Introduction

According to the Scientific Association of Probiotics and Prebiotics (ISAPP) consensus, probiotics are defined as “live microorganisms which when administered in adequate amounts confer a health benefit”. Microorganisms must be well characterized, be safe, and provide a general benefit to humans, supported by scientific evidence, to be considered probiotics ¹. Lactobacillus, Bifidobacterium, and Saccharomyces species are the most commonly used probiotics. Other less frequently used probiotics include species of Bacillus, Propionibacterium, Streptococcus, and Escherichia². Evidence supports that probiotics can improve the immune system response, gastrointestinal health, and vaginal lactobacilli concentration. However, they do not promote persistent changes in gut microbiota³.

Formulations containing probiotics must ensure an effective microorganism dose viable until the expiration date to be considered supplements and be allowed to declare a specific beneficial property. In addition, a risk and benefit assessment and the compliance with regulatory standards in each country are required to register the formulations as drugs¹. In addition to probiotics, many formulations include substrates used selectively by host microorganisms that also confer a health benefit (prebiotics). This combination of probiotics and prebiotics is called a synbiotic⁴.

In Mexico, we can find formulations containing probiotics that have been approved by the Federal Commission for the Protection against Sanitary Risks (COFEPRIS, for its acronym in Spanish; https://www.gob.mx/cofepris)as drugs. Additionally, we can find formulations described as food or dietary supplements that, in some cases, declare the benefit they confer. The population should know what probiotic products can be purchased on the market and the differences among them when selecting them or recommending their use.

In this study, we identified formulations containing probiotics sold in the State of Tabasco, analyzed their composition, and compared them with formulations that can be purchased online.

Materials and methods

Results

It was determined which products formulated with probiotics are sold in 19 pharmacies and two health food stores in Villahermosa by asking the salesmen to identify the probiotic products sold by the establishment. When it was detected that the concept of probiotics was not familiar to the seller, we asked him or her about supplements or medications that help restore or preserve the intestinal flora (the colloquial term used to name the gut microbiota). On some occasions, the best-known probiotic genera (Lactobacillus, Bifidobacterium, and Saccharomyces) were named to guide the seller. In this way, 31 products formulated with probiotics were identified, hereinafter referred to as local products. Likewise, 30 products formulated with probiotics that can be purchased online (products for sale online) were identified and compared with those sold locally.

The establishments visited belong to large pharmaceutical chains that operate regionally (south-southeast of the country) and nationally and have branches in the 17 municipalities of Tabasco. In addition, several of them also offer online sales. Thus, we can assume that the products analyzed as local can be purchased in the 17 municipalities of the State of Tabasco.

Classification of the formulations as drugs or supplements

The 31 local products, along with the 30 products sold online, were classified as supplements or drugs using the label information and verifying their registration by the Federal Commission for Prevention against Sanitary Risks (COFEPRIS) as drugs. Regarding products sold online, 100% (30) were described on the label as a food supplement and contained warnings that they are not regulated by the FDA. In general, these products stated that they contribute to digestive health. On the other hand, 83.87% (26) of local products were food supplements, and only 16.13% (5) were identified as drugs figure 1, some of which specify on the label their functions as regulators of intestinal flora, assistants in the treatment of diarrhea, or both.

Figure 1 Percentage of supplements and drugs formulated with probiotics that are sold locally (31 in total). Fuente: Elaboración propia 

Dosage forms of the formulations analyzed.

Figure 2 shows the dosage forms of the local and online formulations analyzed. The dosage form of 83% (25) of the products sold online was the capsule, and the rest were presented as tablets, chewable tablets, or gummies. By contrast, the variety of dosage forms is better represented in local products: 32% (10) are administered in capsules, 30% (9) in powder, 19% (6) in tablets, and 19% (6) in suspension. The dosage forms common to both types of products were the capsule and the tablet, representing 57% and 13% of the total products, respectively.

Figure 2 Dosage forms of the formulations analyzed (61 in total). Fuente: Elaboración propia 

Genera and species present in the formulations analyzed.

Species belonging to 8 bacterial genera and five yeast genera were identified in the 61 formulations analyzed. As shown in figure 3, local products contain species from only six genera, while online products contain species from 12 genera. In both cases, the most represented genera were Lactobacillus and Bifidobacterium. Some local products [64% (20)] and online products [36% (11)] contain species of a single genus, either Bifidobacterium, Lactobacillus, Saccharomyces, or Bacillus (Figure 3A and 3B). Unlike local products [22% (7)], 47% (14) of online products contained species of 2 genera, which, in most cases, were a mixture of Lactobacillus and Bifidobacterium (Figure 3A). In addition, the species Streptococcus thermophilus was present in 8 products. It is worth noting that Bacillus species were not mixed with species of other genera in any products. Finally, among the online products, there were 2 cases containing species from 10 different genera.

Figure 3 The Venn-Euler diagram represents the number of formulations containing species from the different genera in local (A) and online products (B), where the set called various genera * represents the genera Brettanomyces, Debaryomyces, Kluyveromyces, Torulaspora, and Leuconostoc. The number of products that contain a variable number of species/subspecies in their formulation (C). Fuente: Elaboración propia 

Analyzing the species/subspecies present in the products revealed that 61% (19) of the local products contained a single bacterial species, and 39% (12) contained 2 to 10 different species (Figure 3C). By contrast, 30% (9) of the products sold online contained a single species, while 60% (18) contained 2 to 10 species, and the remaining 10% (3) contained 18 to 31 species from 10 different genera.

Figure 4 shows the 46 species and subspecies identified in the formulations analyzed. Thirty-nine percent (18) were found both in local and in online products, whereas 7% (3) were only present in local products and the remaining 54% (25) were only found in products sold online. Lactobacillus was the genus with the highest abundance of species (18) in the formulations analyzed. Lactobacillus acidophilus, followed by L. rhamnosus, L. plantarum, and L. casei, were the most represented. In all the products, specific strains of each species were mentioned. Although Lactobacillus species diversity in online products was vast, 44% of the species were only present in 3% (2) or less of products.

Figure 4 Species abundance in the probiotic products analyzed. The total number of species in local products was 21, and the total number of species in online products was 43. Fuente: Elaboración propia 

The second most represented genus was Bifidobacterium, with seven species. It is worth pointing out that Bifidobacterium infantis and B. lactis are subspecies of Bifidobacterium longum and B. animalis, respectively. However, they were described with the old classification due to the importance of subspecies in studying and using probiotics. B. infantis is the most abundant subspecies in the infant gut microbiota, and B. lactis was discovered in dairy products and has been widely used in their production. The Bifidobacteria species most represented in local products were different from those most represented in the products sold online. In addition, the number of products sold online containing the most represented species [B. lactis (53% (16)), B. bifidum (40% (12)), and B. longum (33% (10))] was elevated compared to the Bifidobacteria species most represented in local products [B. infantis (13% (4)) and B. lactis (10% (3))]. Few products mentioned specific strains of Bifidobacteria.

The third-most abundant species were yeasts of the genus Saccharomyces (5), and only Saccharomyces boulardii was common to local and online products. Most of the remaining genera (7) were represented by a single species. S. thermophilus stood out for being the ninth most represented species in all of the products, preceded only by Bifidobacteria and Lactobacillus species.

It should be noted that Bacillus coagulans was formerly named Lactobacillus sporogenes⁵, and this name is preserved on product labels.

Classification of formulations as probiotics or synbiotics

The 61 formulations analyzed were classified as probiotics or synbiotics using the nutritional information, the list of ingredients declared on the label, or both figure 5. For this, a synbiotic was considered a mixture containing live microorganisms and substrate(s) used selectively by the host microorganisms that confer a health benefit, according to the consensus reported by ^{Swanson in 2020}. The substrates mentioned in the labels that allowed classifying the products as synbiotics were inulin, fructooligosaccharides, and oligofructose. It was observed that 32% and 23% of local products and online products, respectively, were synbiotic, even though some were declared as probiotics on the label.

Figure 5 . Classification of the analyzed products (61 in total) as probiotics or synbiotics. Fuente: Elaboración propia 

Discussion

The results show that the Tabasco population has access to various supplements and drugs made with probiotics sold by local establishments. Additionally, more complex formulations than those sold locally can be purchased online. It should be noted that only 5 of the 31 local products are registered as drugs by COFEPRIS figure 1. Three of those products contain only Bacillus claussi and are administered in suspension (2) and capsule form (1). The other two products are administered in powder form, with one composed of Saccharomyces boulardii and the other composed of a mix of Lactobacillus fermentum and L. delbrueckii. Interestingly, none of the species contained in these drugs was one of the most represented in the 61 formulations analyzed (Figure 4). Multiple studies have demonstrated the effectiveness of B. claussi in the treatment of acute diarrhea in children⁶ and S. boulardii in reducing the risk of acquiring antibiotic- associated diarrhea in children and adults⁷.

Regulatory approaches in some countries, such as Canada and Italy, agree that products must have a minimum amount of 1 x 10⁹ CFU of well-studied microbial species per serving to be considered probiotic foods or supplements¹. Health Canada lists the following as probiotic species: Bifidobacterium adolescentis, B. animalis subsp. Animalis and lactis, B. bifidum, B. breve, B. longum subsp. longum and infantis, L. acidophilus, L. casei, L. fermentum, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L. rhamnosus, and L. salivarius. Except for L. johnsonii, all these strains were present in at least one formulation that we analyzed (Figure 4). The number of microorganisms declared in the five drugs identified in 64% (17) of local supplements (25) and all products sold online (30) is above 1 x 10⁹ CFU, although some products clarify that this is the amount when manufactured and that the effective number of bacteria until the expiration date is 1 x 10⁷ CFU.

The most commonly used dosage form was the capsule, followed by the tablet figure 2. However, 4 of the five drugs were in powder or suspension form. The strain and the dosage form are critical to ensure that the probiotics remain viable since, after being ingested, the probiotic faces various fluids (bile salts, pancreatic enzymes, gastric juice, among others) during its passage through the gastrointestinal tract⁸. In addition, during encapsulation, factors such as pH, pressure, temperature, moisture content, amount of oxygen, and water activity affect the viability of microorganisms⁹. Improper storage or distribution of these products, or both, can also affect the viability of the microorganisms. Notably, several studies show that probiotics that have been killed, destroyed by heat, or inactivated by ultraviolet light, and even celular fractions, can confer health benefits and be as effective as living organisms and, in some instances, safer for the host in the treatment of some conditions^10,11.

Regarding the complexity of the formulations analyzed, 46% (28) contained only one probiotic strain of the Lactobacillus, Bifidobacterium, Saccharomyces, or Bacillus genera, so they were considered monostrains. In contrast, the rest of the formulations were multistrain and contained two to 32 species from 14 different genera figure 3. The abundance of species of the genera Lactobacillus and Bifidobacterium is evident. These species have been widely studied with favorable results in multiple gastrointestinal tract disorders, such as irritable bowel syndrome, ulcerative colitis, Crohn’s disease, and infectious and antibiotic-associated traveler’s diarrhea¹². The strain included is essential when choosing probiotic formulations for the treatment of gastrointestinal diseases. L. acidophilus LB is effective in the treatment of traveler’s diarrhea, while other L. acidophilus strains are not¹³. For this reason, the strain must be specified in the formulations.

Although monostrain products are the most studied, the efficacy of multistrain formulations in treating different conditions is being increasingly studied. For example, a probiotic composed of 4 strains of Lactobacillus (3) and Enterococcus (1) species was well tolerated and associated with a decrease in intestinal inflammation in patients with ulcerative colitis¹⁴. Likewise, a synbiotic formulation composed of fructooligosaccharides and six strains of Lactobacillus (4), Bifidobacterium (1), and Bacillus (1) genera significantly improved glycemic control in subjects with type 2 diabetes mellitus¹⁵. Another formulation with eight strains of Lactobacillus (4), Bifidobacterium (2), and Lactococcus (2) genera given to patients with type 2 diabetes mellitus for six months without prior medication significantly decreased the HOMA-IR index, reduced inflammation, and improved the cardiometabolic profile of patients¹⁶. These findings show that these types of formulations may be a promising adjuvant in antidiabetic therapy. Treating diseases is not the only benefit that can be obtained from ingesting multistrain formulations. The administration of a probiotic composed of 4 strains of Lactobacillus (3) and Saccharomyces (1) species reduced the toxicity of a mixture of phthalates and bisphenol A in Wistar¹⁷ rats, and very recently, a probiotic formulation of 8 strains of Lactobacillus (4), Bifidobacterium (3) and Streptococcus (1) genera cultivated in consortium from in vitro studies showed that it could absorb toxic elements such as Cd, Hg, and Pb. Additionally, the concentration of Cd in the feces of 15-day- old infants whose mothers consumed this formulation was significantly reduced¹⁸. These findings open the possibility of using probiotic consortiums as a method for bioprotection and detoxification of different pollutants. On the other hand, the addition of different probiotic strains could also be considered a technological strategy. Lactococcus lactis and S. thermophilus were present in some of the products analyzed, always combined with Lactobacillus and Bifidobacteria. L. lactis and S. thermophilus have shown to assist the survival of other lactic acid bacteria in food products¹⁹. In addition, S. thermophilus has high oxygen consumption, so it is added to anaerobic microorganisms to reduce the oxygen considered toxic during encapsulation, avoiding affecting the viability of the microorganisms⁹. It has been shown that L. rhamnosus GG, the second most represented species in the products analyzed, promotes interactions between crucial elements of the microbiota, such as Bifidobacteria and the host epithelium, and significantly modifies the microbiota of healthy individuals²⁰. These properties make it attractive for the formulation of both single and multistrain products.

Among the formulations analyzed, 2 contained 32 species of 10 different genera. The yeasts Brettanomyces anomalus, Debaryomyces hansenii, Kluyveromyces marxianus, and Torulaspora delbrueckii, and the four species of Leuconostoc were exclusive to these products. These yeasts have been identified and used as starters for various dairy products²¹, whereas various species of Leuconostoc have been identified in various fermented products and have shown to inhibit pathogen growth²². In addition to the probiotic strains, these formulations contain some vitamins, minerals, and enzymes that promote digestion. It is important to note that more is not always better. Therefore, it is important to have trials that show that a complex formulation promotes a more significant benefit to consumer health.

Finally, 28% of the products analyzed figure 5 were classified as synbiotic since their components include some prebiotic carbohydrates such as inulin, fructooligosaccharides, and oligofructose. These products are complementary synbiotics since the selective use of prebiotics by autochthonous microbiota has already been established. There are no studies that show that the addition of prebiotics to probiotic formulations leads to a more significant benefit to be considered synergistic synbiotics⁴.

Evidence supporting the benefits conferred by living, dead, or remnants of probiotics, as well as discrepancies in the effectiveness of monostrain and multistrain probiotics in the treatment of several conditions, reinforces the need to have well-characterized formulations supported by in vitro, preclinical and clinical trials to ensure that a formulation is effective to promote health or to assist in the treatment of specific conditions¹¹. In addition, scientific evidence should support that more than one strain, additional ingredients, or both in the same formulation provides more significant benefits. In this sense, probiotics registered as drugs by regulatory agencies such as COFEPRIS in Mexico are safer since this agency certifies that the formulations are supported by clinical trials in humans that prove their benefits and certifies that they have been prepared following good manufacturing practices. The comparison between local and online products shows that although online products widen the variety of formulations that we can purchase, they are marketed in Mexico and abroad following regulations different from those that govern drugs; therefore, they are not required to support the expected benefits with scientific evidence, and consequently, there is no obligation to mention the benefits on the label.

Conclusions

The population of the state of Tabasco can find at least 31 formulations containing probiotics. The variety of products increases if the supplements that can be purchased for sale online are considered, particularly with regard to multistrain supplements. Moreover, online product offerings increase and change quickly. Our findings show that probiotic products that can be purchased in stores and online are not governed by the same health regulations as those found locally. Considering that they are designed with living organisms and that many of their characteristics depend on their viability, health regulations must be reviewed and include aspects that guarantee their quality, especially those declared as supplements. On the other hand, we must not forget that the distribution of any product for sale online does not reach all places, and it is still necessary to have the products available in local establishments. A more in- depth exploration of the local market over time will allow an understanding of if the formulations offered increase and diversify. In addition, exploring the use of probiotics in local clinical practice would be useful to determine if health professionals demand a greater diversity of these products and if they have pertinent information about them to prescribe and recommend them.