The Use of Probiotics Containing Lactobacillus Sp. to A1 Prevent a Primary Salmonella

The Use of Probiotics Containing Lactobacillus sp.To[A1] Prevent a Primary Salmonella sp. Infection in vitro[A2]

Faith Zimmerle

Department of Biological Science

Saddleback College

Mission Viejo, CA 92694[A3]

ABSTRACT

The effectiveness of many commercially available probiotics advertised to treat and prevent gastrointestinal (GI) illnesses is a serious source of skepticism. The main concern with these products is whether or not they are able to survive the highly acidic pH of the stomach in order to reach their target, the small intestine. Due to the vague nature of probiotic efficacy regulation, it was predicted that the efficiency of the inhibition of Salmonella typhimurium exhibited by three popular probiotic brands containing Lactobacillus sp. would decrease as the concentration of the probiotics exposed to a highly acidic environment was decreased (0.1 g/mL, 0.01 g/mL, 0.001 g/mL, C(-) where n=10). It was also predicted that there would not be a statistical difference between the efficiency of the brands at a constant concentration (0.1 g/mL). A modified version of the disc diffusion method was used to analyze the probiotics’ ability to resist a low pH as well as their efficiencies post-acid exposure. An ANOVA and post-hoc test was run[A4] within all three brands as well as between them (0.1 g/mL). The trends in statistical difference of efficacy and acid-resistance between low and high probiotic concentration illustrated throughout all statistical analysis supported the proposed hypothesis. However, protective mechanisms and slight composition differences between brands may have taken precedent at 0.1 g/mL.

INTRODUCTION[A5]

The use of probiotics to treat disease, mainly bacterial infections, is not a new subject of interest. However, their commercial popularity has exploded over the past decade. Though their mode of action, beneficial qualities, possible dangers, and over-all[A6] effectiveness are still debated, many strains of probiotics have been shown to inhibit the invasion and growth of pathogens in the gastrointestinal (GI) tract if they are able to successfully colonize there (Tamayo, 2008).Though various probiotic strains are thought to have health benefits outside of the GI tract, the gut is of special interest because most of the human normal [A7]microbiota or “healthy bacteria” are inhabited there and because it plays a very important role in the functionality of the immune system. The normal microbiota of the stomach is also referred to as “gut flora” and can vary in composition from person to person but is always present and necessary for the body to function normally. When the gut flora is disturbed or depleted due to an infection or the use of strong antibiotics, the human immune center—the stomach and small intestine—is left susceptible to pathogens. The use of probiotics has been shown to work in tandem with the GI tract to make it more difficult for pathogens to colonize and cause infection (Tortora, 2013).

A major issue with commercially available probiotics is whether or not they can even be called “probiotics”. The scientific criteria used to define a bacterial strain as a “probiotic” strain is that the strain must be specifically defined from its genus and species and shown to induce a health benefit at a specific dose and at a specific target.Therefore, in order for a product to scientifically be labeled as a “probiotic”, its suggested dose must contain only specific bacteria strains that have been shown to have entice advertised health benefit and must deliver the strains in a high enough concentration of colony forming units per unit time [A8](cfu/day is most often used) (Sanders, 2008). As of now, the legal definition, detailed and categorized in the Food, Drug, and Cosmetics Act (FDCA), and the scientific definition of “probiotic” are not one in the same. In fact, there is no concrete legal definition for the term “probiotic”. A “non-food” product’s categorization within the FDCA is highly dependent on the specificity of the health claims a company makes about their product, affecting whether or not they are subject to premarket investigation of their claims by the Food and Drug Administration (FDA) or are allowed to generically market their product until its meritor safety comes into question (Degnan, 2008). Since this legal grey area exists, it is likely that there are many products in circulation claiming to be something that they are not.

The importance of determining which bacterial strains can and cannot be called probiotics is extremely relevant when attempting to develop a new product or a treatment plan; however the biggest concern with commercially available probioticscurrently used to combat GI maladies, even if they fit the scientific definition of the term, is whether or not an effective amount of cfu’s can survive the low pH of the stomach (~2.5) to colonize in the small intestine[A9](DoronGorbach, 2006). If the probiotic is unable to reach the target organ and colonize, then probiotic is ultimately useless. Since the legal definition and regulation of probiotics are vague and do not specifically cover the effectiveness of a product’s transmission, it is exceedinglyimportant to investigate the possibility that many popular probiotic brands on the market may be ineffective.

The aim of this study is to analyze the effectiveness ofthree popular probiotic products containing Lactobacillus sp., a widely studied and accepted probiotic-rich genus (NLM, 2015), at preventing the growth of Salmonellatyphimurium, a representative pathogen for gastro-enteric infections, after they have been allowed to cultivate in an acidic environment. The products’ effectiveness will be studied at varied concentrations of 0.1 g/mL, 0.01 g/mL and 0.001 g/mL. It is expected thatthe effectiveness of a probiotic product containing Lactobacillus sp. at inhibiting the growth of S. typhimurium will decrease as the concentration of probiotic used to combat it is decreased. It is also expected that there will not be a statistical difference between the effectiveness of the probiotic brands at a high concentration (0.1 g/mL) due to their similarity in strain composition.

MATERIALS & METHODS[A10]

Nutrient agar (NA) powder, 30 Petri dishes, and a pure culture of S. typhimurium were obtained from Saddleback College’s Microbiology stockroom on 20 October 2015. Two liters of NA were prepared by dissolving 11.5 g of NA powder in 500 mL increments of de-ionized (DI) water. The NA was sterilized and then poured into 30 Petri dishes or plates[A11] using aseptic technique once it was cool enough to handle. Each plate was split into four quadrants; three quadrants for varying the concentration and one for the negative control. Ten plates were designated to each brand.The three Lactobacillus sp. containing probiotic products (Nutrient NOW: PB-8 Veg, Country Life: Power Dophilus, and 365: Daily Probiotic w/ Acinophilus) were purchased at Whole Foods Market in Laguna Niguel, California on 21 October 2015.

On 22 October 2015, 100 mL of pre-made and pre-sterilized nutrient broth (NB) was obtained from Saddleback College’s Microbiology stockroom and acidified to a pH of about 2.5, again using aseptic technique. The NB was allocated into nine sterile test tubes, each containing 10 mL. Each probiotic was then weighed out into three masses relatively close to 1 g, 0.1 g, and 0.01 g: Nutrient NOW(1.0078 g, 0.1024 g, 0.0122 g), Country Life (1.0040 g, 0.1001 g, 0.0126 g),[A12]365 (1.0024 g, 0.1037 g, 0.0132 g). The three masses of probiotic for the three brands were dissolved in the nine acidified NB tubes to create concentrations of 0.1g/mL, 0.01 g/mL and 0.001 g/mL and then incubated at 37°C for 24 hours.The following day, about 150 paper diffusion discs 6.0 ± 0.5 mm in diameter were hole-punched from filter paper and sterilized in an autoclave. The probiotic containing NB tubes were removed from the incubator and placed in a refrigerator to avoid over growth[A13].

On 26 October 2015, lawn spreads of pure S. typhimuriumwere inoculated onto the 30 NA plates using sterile swabs. Sterilized diffusion discs were dipped into the various probiotic solutions for three seconds and placed on top of the S. typhimuriumlawn spread in the correlating quadrant of the appropriate brand’s plate. All 30 plates were allowed to incubate for 48 hours at 37°C. After the incubation period (actual t=50hr[A14]), the zone of inhibition of each quadrant was measured in millimeters[A15]. The resulting data were grouped according to brand and concentration so that they could be compared using four separate single-factor analysis of variance (ANOVA). Threeof the analysis compared the concentrations within each brand and one compared the three brands at 0.1 g/mL. Each ANOVA was followed by apost-hoc Bonferroni Correction in order to identify any sources of statistical difference. Differences were considered significant within brands at p<0.0125 and between brands at p<0.0167.

RESULTS[A16]

The zone of inhibition measured in this experiment was an area surrounding the paper diffusion disc where S. typhimurium growth was entirely inhibited or out-competed by the probiotic. Normally, the disc diffusion method is used to measure a disinfectant’s or an antibiotic’s effectiveness at inhibiting pathogenic growth, therefore the diameter of the diffusion disc is included in the measurement. Because this experiment employed an amended version of this method using live microbes in solution, data including the probiotic growth within the disc diameter is used to discuss probiotic resistance to an acidic environment (Table 1) while data where growth within the disc diameter was subtracted out is used to discuss probiotic effectiveness (Table 2).Negative control values remain at zero in both cases. All statistical analysis were run on raw data sets and expressed in mean ± SEM.

Within Nutrient NOW: PB-8, there was a statistical difference between each concentration and the negative control as well as between [0.1] and [0.001] when disc diameter was included (ANOVA, p<0.0125). There was not a difference between sequential concentrations. When the disc diameter was subtracted, the difference between [0.001] and the negative control became insignificant (ANOVA, p<0.0125, Figure1).

Within Country Life: Power Dophilus, the only source of difference was between [0.1] and all other concentration groups when disc diameter was included (ANOVA, p<0.0125). When the disc diameter was subtracted, all differences were unchanged (ANOVA, p<0.0125, Figure2).

Within 365: Daily Probiotic, there was a difference between all groups except [0.001] and [0.01] as well as [0.001] and the negative control when disc diameter was included (ANOVA, p<0.0125). When the disc diameter was subtracted, the difference between [0.01] and the negative control became insignificant (ANOVA, p<0.0125, Figure 3).

Between the three brands at [0.1] with the disc diameter included, there was a difference between Country Life and Nutrient NOW and between Country Life and 365, yet there was no difference between Nutrient NOW and 365 (ANOVA, p<0.0167). When the disc diameter was subtracted, all differences were unchanged (ANOVA, p<0.0167, Figure 4).

Table 1[A19]. Mean zone of inhibition created by three probiotic brands at varied concentrationin millimeters ± SEM. Data include the diffusion disc diameter (6 mm[A20]). Concentrations are g/mL where a concentration of zero represents the negative control.

Table 2.Mean zone of inhibition created by three probiotic brands at varied concentrationin millimeters ± SEM. Data exclude the diffusion disc diameter (6 mm). Concentrations are g/mL where a concentration of zero represents the negative control.

Figure 1.Mean zone of inhibition created by Nutrient NOW: PB-8 at varied concentration. The blue series includes the disc diameter (6 mm) while the red series does not. *Statistical difference between [0.1] and [0.001] blue. **Statistical difference between [0.001] and negative control blue. ***Statistical difference between [0.01] and negative control red. Error bars are ± SEM.

Figure 3.Mean zone of inhibition created by 365: Daily Probiotic with varied concentration. The blue series includes the disc diameter (6 mm) while the red series does not. *Statistical difference between [0.1] and [0.01] blue. **Statistical difference between [0.01] and negative control blue. ***Statistical difference between [0.1] and negative control red. Error bars are ± SEM.

Figure 2.Mean Zone of inhibition created by Country Life: Super Dophilus at varied concentration. The blue series includes that [A21]disc diameter (6 mm) while the red series does not. *Statistical difference between [0.1] and [0.01] blue. **Statistical difference between [0.1] and negative control blue. Error bars are ± SEM.

Figure 4.Mean zone of inhibition created by all three probiotic brands at [0.1]. The blue series includes the disc diameter while the red series does not. *Statistical difference between NOW and Country Life. **Statistical difference between 365 and Country Life. Error bars are ± SEM.

DISCUSSION[A22]

In order to analyze both the resistance of the probiotic products to acidified NB and their efficacy at inhibiting S. typhimurium, two similar data series were analyzed. The only difference between the data is that in in series one (represented by the color blue in Figure 1-4), the 6 mm diameter of each paper diffusion disc was included in the zone of inhibition measurement while in series two (represented by the color red in Figure 1-4), it was subtracted out.The purpose of devising two data sets was to allow the usual method of disc inclusion data to reveal the consequences of probiotic exposure to a strong acidic environment similar to the human stomach. By allowing the negative control value to equal zero instead of 6 mm, probiotics that colonized on the diffusion discs but did not inhibit the surrounding S. typhimurium lawn could be measured, positively included in the calculated mean, and given the opportunity to contribute to a significant resistance to low pH at that concentration. Note that these measurements are not actual zones of inhibition but will be termed as such for the sake of consistency in this paper.

For Nutrient NOW, the significant difference between the low [0.001] and intermediate [0.01] concentrations and the negative control suggest that the brand is highly resistant to low pH. A lack of difference between sequential concentrations shows that this resistance was relatively unaffected by a change in concentration. These results were unexpected due to the fact that this brand was not encapsulated; a protective mechanism shared by the other two brands[A23]. Country Life exhibited more anticipated results. A significant difference observed only between the high concentration [0.1] and all subsequently lower concentrations[0.01, 0.001, (-)][A24] suggests that this brand is more likely to persist in a strongly acidic environment if a large number of cfu’s is introduced. However, a strange consequence of cultivating such a high concentration of Country Life was that the acidified NB became highly viscous with overgrowth. In 365, a descending trend of acid resistance with respect to concentrationwas demonstrated by the lack of difference between the low concentration [0.001] and the negative control and an observable increase in efficacydirectly related to the increase of probiotic concentration. The difference seen between Country Life and the other two brands at [0.1] was unexpected but irrefutable faced with the extreme over growth of the [0.1] County Life tube. This result may be due to the fact that the Country Life product was protected by a very thick-shelled capsule[A25].

Consequently, in order to discuss the effectiveness of the probiotics and the validity of the proposed hypotheses, the disc diameter had to be subtracted out due to the fact that the “true” zone of inhibition is measured outwardly from the diffusion disc. This is because the inhibiting substance is typically a solution that can readily diffuse out into the surrounding medium, like a disinfectant or an antibiotic (Friedrich et al, 2015). By excluding discs that exhibited growth on the 6 mm disc but did not extend outwardly, the “true” zone of inhibition could be measured and used to gauge the probiotics’ effectiveness against S. typhimurium.

For Nutrient NOW, the amendment of the difference between [0.001] and the negative control from significant to insignificant suggests that although it seemed that all levels of concentration for NOW were significantly resistant to the acid environment, the effectiveness of the brand at a low concentration [0.001] was severely affected by acid exposure. There was no change in the differences within Country Life from the acid-resistance analysis data. This confirms the idea that an adequately high concentration of a probiotic is a useful and often necessary protective mechanism that allows a probiotic product to be effective against pathogens (Sanders, 2008). The change in significance between [0.01] and the negative control within 365 reflect the conclusions suggested by the Nutrient NOW data while the lack of change between probiotic brands at [0.1] reflect the conclusions suggested by Country Life.

Two general conclusions can be drawn from this experiment. The first supports part one of the proposed hypotheses. The effectiveness of a probiotic was shown to decreases[A26] directly with its concentration. This is especially seen within Nutrient NOW: PB-8 and 365. The second conclusion also supports part one of the proposed hypothesis, but also serves to suggest why the null hypothesis or part two must be rejected. The resistance to highly acidic conditions of each of the probiotic brands was shown to be greatest at high concentrations. This conclusion supports part one of the proposed hypothesis because it draws a direct correlation between the acid resistance and effectiveness of the probiotics. This is logical because if a probiotic whose target is the small intestine is able to survive the low pH of the human stomach, it is more likely to reach its target and perform effectively. This same logic also explains why the suggested null hypothesis suggested about the difference in efficacy between brands at high concentrations is rejected. If all three probiotic products containing almost identical strains could be thought to have a “maximum survival concentration” at [0.1] then their slight differences in composition, their protective mechanisms (i.e. capsules versus tablets), their suggested dosage/number of cfu’s per dose, and other possible factors become much more important than the products’ ability to survive the stomach. All of these factors may provide further avenues of research on the mechanisms and effectiveness of probioticsagainst illness.

LITERATURE CITED[A27]

Degnan, Frederick H. 2008. The US Food and Drug Administration and Probiotics: Regulatory Categorization. Clinical Infectious Diseases 46:2 (133-136).

Doron S & Gorbach SL. 2006. Probiotics: their role in the treatment and prevention of disease. Expert Rev. Anti Infect. Ther. 4:2 (261–275).