food supplements are alive in their own way. This is the case also for probiotics included in certain foods such as cereal bars. In case of food products with water activities closer to 1, such as yogurts, fermented milks, cheeses or fruit juices containing probiotics, the factor that limits metabolic activity is the low temperature at which these products are stored, combined in certain cases (yogurts, fermented milks, fruit juices) with the low pH (or high acidity) of these products. The combination of low temperature and acidity is effective in maintaining probiotic cells in a dormant state, impairing any metabolic activity that may lead to cell stress and cell death along the shelf life of the product. Yet, even while tightly controlling factors that impair metabolic activity, some cell death may occur during the shelf life of probiotics in the products that deliver them. In this case, responsible manufacturers are sure to add extra probiotic cells so that the necessary amount of viable cells needed to deliver a health effect are present through the end of the shelf life of the product.

In both probiotic foods and food supplements, the number of viable cells is commonly expressed as a certain number of colony forming units, or by the abbreviation “CFU”. As probiotics are present in high concentrations, the number of viable cells often reaches into the billions within a capsule or in a serving of yogurt. To be able to count such enormous numbers of cells, microbiologists must make serial dilutions of the probiotic product. Then, they will put a small drop of a dilution on the surface of a Petri dish containing a culture medium on which probiotics will grow. Each probiotic cell (or clump of cells) will grow in place and form a visible colony that can be observed to the naked eye, and counted.

Agar plate containing colonies of a probiotic bacteria. Cells deposited on the surface of the agar plate duplicated several times until forming a visible amount of cells: a colony.

In brief, live probiotics are present in food and supplements, but in a state of life different to that of higher organisms where metabolic activity is taking place at all times. During shelf life, the metabolic activity of probiotics is stopped by freeze-drying them (food supplements) or by a combination of low temperature and acidity (yogurts and fruit juices, for example). Active growth returns when these microbes enter out gut and find the proper conditions of nutrients, temperature, acidity and water to be active and deliver their health effects.

Is probiotic colonization essential?

By Prof. Maria Marco, PhD, Department of Food Science & Technology, University of California, Davis

It is increasingly appreciated by consumers, physicians, and researchers alike that the human digestive tract is colonized by trillions of bacteria and many of those bacterial colonists have important roles in promoting human health. Because of this association between the gut microbiota and health, it seems appropriate to suggest that probiotics consumed in foods, beverages, or dietary supplements should also colonize the human digestive tract. But do probiotics really colonize? What is meant by the term “colonization” in the first place? If probiotics don’t colonize, does that mean that they are ineffective? In that case, should we be searching for new probiotic strains that have colonization potential?

My answer to the first question is no – probiotics generally do not colonize the digestive tract or other sites on the human body. Before leaping to conclusions on what this means for probiotic efficacy, “colonization” as defined here means the permanent, or at least long-term (weeks, months, or years) establishment at a specific body site. Colonization can also result in engraftment with consequential changes to the gut microbiota composition and function. For colonization to occur, the probiotic should multiply and form a stably replicating population. This outcome is distinct from a more transient, short-term (a few days to a week or so) persistence of a probiotic. For transient probiotics, it has been shown in numerous ways that they are metabolically active in the intestine and might even grow and divide. However, they are not expected to replicate to high numbers or displace members of the native gut microbiota.

Although some studies have shown that digestive tracts of infants can be colonized by probiotics (weeks to months), the intestinal persistence times of probiotic strains in children and adults is generally much shorter, lasting only few days. This difference is likely due to the resident gut microbiota that develops during infancy and tends to remain relatively stable throughout adulthood. Even with perturbations caused by antibiotics or foodborne illness, the gut microbiome tends to be resilient to the long-term establishment of exogenous bacterial strains. In instances where probiotic colonization or long-term persistence was found, colonization potential has been attributed more permissive gut microbiomes specific to certain individuals. In either case, for colonization to occur, any introduced probiotic has to overcome the significant ecological constraints inherent to existing, stable ecosystems.

Photo by http://benvandenbroecke.be/ Copyright, ISAPP 2019.

This leads to the next question: Can probiotics confer health benefits even if they do not colonize? My answer is definitely yes! Human studies on probiotics with positive outcomes have not relied on intestinal colonization by those microbes to cause an effect. Instead of colonizing, probiotics can alter the digestive tract in other ways such as by producing metabolites that modulate the activity of the gut microbiota or stimulate the intestinal epithelium directly. These effects could happen even on short-time scales, ranging from minutes to hours.

Should we be searching for new probiotic strains that have greater colonization potential? By extension of what we know about the resident human gut microbiota, it is increasingly attractive to identify bacteria that colonize the human digestive tract in the same way. In some situations, colonization might be preferred or even essential to impacting health, such as by engrafting a microbe that performs critical metabolic functions in the gut (e.g. break down complex carbohydrates). However, colonization also comes with risks of unintended consequences and the loss of ability to control the dose, frequency, and duration of exposure to that particular microbe.

Just as most pharmaceutical drugs have a transient impact on the human body, why should we expect more from probiotics? Many medications need to be taken life-long in order manage chronic conditions. Single or even repeated doses of any medication are similarly not expected to cure disease. Therefore, we should not assume a priori that any observed variations in probiotic efficacy are due to a lack of colonization. To the contrary, the consumption of probiotics could be sufficient for a ripple effect in the intestine, subtly altering the responses of the gut microbiome and intestinal epithelium in ways that are amplified throughout the body. Instead of aiming for engraftment directly or hand-wringing due to a lack of colonization, understanding the precise molecular interactions and cause/effect consequences of probiotic introduction will lead to a path that ultimately determines whether colonization is needed or just a distraction.