Abstract
Graphical abstract
Abstract
Objective
Significant scientific and industrial research on probiotic strains is influencing the growing size of the market. This underlines the ongoing need for an accurate declaration of identification, with proven safety and functionality of the incorporated strains.
Methods
The research was designed to address two key issues, first, to assess the viability of strains of commercially available probiotic formulation, and second, to test the survival of probiotics in the gastrointestinal milieu in vitro. Therefore, we sought to validate the live cell counts and bacterial strains in probiotic products and compare the values with those declared on labels. The viable plate count was used to determine the number of viable microbes present in the oral probiotic suspension. To assess the capacity of the strains contained in the probiotic product to survive the extreme environment of the gastrointestinal tract (GIT), an in vitro approach involving the simulated gastric and intestinal fluids was applied. As the tested product is intended for newborns and infants, we simulated infant GIT conditions.
Results
The analysis revealed that probiotic product samples matched the label data regarding viable cell counts and contained probiotic species. Microbiological analysis and next-generation sequencing (NGS) long read excluded the presence of contaminating non-probiotic bacteria in the probiotic product.
Conclusions
The probiotic strains, contained in the formulation, effectively survived simulated intestinal conditions in vitro, fulfilling one of the first requirements in the quality assurance assessment of probiotics.
Significance statement
These findings support the importance of probiotics as a microbiota-modulating strategy.
Introduction
Probiotic supplementation extends from its use as dietary supplements to biotherapeutics for alleviating various health disorders by balancing the composition of the gut microbiome (Novak et al. 2021). Probiotics are taxonomically defined by their specific strain, which includes the genus, the species, the subspecies (if applicable) and an alphanumeric strain designation. A key quality requirement is that probiotic formulation contains defined numbers of living cells, as stated in the declaration (Tuomola et al. 2001, Davis 2014). Safety, identification and compliance of the concentration of probiotic cells with the recommended daily dose are considered priority in the context of quality assurance of the formulation. It is well-established that the health promoting effects of probiotics are dose-dependent and the minimum recommended amount to be consumed is often suggested at the level of 109 CFU per day (Wendel 2021). For the quality assessment, a crucial requirement to be verified is that probiotic formulation contains a defined number of living cells, that is compliant with those declared on the label (Tuomola et al. 2001, Weitzel et al. 2021). The probiotic strains included in probiotic products (PPs) must fulfill strict criteria and guarantee their safety, quality and functionality. The quality assurance and compliance of PPs are governed by global government and non-governmental regulatory organizations (FAO/WHO 2002, Swanson et al. 2020), who continuously emphasized the significance of standardized and precise quality monitoring, setting up the highest standards to uncompromisingly ensure the viability and identification of the contained strains.
These products are exclusively marketed as dietary supplements, also as therapeutics, but not as drugs, and are therefore subject to different manufacturing and quality control standards than approved drugs are. A large proportion of PPs are implied for use in infants, from birth onward, both in preventive and therapy purposes (Depoorter & Vandenplas 2021). As use of probiotics in infants and children is, for the past decade, very popular, the market of these kind of products is growing very fast. Health benefits of probiotics are species- and strain-specific, which is why the identification and number of the labeled microbes is a very important issue (Kolaček et al. 2017). A rather large number of studies showed irregularities in PPs quality. Davis (2014) provides an overview of several studies that reported that the PPs did not contain a declared probiotic count, but had significantly lower levels than declared. And more recent reports showed that content of several PPs on market did not correspond to the product claim regarding the species/ strain, number of living organisms and purity (Mazzantini et al. 2021). Important aspects to consider when determining the efficacy of probiotics are the accurate taxonomic identification and labeling of applied strains, the viability of strains administered and monitoring of product formulation over time (Patro et al. 2016). A wide range of different PPs are available on the market. These products include fermented food products or pharmaceutical supplements, which contain one or more probiotic strains. However, there is no uniform validation of these preparations at the national level. Hence, the objective of this work aimed at providing information on the analysis of the total cell counts of the component strains in probiotic formulation by 4UPharma, BABYTOL NEONATE FOR YOU® (later on in the text probiotic formulation designed as PP) and their survival in the simulated intestinal tract. The research was designed to address two key challenges associated with the use and administration of probiotics. The priority was to assess the viability of the strains of commercially available probiotic formulation and second to evaluate the survival of probiotics in the simulated infant gastrointestinal tract (GIT) conditions without and with infant formula.
Materials and methods
Quantification of probiotic cells
The quantification of probiotic cells was evaluated to assess the quality of selected probiotic preparations, primarily by counting the bacteria present in the different batches of probiotic formulation. In addition to nutrient agar, after dilution, the product content from two different batches in three to four biological replications was plated on the appropriate selective media under standardized cultivation conditions for the growth of labeled strains: heterotrophic plate, MRS agar (Oxoid, UK) for Lactobacillus species, TOS MUP propionate agar medium (Merck, Germany) for Bifidobacterium, but also to M17 agar and BHI medium for the detection of Gram-negative contaminants. Bacteria quantifying was based on the determination of the total number of colony-forming units (CFU) grown on a selective medium from serial dilutions and expressed as the number of CFU per milliliter of the original sample. The first dilution prepared in sterile physiological solution was serially diluted and inoculated onto media agar plates to validate the label claim count of CFU per mL of sample. The final dilutions ranging from −5 to −10 were inoculated to triplicate and incubated at 37°C for 48 or 72 h in anaerobic conditions. The counting of bacterial colonies was validated using the BZG30 Colony Counter (WTW, UK). An overview of the results of the microbiological analysis was summarized as detection (√) or no detection (x). The probiotic bacteria analyzed in this study were a consortium of Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus reuteri, Bifidobacterium infantis, Bifidobacterium breve and Bifidobacterium bifidum, with a declared total bacterial count 1,225 × 1010 CFU/mL.
Determination of colony morphology
Colony morphology and microscopic details such as Gram stain reaction and cell shape and lactic acid fermentation patterns and bile salt tolerance were evaluated. Individual bacterial colonies were picked from the MRS agar and stained by the Gram method. High magnification objectives were applied to examine bacteria by oil immersion microscopy using 100×. In addition, to confirm the physiological characteristics typical of Lactobacillus spp., but also of Bifidobacterium spp., as major short-chain acid producers, the acidification capacity and acid production were assessed.
In vitro assays for tolerance to bile
The in vitro methodology described by Charteris et al. (1998) was used to test strains for resistance to bile. The growth of strains in a MRS broth medium with (0.3% ox gall) and without bile acids was compared. Briefly, a 1% probiotic inoculum was grown overnight in MRS broth, centrifuged and washed three times in sterile distilled water. The tolerance of strains was determined by sampling aliquots and monitoring OD620 over time (Kozak & Cuthbert 2016).
Assessment of probiotic viability in vitro by stimulating the digestive tract of infants
The in vitro methodology was used, which mimics conditions encountered during in vivo human gastrointestinal transit. Simulated gastric fluid (SGF) was prepared by suspending pepsin (3 g/L) (Sigma, USA) in a 0.5% (w/v) sterile sodium chloride (Kemika, Croatia), followed by pH adjustment to 2.0 by adding concentrated HCl (Sigma-Aldrich, USA). Simulated small intestinal fluid (SSIF) was prepared by suspending 1 g/L pancreatin (Fluka, Switzerland) and 3.0 mg/mL bile salts (Difco, USA) in 0.5% (w/v) sterile sodium chloride and adjusting the pH to 8.
Contained probiotic strains were cultivated overnight in 5 mL MRS medium at 37°C. After washing in 1 mL physiological solution (0.9% w/w), the bacterial debris was resuspended in infant formula and in SGF and incubated at 37°C. Aliquots (100 μL) taken after 0, 1 and 2 h were plated on MRS agar or TOS agar for colony enumeration. After 2 h incubation in SGF, bacterial cells were separated by centrifugation (3,500 g , 5 min) and transferred into SSIF and incubated another 4 h at 37°C. Bacterial aliquots were sampled after 1, 2, 3 and 4 h for plate count by an indirect method. The experiment was carried out in several replicates.
DNA isolation and sequence analysis
After removing the supernatant and fat layer, the bacterial cells were incubated for 2 hours in 200 μL TE buffer supplemented with lysozyme (5 mg mL−1). Afterward, the samples were immersed in an ice bath to prevent heating during sonication. Cell lysates were sonicated trice (30 s with a 15 s pause) using the Sonopuls mini20 homogenizer (Bandelin, Germany) at 4°C. Genomic DNA was extracted with the Maxwell 16 Tissue DNA purification kit (Promega, USA) by using the Maxwell 16 Research System for automated DNA isolation. The DNA concentration was determined spectrophotometrically and the samples were stored at −20°C until further analysis.
Determination of DNA concentrations by NanoDrop spectrophotometry
DNA concentration and purity were measured from a 2 μL sample using the BioSpec-nano spectrophotometer (Shimadzu, Japan) at a wavelengths of 260 and 280 nm, with optical path length 0.7 nm. An elution buffer was used as a blank test. The 260/280 ratio was used as an indicator of the purity of the extracted DNA. The concentrations of the total DNA extracted from the contained probiotic strains from three lots were in the range of 105.98–163.12 ng/μl. DNA purity was also analyzed by UV absorbance on a BioSpec-nano spectrophotometer and the DNA with OD260/280 ratios of 1.8 was prepared for sequencing.
Sequence analysis
Incorporating strains were analyzed using bacterial tag-encoded flexible-titanium (FLX) amplicon pyrosequencing (bTEFAP®) at MR DNA Shallowater, TX (Dowd et al. 2008, Glassing et al. 2015). Briefly, the full-length 16S rRNA gene was amplified using degenerated versions of the universal bacterial 16S rRNA gene primers 27F (5′-AGRGTTTGATYMTGGCTCAG) and 1492R (5′-GGYTACCTTGTTACGACTT3′) in 35 cycles using the HotStarTaq Plus Master Mix Kit (Qiagen, USA) under the following conditions: 94°C for 30 s, followed by 53°C for 40 s and 72°C for 90 s, followed by a final elongation step at 72°C for 5 min. Amplified PCR products were confirmed by gel electrophoresis in a 2% agarose gel. The SMRTbell libraries (Pacific Biosciences, USA) were prepared, following the manufacturer’s user guide, and sequencing performed at MR DNA (www.mrdna.labcom, USA) on the PacBio Sequel, following the manufacturer’s guidelines. Sequence data was then processed using the MR DNA analysis pipeline (MR DNA, USA). Final OTUs were taxonomically classified using BLASTN against a curated database derived from NCBI (www.ncbi.nlm.nih.gov.) and compiled into each taxonomic level into ‘counts’ and ‘percentage’ files.
Results
Evaluation of the probiotic cell counts
In addition to ingredient labeling, most PPs contain information about the active-strain(s) they contain. To test the validity of claims of selected PP, the analysis of total cell counts of the constituent strains and assessment of their survival in the simulated conditions of the intestinal tract was performed. The types of media used to test for the presence of bacteria allow for the determination of the number of CFU per gram or ml of the product. To calculate the final CFU, the resulting colonies were multiplied by the dilution factor and averaged between the replicates. Selective media for the isolation of Lactobacillus spp., MRS agar, and TOS-MUP for the selective isolation of Bifidobacterium spp., were used and the number of probiotic viable cells was determined. In addition, nutrient agar, M17 agar and BHI agar were selected for preliminary screening for the presence of bacterial growth (Fig. 1).
Cultivation of the probiotic cultures on nutrient agar and selective growth media. Four independent biological replicates, with two technical replicates each, were performed.
Citation: Microbiota and Host 3, 1; 10.1530/MAH-24-0010
As expected, the growth was not observed (BHI) or was observed at significantly lower densities (M17, nutrient agar) when media other than MRS agar or TOS were applied for the inoculation of overnight grown probiotic culture (Fig. 1). After the product content was plated directly onto a selective MRS medium, the strains grew efficiently when incubated overnight at 37°C anaerobically (Fig. 2A). The determined number of cultivable probiotic cells was also high when plating on TOS MUP medium and counted after 48 and 72 h of incubation at 37°C (Fig. 2A and B). The colony counts ranged to high levels of 1011 to 1012 CFU/mL (Fig. 2A and B). The average value (
(A) Analysis of the number of grown colonies from the probiotic formulation PP on MRS agar after anaerobic incubation at 37°C (averaged two lots), expressed as Log(CFU/mL). (B) Analysis of the number of grown colonies from the probiotic formulation PP on TOS MUP medium after anaerobic incubation at 37°C. Data are expressed as the mean ± SD.
Citation: Microbiota and Host 3, 1; 10.1530/MAH-24-0010
Next, morphologically distinct, well-isolated single colonies were handpicked and further streaked into MRS broth and subjected to Gram staining after overnight growth. Two types of colony morphotypes were observed on MRS agar, those that form convex, white colonies that grow well and other spiky colonies significantly of a much smaller size. Upon Gram staining, microscopic analysis under a light microscope (Olympus CH20, Japan) at 100× magnification revealed that the bacteria present were Gram-positive, long, nonmotile, narrow, non-spore-forming rods that were either single cells or in clusters or chains typically for lactic acid bacteria. To confirm lactic acid bacteria characteristics, the growth of strains was monitored by measuring the drop in pH and increase in lactic acid production, which was significantly diminished as the stationary phase was approached, approximately 12–16 h after inoculation (Table 1).
Analysis of the pH decline of the overnight grown probiotic strains in MRS broth and quantification of the produced acids in the culture supernatant.
| Probiotic sample | pH | % SH | % acids |
|---|---|---|---|
| Sample 1 | 3.83 | 76 | 1.71 |
| Sample 2 | 3.84 | 76 | 1.71 |
| Sample 3 | 3.82 | 80 | 1.80 |
| AVR | 3.83 ± 0.01 | 77 ± 2 | 1.74 ± 0.05 |
Sequencing analysis
Phenotypic methods that allow the identification of LAB at the genus-species level are based on the morphological and physiological characteristic and therefore are limited in precise taxonomic identification. However, these approaches are rapid, affordable and are valuable tool for the characterization of specific physiological properties of probiotic strains and their preliminary classification. Therefore, here, in support to phenotypic analysis, a NGS approach was used to assess the contained strains. Several authors have investigated the potential of modern molecular methodologies for rapid labeling of PPs and identification of possible contaminants to meet quality control and safety (Kim et al. 2022, Tracey et al. 2023, Shehata & Newmaster 2023). NGS appears to be an excellent method of choice as a screening tool to determine whether microbial contaminants are present in PPs. bTEFAP® has been used to determine the Lactobacillus bacteria present and sequencing analysis confirmed that the probiotic formulation did not contain any other microbial contamination, filtered to 0.1%. High-throughput sequencing of the sample confirmed Lactobacillus rhamnosus GG, Lactobacillus acidophilus and Lactobacillus reuteri declared on its label. Sequencing analysis revealed that the product was qualitatively consistent across lots, with only limited apparent quantitative differences.
Assessment of probiotic cell viability in vitro by stimulating the digestive tract conditions
In vitro tests for evaluating acid or bile resistant strains are simple and important for predicting survival in the digestive system. First, the potential of strains to survive GI transit was assessed based on the in vitro susceptibility to 0.3% (w/v) bile salts, SGF and SSIF. In vitro sensitivity to 0.3% bile was assessed by monitoring growth in the presence of bile salts by measuring the optical density at 620 nm (Fig. 2).
Growth curves obtained by measuring the OD620 of probiotic cell suspension in the presence of bile for 7 h were prepared and a difference in the time required to reach the stationary phase was observed, compared to control growth. Exposure to bile salt reduced the survival of the strains; however, the effect is not so drastic during the first 3 hours of the exposure, and it is worth noting that the OD620 values still were high (Fig. 3). The typical transit time of consumed food, which converts in a lower parts of the intestine to bolus, to reach the colon is determined by the rate of gastric emptying time and the time it takes to pass through the small intestine. The transit time through the small intestine is relatively constant (approximately 2 h) (Qureshi et al. 2013). This period was therefore chosen to test the resilience of probiotic strains to simulated gastric contents. The multistrain probiotic formulation exhibited an appreciable level of survival (approximately 86.90% of initial count) and is considered tolerant to gastric transit. Survival was maintained during passage through simulated small intestinal transit (71.25%) during an additional 4 h of incubation, implying that the ingredient strains are tolerant (Fig. 4A and B).
Survival of strains from probiotic formulation PP during exposure to simulated 0.3% bile solution. The black box bars inside show a comparison of how given values, control growth versus growth in the presence of bile salts, reflect a decrease in OD620 nm.
Citation: Microbiota and Host 3, 1; 10.1530/MAH-24-0010
Analysis of the results obtained by biological replicates after performing microbiological analysis by plating the cell suspensions on (A) MRS agar.
Citation: Microbiota and Host 3, 1; 10.1530/MAH-24-0010
Cell mortality of probiotic strains after 6 h of incubation ranged between 1 to 3-log reduction in viability (ΔlogCFU/mL), indicating capacity to survive in simulated GIT conditions (maximum reduction after 6 h, Δlog CFU/mL = 3.161).
Discussion
In this research, the calculation of bacteria in the probiotic formulation was estimated by routine counting the total number of CFU grown on an agar plate from serial 10-fold dilutions, expressed as CFU per mL of the original sample. Through microbiological analysis, using the indirect method of counting bacterial colonies, it was established that the probiotic formulation contains significantly more than 106 CFU/mL, which, according to the recommendation, is the minimum prescribed concentration of bacterial cells per gram or milliliter of content for a particular formulation to express probiotic activity (FAO/WHO 2002). The number of probiotic cells complies with the declaration and exceeds the value prescribed as the minimum concentration of bacterial cells per milliliter or gram of the probiotic formulation. The numbers are estimated because only live cells capable of forming colonies, which is determined by the experimental conditions such growth media, incubation temperature, time and oxygen availability, are counted. To ensure quality control and to exclude that probiotic formulations contain contaminating microbes such as yeast and molds, their total viable counts, after incubation under aerobic conditions using selective media, should also be addressed. Furthermore, culture-based plating is the gold standard for microbiological analyses and is indispensable. However, as many studies suggest, these classical approaches should be integrated with molecular methods for precision analysis. Recent studies used culture-independent methodologies such as flow cytometry to enumerate individual probiotic strains or multistrain probiotics (Davis 2014, Ilango et al. 2016, Tracey et al. 2023). Tracey et al. (2023) highlighted the importance of further studies on determining the probiotic counts in the multistrain PPs by advanced approaches such as the application of flow cytometry and comparison of the results to the plate counts.
Further in this study, using bacterial tag-encoded FLX-titanium amplicon pyrosequencing (bTEFAP®), diversity of bacterial species in probiotic formulation was investigated. Specifically, the full-length 16S rRNA gene was amplified because the goal was to generate the long-read amplicons to encompass almost the entire 16s gene ∼1,400 bp and with an aim that this approach can result to define to genus and even to species level. Using this method, we tested whether the probiotic formulation contained a qualitative list of labeled Lactobacillus species. No undeclared species were detected above the threshold of 0.2% of total reads per sample. Shehata & Newmaster (2023) applied untargeted amplicon-based HTS of V3–V4 region of 16S rRNA gene and found that 95–97% of total reads per sample matched the target species. The analysis also revealed that amplicon-based HTS of the V5–V8 region of the 16S rRNA gene resulted in 99% of total reads per sample matching the target species. This integrated methodological approach, which combines culture-dependent and culture-independent techniques, is valuable for ensuring high-quality control of probiotic biomanufacturing production, monitoring the application of proprietary strains and establishing the relatedness between different research and commercial probiotic strains. Jackson et al. (2019) indicated the need for the establishment of validated methodologies for assessing the quality of probiotics, proposing whole-genome sequencing as an optimal solution, emphasizing the need for high-quality sequencing, which in turn affects technological feasibility and increases the cost of analysis; however, it offers a possibility to distinguish a single base pair. Regulatory standards and their enforcement are unequal, focused rather on safety than accuracy in labeling. Therefore, there is still a need to implement comprehensive quality control approaches to ensure that the PPs address the label claims (Jackson et al. 2019).
In order to survive intestinal transit, probiotic strains must tolerate the acidic conditions and released proteases and in situ concentrations of bile acids. Compared to gastric acid, bile is more harmful to probiotic cells due to its membrane-disrupting detergent-like properties (Ilango et al. 2016). Exposure of probiotic cells to bile affects the change in lipid structure in the cell membrane, and thus the permeability of the probiotic cell, which reduces the cell’s tolerance to environmental conditions. The survival was assessed by microbiological analysis of the ingredient probiotic strains based on the estimation of the number of viable probiotic cells. The viability of the component strains has been shown as greater than 7 log CFU/mL after 6 h of exposure to simulated conditions of GIT, and according to empirical recommendations, it must be higher than 6 log CFU/mL. Preliminary in vitro analysis has shown that contained strains exhibited effective survival capacity in simulated GIT conditions. It is important to emphasize that the survival of the probiotic strain through the digestive tract is only a preliminary step toward the analysis of the potential beneficial responses by the host. However, on the other hand, the milk proteins contained in human milk or infant formulas and intestinal lining may alleviate the adverse effects of upper intestine contents when probiotic formulations are ingested. Besides, the networked interaction between probiotic strains can impact the increase in survival and functional performance. In vitro analysis demonstrated that PPs performed well in survivability tests, both in simulated gastric and intestinal conditions of infant. PPs must be continually monitored and testing results should be available for review before PPs are recommended to the target populations. Although the results indicate a high quality of the tested probiotic formulation, it is important to emphasize that strict, detailed quality controls of PPs should always be implemented to ensure optimal health benefits for the target host.
Conclusion
The quality control of commercial PPs, with special emphasis on at-risk populations, still needs to be improved from the aspect of transparency. Therefore, PPs should meet rigorous quality standards, especially if they are intended for newborns, infants and children. According to our results, the tested product contains high cell numbers of probiotic strains as labeled. Moreover, the high cell number of probiotics was detected after the gastrointestinal in vitro trial, fulfilling one of the first requirements in the quality assurance assessment of probiotics.
Declaration of interest
The authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
Funding
4U Pharma Gmbh, Herisau, Switzerland, a limited liability company, funded this research, and its fund number is 44359.
Author contribution statement
Conceptualization was done by TN and JN. Methodology was given by MZ, MB and JN. TN and JN helped with software. Validation was done by TN and JN. Investigation was done by MB, MZ and JN. JN helped in data curation. TN and JN helped in writing of the original draft. JN helped in review writing and editing. Visualization was done by MB, MZ and JN. JN helped in supervision. TN helped with funding acquisition. All authors have read and agreed to the published version of the manuscript.
Data availability
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding author.
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