Probiotics, what are they and what types are there

Published: 11 September, 2023 | 10'

What are probiotics?

Probiotics are defined by both the FAO¹ and the WHO¹ as "live microorganisms that, when consumed in appropriate amounts as part of a food, confer a health benefit on the host".

The intake of probiotics in our daily diet provides direct benefits to our intestinal flora or microbiota, maintaining it in balance and preventing intestinal imbalances such as diarrhea, flatulence, abdominal bloating, malfunction of the intestinal barrier affecting the immune system, among other consequences.

How are probiotics classified?

From a scientific point of view, the classification of any biological organism is the arrangement of organisms into taxonomic groups (taxa) based on similarities or relationships.

Taxonomy of probiotic organisms.

Probiotic organisms are classified according to their genus, species, subspecies, and an alphanumeric designation that identifies a specific strain, according to the World Gastroenterology Organisation².

Denomination of different microorganisms.

An example of nomenclature applicable to microorganisms is: Lactobacillus (genus) casei (species) DN-114 001 (strain).

According to the guidelines of the WHO/FAO¹, probiotic manufacturers must register their strains with an international depository, which assigns an additional designation to the strains.

The following table, taken from the World Gastroenterology Organisation Global Guidelines², shows some examples of commercial strains and their corresponding names.

What is a probiotic strain?

A strain is defined as a group of microorganisms, such as probiotic bacteria, that belong to the same species and share certain characteristics that are not found in other members of the species.

These characteristics may vary from one species to another, which means their effects can be different.

Importance of strain designation.

In the case of probiotics, it is important to use strain designations because the strongest approach to probiotic evidence is being able to attribute benefits to specific strains or combinations of probiotic strains at an effective dose².

The FAO and the WHO propose that since probiotic properties are strain-specific, strain identification (genetic typing) should be performed using methods such as pulsed-field gel electrophoresis (PFGE) as well as genetic identification using DNA or RNA, or other internationally recognized methods.

CFU (Colony Forming Unit)

A CFU is a unit of measurement used to quantify microorganisms, i.e., to count the number of viable bacteria in a liquid or solid sample. Colony Forming Units are measured in units of volume (CFU/ml) or mass (CFU/g).

What is the viability of microorganisms?

The viability of a microorganism is defined as the ability to multiply under controlled conditions. The probiotic quality is primarily measured by the maintenance of viability (expressed as Colony Forming Units or CFU) until the end of the product's shelf life, and the genus, species, and strain of all organisms included in the product should be identified.

Are all probiotics the same?

The FAO and the WHO have defined (see figure taken and translated from Pandey, 2015⁴) a list of parameters that provides a systematic approach for an effective assessment of probiotics that substantiates health claims and benefits.

These parameters determine the obligations for developing probiotic products, which include the following activities:

1. Strain identification.
2. Functional characterization of the strain(s) in terms of safety and probiotic attributes.
3. Validation of health benefits in human studies.
4. Correct labeling of efficacy claims and content throughout the shelf life.
5. The recommended dosage, which should be based on the induction of the declared physiological effect.

Types of probiotics

As mentioned, the probiotic potential of different bacterial strains, even within the same species, can vary.

This means that probiotic strains of the same species are always unique and can have different sites of action, as well as specific immunological effects, and can differ in their effects on both a healthy and inflamed intestinal system.

The most popular strains are represented by the following bacterial genera:

• Bifidobacterium
• Lactobacillus
• Streptococcus

But other organisms, including

• Yeast
• Enterococci

have also been used as probiotics.

Bacteria as probiotics.

Genus Bifidobacterium.

Bifidobacteria are anaerobic Gram-positive (grow in the absence of oxygen), rod-shaped, non-gas producing, non-sporulating, and non-motile.

This genus includes 30 species:

• 10 of which are derived from human sources (dental caries, feces, and vagina),
• 17 from the animal intestinal tract or rumen,
• 2 from wastewater, and
• 1 from fermented milk.

Bifidobacteria are microorganisms of great importance in the gastrointestinal and genitourinary tracts, whose exact proportion is mainly determined by age and diet.

The number of bifidobacteria decreases as age increases and ends up becoming the third most abundant genus (representing approximately 25% of the total adult intestinal flora) after the Bacteroides and Eubacterium genera.

The clinical data in the following table mentions some of the positive actions of bifidobacteria.² 

Genus Lactobacillus.

Lactobacilli are characterized by being Gram-positive bacilli or cocobacilli, non-sporulating, non-flagellated, able to grow in oxygenated or non-oxygenated environments, and strictly fermenting.

There are 56 species of the Lactobacillus genus that have been identified.

Lactobacilli are distributed along the gastrointestinal and genital tracts. Their distribution is affected by various environmental factors, such as pH, oxygen availability, level of specific substrates, presence of secretions, and bacterial interactions.

Positive Effects of Lactobacilli:

The table below shows some clinical results of the positive effects of lactobacilli.^2,7

Other probiotic bacteria:

Genus Bacillus.

The species of Bacillus have been used as probiotics for at least 50 years in an Italian product marketed as Enterogermina® (2x10⁹ spores). Some advantages of bacterial spores are their resistance to heat, which allows them to be stored at room temperature and in a desiccated form.

Furthermore, these bacteria are capable of reaching the small intestine as they survive the acidic pH of the stomach.

The most studied species is B. subtilis, because its consumption in some probiotic products has shown evidence of stimulating the immune system, through action on the gut-associated lymphoid tissue and secretion of antimicrobial substances.

Genus Enterococcus.

Among the species of Enterococcus, Enterococcus faecium is the most commonly used in probiotics. The presence of E. faecium is important in the prevention of infection by Salmonella enterica.

Interesting characteristics of the Enterococcus group are survival on dry surfaces for extended periods and resistance to antibiotics.

Probiotic Yeasts

They are a group of unicellular fungi that primarily grow and ferment sugars. There are different families, but the most important from the point of view of nutrition and health are the so-called ascomycetes, which include the genus Saccharomyces, which are unicellular fungi that form spores and are heat resistant. Well-known examples are yeast for bread and beer.

Brewer's Yeast, Sacharomices cerevisiae

The potential probiotic effect of S. cerevisiae and S. cerevisiae var. boulardii has been demonstrated, as they are able to tolerate the acidic environment and bile, and may have effects against bacterial infections by reducing intestinal proinflammatory response.

The table below shows clinical data of these effects.^2,4,7

* ESPGHAN: European Society for Paediatric Gastroenterology, Hepatology, and Nutrition; ESPID: European Society for Paediatric Infectious Diseases.

How are probiotics produced?

The manufacturing process of food-type probiotic products includes a series of stressing factors that challenge their potential benefit.

One of the most important is ensuring a high microbial load in the final product throughout its shelf life, and for this reason probiotics are first cultivated in large quantities, on an industrial scale, using appropriate culture media.

The second factor is that microbial inocula are usually stored frozen, to avoid subjecting them to low or changing temperatures.

Drying Techniques

Taking these factors into account, two drying techniques are generally used, spray drying and freeze-drying, which generate high-density probiotic powders that can be added to various types of food products, including dietary supplements.

• Spray Drying: It is a cost-effective technique that produces stable microbial powders with high cell populations. However, it often leads to a significant loss of cell viability due to the severe stress that especially affects the structure and functionality of probiotic cells, due to the high air temperature (evaporation of water in the drying chamber) and water removal.

• Freeze-Drying: This technique requires gentler conditions and is usually better tolerated by bacteria. Similarly, the process subjects the probiotic inoculum to very low temperatures and dehydration, which affect both bacterial or fungal integrity and viability. Likewise, the subsequent storage of dried probiotic biomass, loss of viability and activity is quite common. Probiotic products containing bifidobacteria are developed using either of these two techniques.

• Food Matrix: Another technique for producing food-type probiotics is especially for fermented products and their derivatives. It involves a direct inoculation into the food matrix that enables the growth of probiotic species through fermentation. This technique provides the advantages of avoiding the dehydration and inoculation steps in a different food matrix; however, the final product with incorporated probiotics may undergo variations depending on the specific composition and intrinsic physicochemical parameters of the food matrix itself, as well as its storage conditions. Probiotics containing lactobacilli generally use this technique for their production.

Strategies to improve viability of probiotic products.

The finding that probiotic products may have deficiencies in the number of truly viable cells, both in foods and dietary supplements, has prompted the development of new strategies to improve probiotic properties and response to stressors. Let's see some of them.

Encapsulation.

It serves two purposes, to protect probiotics from gastric stress after oral intake, and to improve survival during the drying stages of manufacturing, and stability during storage in the finished food product. Chitosan and alginate are generally used as encapsulating agents.

Carrier media and protective agents.

The use of complex matrices, such as skim milk, significantly improves the survival and stability of probiotics, especially after drying and during storage.

Prebiotic fiber.

The use of fibers from various plant sources enhances both the survival of probiotics during the drying process of the inocula and the storage stability of cells within food products. Fructooligosaccharides (FOS) are the most commonly used prebiotic ingredients for this purpose.

Addition of protective agents to counteract acidic pH.

Acidic pH is considered the main threat to probiotics, especially in the gastrointestinal tract. For this purpose, the addition of saccharides, amino acids, and fatty acids effectively protects probiotics against the acidic environment. This strategy is beneficial for probiotics containing yeasts.

Adaptation to stress and cross-protection.

In this strategy, bacteria are pre-exposed to sublethal levels of a specific stressor, which induces an adaptive response of increased resistance to subsequent doses of the stressor or even to a different type of stress. This process enhances probiotic survival.

Probiotic Dietary Supplements

There are probiotic dietary supplements available on the market that mainly contain strains of Lactobacillus, Streptococcus, Bifidobacterium, and yeasts (S. boulardii).

Probiotic dietary supplements are generally available in sachets, tablets, or capsules. There are three main factors that influence the viability of probiotic dietary supplements during storage: temperature, oxygen, and relative humidity.

Therefore, it is important to consider three aspects to ensure the viability of probiotics contained in dietary supplements:

• Store the products refrigerated, even if the label indicates that the cultures are stable at room temperature. In traditional lyophilization processes, increasing the storage temperature from 4 to 25 °C reduces stability by ten times.
• Close the container as quickly as possible after taking the supplement to reduce the entry of oxygen and moisture into the container. Many probiotic products design their storage formats with water-impermeable jar packaging or laminated packaging films, or by adding small moisture-absorbing sachets to the containers.
• If the container contains moisture- or oxygen-absorbing sachets, do not remove them. Oxygen is harmful to the viability of probiotics during storage.