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Article

In Vitro Evaluation of Commercial Probiotic Products Containing Streptococcus salivarius K12 by Assessment of Probiotic Viability and Inhibitory Potency against Respiratory Pathogens

by
Medana Zamfir
,
Iulia-Roxana Angelescu
* and
Silvia-Simona Grosu-Tudor
Institute of Biology Bucharest of the Romanian Academy, Splaiul Independentei No. 296, 060031 Bucharest, Romania
*
Author to whom correspondence should be addressed.
Processes 2023, 11(2), 622; https://doi.org/10.3390/pr11020622
Submission received: 9 January 2023 / Revised: 16 February 2023 / Accepted: 17 February 2023 / Published: 18 February 2023
(This article belongs to the Special Issue Microbiotechnology in Cosmetics, Pharmaceuticals and Food)
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Abstract

:
Upper respiratory infections (URI) are the most frequent illnesses, especially in children. The majority of those infections are prescribed broad-spectrum antibiotics, which are associated with various side effects and with the increase in multi-drug-resistant strains. A promising alternative approach is the administration of the probiotic strain Streptococcus salivarius K12 (SSK12) that colonizes the upper respiratory tract (URT) and produces the salivaricins A2 and B, which strongly antagonize the growth of key respiratory pathogens. However, since for food supplements no quality controls of the active probiotic ingredient are mandatory, the efficacy of commercial products containing SSK12 may vary. This study aimed to investigate the in vitro efficacy of several commercial SSK12-containing probiotics, positioned for the prevention of respiratory infections. The parameters evaluated to determine the in vitro efficacy included the viability of the probiotic bacterial strain and the minimum inhibitory dilution (MID) of the probiotic, determined by the agar spot method, against the pathogenic/potential pathogenic bacterial strains Streptococcus pyogenes FF22 and Micrococcus luteus T18. All tests were carried out both 12 and 24 months after manufacturing (AM) for each commercial product. The viability ranged from 9 × 108 to 4.4 × 109 CFU/serving at 12 months AM and from 8.5 × 107 to 2.8 × 109 CFU/serving at 24 months AM. The MID was, in general, positively correlated with the probiotic bacterium viability and varied between the commercial products, ranging from 10−5 to 10−7 at 12 months AM and from 10−4 to 10−7 at 24 months AM. Moreover, the inhibition zones related to the two indicator strains were variable in diameter for different products. The high variation of the in vitro efficacy of commercial products containing SSK12 may explain the different results reported in the literature regarding the clinical benefits of these preparations, and the determination of this parameter may be useful to evaluate the quality of probiotic products containing this bacterial strain.

1. Introduction

URI such as common cold, pharyngotonsillitis and otitis media are the most frequent illnesses worldwide [1,2]. Globally, the incidence rate of URI reached more than 17 billion in 2019, accounting for almost 43% of all disease and injury cases in the Global Burden of Diseases, Injuries, and Risk Factors Study (GBD 2019) [3]. Global data on the incidence rate of URI in 204 countries and territories from 1999 to 2019 show the highest incidence rate in children under 5 years old [3]. Moreover, a high share of those children suffers from recurrent respiratory infections (RRIs), with an incidence of 25% of all children under 1 year and of 6% of children during the first 6 years of life [4]. URI are the most important cause of hospitalization of children under 4 years of age [5]. The treatment options for respiratory tract infections often include cough suppressants, steam inhalation and anti-inflammatories for pain relief. However, antibiotics are administered to children who have persistent bacterial infections and to those at risk of complications. In addition, a high share of those infections are prescribed broad-spectrum antibiotics [6,7], which are associated with various side effects such as gastrointestinal effects (e.g., bloating, diarrhea, abdominal pain), headache, candidiasis, rhinitis and dizziness [8]). Moreover, the high consumption of broad-spectrum antibiotics has led to an increasing prevalence of Multi-Drug-Resistant (MDR) pathogens worldwide [9]. A multinational surveillance study showed that the percentage of MDR Streptococcus pneumoniae and beta-lactamase-producing Haemophillus influenzae or Moraxella catarrhalis, group A β-hemolytic streptococci strains, reached in some countries 25% of all identified strains [9]. In the past 15 years, the rate of penicillin failure has dramatically increased to almost 40% in some regions of the world [10].
A promising alternative approach for the prevention and treatment of URI is the probiotic strain Streptococcus salivarius K12 (SSK12) that colonizes the URT and produces the salivaricins A2 and B [11], both strongly antagonizing the growth of key respiratory pathogens such as Streptococcus pyogenes and Staphylococcus aureus, but also the growth of Micrococcus luteus. Moreover, these bacteriocins are partially antagonistic to Moraxella catarrhalis, Haemophilus influenzae and Streptococcus pneumoniae and may also have antiviral action [7].
The high prevalence of upper respiratory diseases, especially during the COVID-19 pandemic, and also the efficacy of Streptococcus salivarius K12 make the use of oral probiotics, both for the prevention and for the treatment of URI, a good alternative for both children and adults [12].
Several commercial probiotic products containing SSK12 have been marketed over the last decade. The clinical efficacy of the probiotic strain in reducing the incidence of respiratory infections has been reported in numerous clinical trials [7,13], although some found no significant benefits [14,15]. The clinical benefits of SSK12 include, among others, a significant decrease of the incidence rate of bacterial and viral infections of the URT, with a concomitant decrease in antibiotic consumption, a reduction of key pathogens in the oral cavity and a significant reduction of tonsillectomies [16,17,18,19,20,21]. In addition, clinical trials conducted in both adults [16] and children [17] demonstrated the reduction of the recurrences of bacterial pharyngotonsillitis by approximately 80% and 90%, respectively.
Previous studies have shown a dose–response relationship for the colonization of SSK12 at a critical level of 1 × 109 CFU/g [22], and there also seems to be a critical minimum concentration of SSK12 necessary to obtain significant inhibitory activity [23]. The majority of the commercial probiotic products are marketed as food supplements in the European Union, with no mandatory tests required to prove the viability of the probiotic strains claimed in clinical studies or on the packaging. Since other studies have shown significant differences in the quality of commercial probiotics in terms of probiotic viability and strain identification [24,25], an inconsistent quality of commercial products containing SSK12 could be an explanation for the few studies with SSK12 which could not find any clinical benefits.
Starting from this assumption, our study investigated the in vitro efficacy of commercial probiotics containing SSK12, positioned for the prevention and treatment of respiratory infections in both children and adults. The in vitro efficacy was assessed by analyzing the cell viability and antimicrobial activity of SSK12 present in the commercial products. Since water activity is a crucial aspect for the stability of probiotic products [26,27], we also determined this parameter.

2. Materials and Methods

2.1. Commercial Products Containing SSK12

Seven commercial products containing SSK12 were purchased from different online pharmacies and were analyzed at 12 months and 24 months AM. All products were stored under room temperature (<25 °C) by strictly following the advice on the product packaging. Table 1 provides an overview of the commercial products tested, including their matrix, packaging, direction of use and CFU counts claimed on the labels. Five of the products are delivered as a lozenge which should slowly dissolve in the mouth in order to ensure colonization of the oral cavity with the probiotic strain SSK12. Only two products are delivered as a powder for small children who are not yet able to consume a lozenge. The CFU counts claimed on the packaging vary between 500 million and 2.5 billion per serving. For most of the products, a daily dosage of 1 lozenge/sachet is recommended, which can be increased in times of high need. Now Foods OralBiotic® is the only product for which the daily dosage starts from 2 lozenges.

2.2. Average Weight

The average weight was determined by following the method described in the Pharmacopoeia 2.9.5. Basically, 20 lozenges/sachets were measured to determine their average mass.

2.3. Water Activity

The water activity of all products was determined at 12 and 24 months AM, using the Rotronic probe type HC2-AW-(USB) (Rotronic Instrument Corp., Hauppauge, NY, USA) and following the procedure described in the product manual.

2.4. In Vitro Efficacy of SSK12 from Commercial Probiotic Products

The parameters to evaluate the in vitro efficacy included the viability of SSK12 in the commercial products and its inhibitory activity against a typical pathogen species involved in URI, namely, Streptococcus pyogenes, and a strain of Micrococcus luteus, an opportunistic pathogen in immunocompromised patients, causing various infections, including pneumonia [28,29].
A first dilution of each product was prepared by aseptically suspending 5 times the minimum daily recommended dosage in 45 mL of PBS (Oxoid) and homogenizing for 5 min in a stomacher (MiniMix, Interscience, Saint-Nom-la-Bretèche, France). All tests were carried out at 12 and 24 months AM.

2.4.1. Viability Tests

From the first dilution of each tested product, serial 10-fold dilutions were plated on Columbia Blood Agar Base medium (BD Difco, Winnersh, UK) supplemented with 5 g/L of yeast extract, 2.5 g/L of glucose and 1 g/L of calcium carbonate [30], further referred to as CAB K12 medium, and incubated at 37 °C, 5% CO2 in air, for 24 h. The colony-forming units (CFU) were counted in the appropriate dilution and calculated per serving.

2.4.2. Inhibitory Efficacy

The Minimum Inhibitory Dilution (MID) was determined to evaluate the antimicrobial activity of the SSK12 strain in the commercial products against the bacterial indicator strains Micrococcus luteus T18 and Streptococcus pyogenes FF22 [31]. Both strains were purchased from the New Zealand Reference Culture Collection: Medical Section (NZRM 2283 and NZRM 2284, respectively).
The agar spot method [32] was used in order to determine the MID. Ten microliters of the serial 10-fold dilutions of each product were placed on the surface of CAB K12 agar plates and incubated at 37 °C, 5% CO2 in air, for 24 h, to allow the growth of SSK12. The agar medium was then covered with a thin layer of indicator lawn, i.e., 5 mL of Brain Hearth Infusion medium (BHI, Roth, Karlsruhe, Germany), supplemented with 0.7% agar and inoculated with 100 µL of the indicator strain. The plates were then incubated for another 24 h under the same conditions.
The MID was defined as the lowest probiotic dilution that resulted in no visible growth of the indicator strains around the SSK12 spots. The MID can therefore work as an indicator of the efficacy of the various commercial products analyzed in this study. Moreover, as a second indicator of the activity of the probiotic strain, the inhibition zones of all active dilutions of the SSK12 samples from each commercial product, was measured with a ruler. The tests were performed for three repetitive samples of each product, with three simultaneous tests of each dilution, and the results are presented as mean values ± standard deviation (SD).

2.5. Statistical Analysis

GraphPad Prism (GraphPad Software LLC, Boston, MA, USA) was used for the statistical analysis of the results. The significance level (alpha) was set at 0.05.

3. Results

3.1. Average Weight

As seen in Table 2, the average weight was, in general, close to the net weight claimed by the manufacturer. Higher differences between the claimed and the analyzed weights (of about 10%) were detected for the products Daily Defence-BLIS K12TM and Throat Guard PRO-BLIS K12TM.

3.2. Water Activity

The water activity at 12 months AM varied among the commercial products containing SSK12, ranging from 0.099 aw for Daily Defence Junior-LIS K12TM and 0.097 aw for Now Foods OralBiotic® to 0.245 aw for Throat Guard PRO-BLIS K12TM (Figure 1). The analysis further showed that the water activity values increased over time for all products. At 24 months AM, the Bactoblis® lozenges had the lowest water activity (0.137 aw), while the Daily Defence-BLIS K12TM product had the highest water activity (0.373 aw) among all products tested in this study.
The relative increase in water activity was, however, the highest for the Daily Defence Junior-BLIS K12TM product, corresponding to about 88%, while the lowest relative increase was observed for the Bactoblis® lozenge, corresponding to only 7.5%.

3.3. In Vitro Efficacy of the Commercial Products Containing SS K12

3.3.1. Viability Tests

The enumeration of viable SSK12 cells showed significant variations among the products (p < 0.0001) and at different timepoints. The viable cell counts ranged at 12 months AM from 9.0 × 108 CFU/serving for for Difflam® to 4.4 × 109 CFU/serving for Bactoblis®. In general, the viability of SSK12 decreased over time in all the tested products, but at a variable degree. At 24 months AM, the lower viability was detected for Daily Defence-BLIS K12TM, while the highest viability was found for the Bactoblis® lozenges, followed by Bactoblis® Sachet (Table 3). The latter two products were the only ones with CFU counts at 24 months AM still in the range claimed by the manufacturer.

3.3.2. Inhibitory Efficacy

Figure 2 shows two examples of the results obtained by the agar spot method used in order to determine the MID. A clear inhibition of the two indicator strains was detected for the Bactoblis® product up to a dilution of 10−7, both at 12 and 24 months AM. For the Daily Defence-BLIS K12TM product, a clear inhibition could only be observed at a dilution of 10−6 at 12 months AM and of 10−4 at 24 months AM, respectively.
A summary of the results for all the tested products is presented in Table 4. At 12 months AM, the highest inhibitory activities were detected for Bactoblis® and Bactoblis® Sachet. The MID in these cases was 10−7, and the inhibition zones were the largest recorded among the products, in relation to both indicator strains. These two products also maintained a MID of 10−7 up to 24 months AM, although the diameters of the inhibition zones were slightly smaller at the end of their shelf life.
Most of the other products showed clear inhibition zones up to a dilution of 10−6 (MID = 10−6) at 12 months AM, except for Now Foods OralBiotic®, with an MID of 10−5 at this time point. However, there were significant differences (p < 0.005) in the diameters of the inhibition zones among the products as well as in relation to the indicator strains used. In general, the inhibition zones obtained with Micrococcus luteus T18 were larger compared to those obtained when using Streptococcus pyogenes FF22.
At 24 months AM, except for the two Bactoblis® products, only Difflam® 1st Signs Defence Probiotic had the same MID as that at 12 months AM (MID = 10−6). For all the other products, we could observe a decrease in inhibitory activity over time, in terms of both the MID value (1–2 points lower compared to the MID at 12 months AM) (Table 4) and the diameter of the inhibition zones (results not shown).
A statistical analysis of all the results (Figure 3) showed that the alu–alu packed Bactoblis products, both at 12 and 24 months AM, grouped together, having the highest CFU values of SSK12 and the lowest MID, but also showing the lowest increases in water activity at 24 months. For the two products packed in plastic/glass bottles, namely, Now Foods Oral Biotics and Daily Defence Junior, we observed a high increase in water activity at 24 months, with a concomitant decrease of probiotic viability and an increase in MID. A similar pattern was observed for Daily Defence and Throat Guard PRO, packed in alu–PVDC. In the case of Difflam, however, an increase in water activity did not have a significant effect on probiotic viability and MID.

4. Discussion

The number of clinical trials using oral probiotics is still limited, but the field is rapidly expanding. In general, the published studies recognize that S. salivarius K12 has a low pathogenicity and is well tolerated, but the results regarding its clinical benefits differ from one study to another [7,13,14,33]. For instance, in the presence of acute pharyngotonsillitis treated with penicillin, SSK12 probiotics are unlikely to be effective [14] but may be used as an alternative to antibiotics in non-severe cases or for the prevention of disease recurrence [7]. On the other hand, the prophylactic administration of SSK12 seems to reduce streptococcal and viral infections in children with a history of recurrent oral streptococcal disease [17].
The variable results obtained in different clinical trials may be explained by the use of different SSK12 formulas. Since all these probiotics contain the same active bacterial strain, the reason for the variable efficacy may be found in the different efficacy of this strain in the particular formulation of each product, but also in the concentration and stability of the product. In this context, we aimed to study the differences among several commercial probiotics containing SSK12 in terms of in vitro efficacy. Probiotic viability and inhibitory efficacy were evaluated both in the middle (12 months AM) and at the end of the claimed products’ shelf life (24 months AM).
Recent studies showed that stability is a critical issue for commercial products containing live probiotics, and often only freshly produced products contain the number of viable cells claimed on the product packaging [34,35]. In our study, for most products, the number of viable cells claimed by the manufacturer could be confirmed at 12 months AM. The number of live probiotic bacteria decreased over time in all products. However, the decrease varied among the products, and we could observe an inverse correlation between the viability of the active strain and the water activity of the product (r = −0.54). It was previously suggested that water activity is one of the main factors influencing a probiotic stability over the shelf life of a product [27]. The lowest SSK12 viability at 24 months AM was observed for DailyDefence-LIS K12TM, which also appeared to be the product with the highest water activity (0.373 aw). In general, the water activity highly differed between the commercial products and increased over time. In order to reach a high stability of a probiotic, a low water activity of the finished product is one of the most crucial aspects. According to Fenster et al. (2019) [36], once a low-water-activity probiotic product has been produced, a low water activity can be maintained by choosing a type of packaging with adequate moisture vapor transmission rate (MVTR). The lower the MVTR, the slower the moisture increase, and the better can the viable cell count be maintained over the shelf life of the product [36]. Therefore, ensuring a low water activity during manufacturing and throughout the shelf life of a probiotic product is a key factor for the viability of the product. Moreover, the oxygen transmission rate (OTR) is the second crucial aspect when discussing different packaging options. The packaging of the commercial products tested in this study was very diverse, ranging from high-quality alu–alu-blisters to alu–PVDC blisters or plastic bottles for the lozenge products and from alu sachet packaging to glass bottles for the powder products. The products with the highest SSK12 viability at 12 months AM were Bactoblis® and Bactoblis® Sachet, with CFU counts of 4.4 × 109 and 2.9 × 109, respectively. These products also presented the highest cell counts at 24 months AM, namely, 2.8 × 109 and 2.4 × 109, respectively. Both products are packed in alu–alu blisters/sachet, a packaging option which is usually more expensive but offers low MVTR and low OTR and guarantees a high viability of the probiotics over the product’s shelf life [37]. For all the other products, the viability at 12 months AM was, in general, close to that mentioned by the producer, but it decreased in the following 12 months below the critical level of 1 × 109. The viable cell counts of DailyDefence-BLIS K12TM even dropped to 8.5 × 107 CFU/serving. The alu–PVDC packaging of this product, with its higher MVTR and OTR, might be an explanation for this significant drop in CFU counts over time.
The MID against the two bacterial strains Micrococcus luteus T18 and Streptococcus pyogenes FF22 was the second parameter we examined to evaluate the in vitro efficacy of the tested commercial products. Since Streptococcus pyogenes and Micrococcus luteus are among the pathogenic/opportunistic pathogenic species causing bacterial URI, the MID can be used as an indicator of the antimicrobial activity of commercial probiotic products against bacterial pathogens involved in URI. The lower the MID is, the more potent may the SSK12 strain be in the probiotics. We observed that the MID was, in general, directly related (r = 0.82) to the viability of SSK12 in the commercial products. The two Bactoblis® products, with a viability of over 2.4 billion CFU/dose, both at 12 and 24 months AM, had the lowest MID, of 10−7, during the entire shelf life. In addition, the inhibitory activity of SSK12 evaluated by measuring the diameters of the inhibition zones was the highest for these two products. Decreased functional properties, such as the inability to inhibit a certain pathogen, have been previously described and related to the manufacturing processes and the probiotic matrix [38].
Having in mind that previous studies showed a dose–response relationship for the colonization of SSK12 at a critical level of 1 × 109 CFU/g [22] and that a critical minimum concentration of SSK12 seems to be required to obtain significant inhibitory activity [23], it is very likely that most of the commercial products tested in this study do not provide clinical benefits associated with SSK12 such as a reduction in the incidence rate of bacterial and viral URI (pharyngitis, tonsilitis, otitis media, influenza, COVID-19) and consequently a reduction in antibiotic and anti-inflammatory intake, when taken later than 12 months AM.
Moreover, for two of the products, namely, Daily Defence-BLIS K12TM and Throat Guard PRO-BLIS K12TM, a difference of about 10% between the claimed and the measured net weight was observed, which is above the maximum percentage deviation of 5% for lozenge products of 250 mg or more, defined in the European Pharmacopoeia (European Pharmacopoeia 10.0; 2.9.5 Uniformity of Mass of Single Dose Preparations).

5. Conclusions

The results showed a high variation in the concentration and inhibitory activity of SSK12 found in commercial products. Many of the products provided, at 24 months AM, a concentration of live SSK12 below the critical level of 1 × 109, which may be an explanation for the lack of benefits reported by the few clinical trials with SSK12 for the prevention of URI. Moreover, most of the commercial products showed a low in vitro inhibitory activity of SSK12, which again may explain the different clinical efficacies reported.
This study clearly demonstrates that the quality of commercial supplements containing SSK12 highly varies, as it has been shown for many other commercial probiotic supplements [39]. In order to ensure the outstanding clinical efficacy reported for the probiotic strain SSK12 in more than 20 clinical trials, the commercial products containing SSK12—or any other commercial probiotic—should be tested much more rigorously in terms of quality before being marketed. More rigorous quality control is also required to ensure that the products contain the viable cell counts claimed by the manufacturer over the period they are available in the pharmacy.
In this respect, the probiotic viability and antimicrobial potency might be important criteria to evaluate the quality and clinical efficacy of probiotic products containing SSK12. They are simple parameters that may be evaluated in systematic quality control procedures of commercial probiotics which claim a mode of action based on an antibacterial activity.

Author Contributions

Conceptualization, M.Z.; methodology, I.-R.A. and S.-S.G.-T.; formal analysis, I.-R.A., S.-S.G.-T. and M.Z.; writing—original draft preparation, I.-R.A. and S.-S.G.-T.; writing—review and editing, M.Z.; supervision, M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This research received no external funding.

Data Availability Statement

The data presented in this study are available in the article.

Conflicts of Interest

The authors declare no conflict of interest.

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Processes 11 00622 g001 550
Figure 1. Water activity of the analyzed commercial products, measured at 12 and 24 months AM. The results are presented as mean values ± standard deviation.
Figure 1. Water activity of the analyzed commercial products, measured at 12 and 24 months AM. The results are presented as mean values ± standard deviation.
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Figure 2. Inhibitory activity of Bactoblis® (a) and Daily Defence-BLIS K12TM (b) at 12 and 24 months AM. The inhibitory activity was determined by the agar spot method, using Micrococcus luteus T18 and Streptococcus pyogenes FF22 as indicator strains. The numbers represent the decimal dilutions of each tested sample.
Figure 2. Inhibitory activity of Bactoblis® (a) and Daily Defence-BLIS K12TM (b) at 12 and 24 months AM. The inhibitory activity was determined by the agar spot method, using Micrococcus luteus T18 and Streptococcus pyogenes FF22 as indicator strains. The numbers represent the decimal dilutions of each tested sample.
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Figure 3. Principal component analysis (PCA) of the probiotic products at 12 and 24 months after manufacture (AM). BL = Bactoblis Lozenge; BS = Bactoblis Sachets; DD = Daily Defence; DDJr = Daily Defence Junior; Dif = Difflam; TG = Throat Guard PRO; NF = Now Foods Oral Biotics. The numbers (12/24) indicate the time of testing, namely, 12/24 months AM.
Figure 3. Principal component analysis (PCA) of the probiotic products at 12 and 24 months after manufacture (AM). BL = Bactoblis Lozenge; BS = Bactoblis Sachets; DD = Daily Defence; DDJr = Daily Defence Junior; Dif = Difflam; TG = Throat Guard PRO; NF = Now Foods Oral Biotics. The numbers (12/24) indicate the time of testing, namely, 12/24 months AM.
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Table
Table 1. Commercial probiotics containing SSK12 used in this study.
Table 1. Commercial probiotics containing SSK12 used in this study.
ProductManufacturerMatrixPrimary Packaging of the Products §Direction of UseCFU Claimed per Serving *
Bactoblis®Bluestone PharmaLozengealu-alu blister 1 lozenge per day1 billion CFU/lozenge 1
Bactoblis® SachetBluestone PharmaPowderalu-sachet1 sachet per day1 billion CFU/sachet 1
DailyDefence-BLIS K12TMBLIS TechnologiesLozengealu-PVDC blister1–2 lozenges per day. In times of high need, take a maximum of 4 lozenges per day 1.25 billion CFU/lozenge 2
DailyDefence Junior-BLIS K12TMBLIS TechnologiesPowderglass bottle1–2 times per day1.25 billion CFU/dose-powder 2
ThroatGuard PRO-BLIS K12TMBLIS TechnologiesLozengealu-PVDC-blister1–2 lozenges per day. In times of high need, take a maximum of 4 lozenges per day2.5 billion CFU/lozenge 2
Difflam® 1st Signs Defence ProbioticDifflam®Lozengealu-PVDC blister1 lozenge, two times per day500 million CFU/lozenge 2
Now Foods OralBiotic®Now FoodsLozengeplastic bottle2 lozenges, 1–4 times per day1 billion CFU/2-lozenges 2
§ alu = aluminum; PVDC = polyvinylidene chloride; * minimum intake recommended by the manufacturer; 1 CFU claimed after 24 months shelf live; 2 CFU claimed at the date of manufacturing.
Table
Table 2. Claimed and analyzed net weights of the commercial products.
Table 2. Claimed and analyzed net weights of the commercial products.
ProductNet Weight Claimed by ManufacturerNet Weight Analyzed
Bactoblis®950 mg/lozenge950 ± 16 mg/lozenge
Bactoblis® Sachet1.5 g/sachet1.47 ± 0.01 g/sachet
Daily Defence-BLIS K12TM1 g/lozenge902 ± 18 mg/lozenge
Daily Defence Junior-BLIS K12TMabout 1 g/powder dosen.a.
Throat Guard PRO-BLIS K12TM1 g/lozenge901 ± 3 mg/lozenge
Difflam® 1st Signs Defence ProbioticNo information on the packaging903 mg ± 5 mg
Now Foods OralBiotic®500 mg/lozenge506 ± 4 mg/lozenge
n.a. = not applicable, since the product is delivered as a powder and has to been taken with a measuring spoon provided by the distributor.
Table
Table 3. Probiotic viability in the examined commercial products at 12 and 24 months AM.
Table 3. Probiotic viability in the examined commercial products at 12 and 24 months AM.
ProductCFU/Serving at
12 Months AM		CFU/Serving at
24 Months AM
Bactoblis®4.4 × 109 ± 2.8 × 108 2.8 × 109 ± 1.4 × 108
Bactoblis® Sachet2.9 × 109 ± 1.1 × 1082.4 × 109 ± 1.5 × 108
Daily Defence-BLIS K12TM1.4 × 109 ± 5.0 × 1078.5 × 107 ± 2.6 × 106
Daily Defence Junior-BLIS K12TM1.2 × 109 ± 6.9 × 1077.0 × 108 ± 6.9 × 107
Throat Guard PRO-BLIS K12TM1.3 × 109 ± 7.8 × 1073.7 × 108 ± 2.5 × 107
Difflam® 1st Signs Defence Probiotic9.0 × 108 ± 5.0 × 1078.5 × 108 ± 9.7 × 107
Now Foods OralBiotic®1.6 × 109 ± 9.5 × 1072.6 × 108 ± 8.7 × 106
Table
Table 4. Minimum inhibitory dilution (MID) of the commercial products and diameters of the inhibition zones they produced when tested against Micrococcus luteus T18 and Streptococcus pyogenes FF22 at 12 months and 24 months AM.
Table 4. Minimum inhibitory dilution (MID) of the commercial products and diameters of the inhibition zones they produced when tested against Micrococcus luteus T18 and Streptococcus pyogenes FF22 at 12 months and 24 months AM.
ProductDiameter of the Inhibition Zones (mm) *
12 Months AM24 Months AM
M. luteus T18S. pyogenes FF22M. luteus T18S. pyogenes FF22
10−610−710−610−710−610−710−610−7
Bactoblis®14 ± 0.310 ± 0.715 ± 0.66 ± 0.515 ± 0.56 ± 0.614 ± 0.95 ± 0.9
Bactoblis® Sachet20 ± 1.114 ± 0.817 ± 0.411 ± 115 ± 0.29 ± 1.215 ± 1.14 ± 1.8
Daily Defence-BLIS K12TM12 ± 2.7n.a.5 ± 1.2n.a.MID = 10−4
Daily Defence Junior-BLIS K12TM13 ± 0.4n.a.13 ± 0.1n.a.MID = 10−5
Throat Guard PRO-BLIS K12TM13 ± 1.2n.a.9 ± 0.9n.a.MID = 10−4
Difflam® 1st Signs Defence Probiotic4 ± 0.4n.a.5 ± 0.8n.a.10 ± 1.8n.a.9 ± 2.8n.a.
Now Foods OralBiotic®MID = 10−5MID = 10−4
* n.a. = no activity (no inhibition could be observed at this dilution); MID = minimum inhibitory dilution. * for a comparison, the diameters of the inhibition zones at the dilutions of 10−6 and 10−7 are shown in the Table; when no inhibition was detected at any of these two dilutions, the MID value is mentioned in the table; no inhibition could be detected at dilutions higher than 10−7 for any of the tested products.
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Zamfir, M.; Angelescu, I.-R.; Grosu-Tudor, S.-S. In Vitro Evaluation of Commercial Probiotic Products Containing Streptococcus salivarius K12 by Assessment of Probiotic Viability and Inhibitory Potency against Respiratory Pathogens. Processes 2023, 11, 622. https://doi.org/10.3390/pr11020622

AMA Style

Zamfir M, Angelescu I-R, Grosu-Tudor S-S. In Vitro Evaluation of Commercial Probiotic Products Containing Streptococcus salivarius K12 by Assessment of Probiotic Viability and Inhibitory Potency against Respiratory Pathogens. Processes. 2023; 11(2):622. https://doi.org/10.3390/pr11020622

Chicago/Turabian Style

Zamfir, Medana, Iulia-Roxana Angelescu, and Silvia-Simona Grosu-Tudor. 2023. "In Vitro Evaluation of Commercial Probiotic Products Containing Streptococcus salivarius K12 by Assessment of Probiotic Viability and Inhibitory Potency against Respiratory Pathogens" Processes 11, no. 2: 622. https://doi.org/10.3390/pr11020622

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