The probiotic Lacticaseibacillus paracasei strain Shirota (LcS) in a fermented milk beverage survives the gastrointestinal tract of generally healthy U.S. Adults

Abstract The probiotic strain Lacticaseibacillus paracasei (previously Lactobacillus casei) strain Shirota (LcS) has demonstrated its survivability in the gastrointestinal tract across populations in different countries. The objective of this study was to validate this survivability in the United States, where evidence is lacking. Faecal samples were collected from 26 healthy individuals (age: 32.0 ± 5.9 years) at baseline, after 7 and 14 days of daily consumption of 80 mL fermented milk containing 108 colony forming units (CFU) LcS/mL, and after a subsequent 14-days of no product consumption. Live LcS counts significantly (p < 0.001) increased after 7 and 14 days of product consumption (6.37 ± 1.18 and 5.24 ± 1.81 log10 CFU/g faeces, respectively) and returned to baseline in 87% of participants. These results indicate LcS survives passage through the gastrointestinal tract of generally healthy U.S. adults, providing support for its uniquely accumulated evidence of universal survival capacity in the gastrointestinal tract.


Introduction
Probiotics, found predominantly in fermented foods and dietary supplements, are live microorganisms (mostly bacteria) that confer a health benefit on the host when consumed in adequate amounts (FAO/ WHO 2001;Hill et al. 2014). Different probiotics have been linked to a range of health benefits, with some of the more promising areas including digestive health and immune system support (Wilkins and Sequoia 2017;Khalesi et al. 2019;Maldonado Galdeano et al. 2019). Indeed, the utilisation of probiotics has been increasing in the general population, especially in the United States (O'Connor et al. 2021), as a growing body of evidence supports the beneficial impact of probiotics on human health. This growing demand for probiotics has put an emphasis on the proper selection of bacterial strains that might be defined as probiotic bacteria. One of the most common commercially available probiotics for human consumption belongs to the Lactobacillus genus (Kligler and Cohrssen 2008). Recently, the genus Lactobacillus has been reclassified into 25 genera including Lacticaseibacillus, due to the number of species that are extremely diverse at the phenotypic, ecological, and genotypic levels (Zheng et al. 2020). The effectiveness of these bacteria to confer a health benefit to the host is contingent on their ability to survive and proliferate in the gastrointestinal tract, but survival can vary considerably among the different strains (Yuki et al. 1999;Guergoletto et al. 2010;Sahadeva et al. 2011;Naissinger da Silva et al. 2021).
Many factors can influence survival, in particular variability in the resistance to gastric acid and interaction with bile salts (Ruiz et al. 2013). When probiotics are ingested, they encounter a hostile environment beginning in the stomach. The duration of time spent in the stomach affects survival; thus, the gastric emptying rate is an important factor that influences the survival of bacteria by limiting exposure to gastric acid. Gastric emptying rate can vary both within and between individuals due predominantly to differences in the type and composition of food consumed in the habitual diet (Welch et al. 1988;Doran et al. 1998), but also to other biological factors such as health status (e.g. presence of gastrointestinal disease) (Qu et al. 2013;Vijayvargiya et al. 2019), age (Horowitz et al. 1984), sex (Datz et al. 1987), and/or race (Qu et al. 2013). Additionally, the small intestine, especially the proximal portion, contains hydrolytic enzymes and bile salts that can be lethal to microorganisms. Consequently, it is clear that the ability to persist through the gastrointestinal tract significantly influences the survival of ingested probiotic bacteria (Berrada et al. 1991;Oozeer et al. 2004;. Importantly, Lacticaseibacillus paracasei (the previous taxonomic nomenclature was Lactobacillus casei) strain Shirota (LcS) is one such probiotic that has been shown in multiple studies to survive passage through the gastrointestinal tract of several different human races and ethnicities (Yuki et al. 1999;Tuohy et al. 2007;Sakai et al. 2010;Tiengrim et al. 2012;Utami et al. 2015;Wang et al. 2015;Mai et al. 2017;Cox et al. 2019;Khine et al. 2019).
Given that the effects of probiotics are strain specific and may vary between different population groups, it is important to confirm the results of prior studies demonstrating that LcS is recoverable in previously unstudied cohorts. Therefore, the objective of this study was to investigate the survival of LcS in the gastrointestinal tract of generally healthy adults in the United States after consumption of a fermented milk containing viable 8 billion LcS per 80 mL.

Participants
Men and women, 18-40 years of age (inclusive), each with a body mass index (BMI) between ≥18.5 to <30.0 kg/m 2 who were in good general health based on medical history and had regular bowel habits that included consistently having a bowel movement daily (preferably in the morning) were eligible for the study. Important exclusion criteria with respect to potential confounding effects on outcomes of interest included the presence of any clinically important gastrointestinal condition(s) that would potentially interfere with the evaluation of the study product, in particular a history of functional constipation or diarrhoea as defined by the Rome IV criteria (Drossman and Hasler 2016), within 6 weeks of screening. The use of proton pump inhibitors, H2 receptor antagonists, anticoagulants (except for 81 mg aspirin), corticosteroids, antibiotics, antifungals, antiparasitics, antidiarrheals, laxatives, or regular (>3 d/ week) use of non-steroidal anti-inflammatory drugs was not allowed within 30 days of screening and throughout the study period. Additionally, unstable use of certain medications was also considered exclusionary, including a change in dose or medication of anti-hypertensives within 90 days of screening. Individuals with a known allergy or sensitivity to any ingredient in the study product were also excluded.

Study design
This was a single-arm, open-label study design ( Figure  1). Briefly, following an in-clinic screening visit (Day 0), eligible participants began a 14-day run-in period with no product consumption, followed by a 14-day period of daily study product consumption, and a final 14-day period with no product consumption. For each clinic visit on days 14, 21, 28, and 42, participants collected a single stool sample sometime after 7:00 pm in the evening prior to the visit and submitted the sample in a cooler with ice packs maintained cold documented by a temperature logger. Throughout the entire 42-day study period, participants were instructed to maintain daily habitual diet, body weight and lifestyle patterns, but to avoid consuming any dairy or non-dairy fermented products (examples included probiotics, supplements, and fermented foods such as kefir, cultured dairy products, unpasteurised cheeses, kimchi, kombucha, natto, and yogurts with probiotics.) A daily electronic log was completed to document 1) compliance with daily study product consumption, 2) compliance with instructions to avoid fermented products, and 3) record any changes in concomitant medication/supplement use. Adverse events were assessed by an open-ended question at the beginning and end of each clinic visit and participants were counselled to contact the clinic with concerns or discomforts between visits.
The protocol was approved by Sterling Institutional. Review Board (IRB) (Atlanta, GA) on 25 July 2022, and all subjects provided informed consent prior to study commencement. Sterling IRB is fully accredited by the Association for the Accreditation of Human Research Participation Protection Programs (AAHRPP). This study was registered in the US ClinicalTrials.gov (identifier NCT05522777) prior to subject enrolment.

Study product
The study product was a fermented milk containing viable 8 billion LcS per 80 mL (10 8 colony forming units [CFU]/mL) produced by Yakult U.S.A. Inc. and commercially available under the brand name Yakult. Study products were provided as ready-to-drink beverages in individual bottles containing 12 g carbohydrate, 0 g fat, and 1 g protein (50 kcal/80 mL serving). Participants were instructed to maintain study product under refrigerated conditions (<10 °C) with storage conditions confirmed via a temperature logger.
At the baseline visit, participants began consuming one serving (one 80 mL bottle) of study product in the clinic within 1 h of breakfast and were instructed to begin consuming one serving/day within 30 min of breakfast thereafter for 14 days. If participants missed consuming study product in the morning, they were counselled to consume the product as soon as possible on the same day but cautioned not to consume more than one serving/day.

Quantification of LcS in faecal samples
Bacterial enumeration was conducted using the lactitol-LBS vancomycin plus fosfomycin medium (LLV-FOM) culture method and polymerase chain reaction (PCR) approaches, reported as log 10 CFU/g faeces (Tiengrim et al. 2012). Faecal samples were homogenised in nine volumes of transport medium consisting of 0.23 g KH 2 PO 4 , 0.23 g K 2 HPO 4 , 0.45 g NaCl, 0.23 g (NH 4 ) 2 SO 4 , 0.02 g CaCl 2 , 0.02 g MgSO 4 , 0.5 g L-cysteine hydrochloride, 0.5 g agar, 10 g Lab-Lemco powder, and 100 mL glycerol per litre. A serial dilution (10-fold) of each faecal suspension was created using phosphate buffered saline (PBS) to 10 −6 . A 0.1 mL aliquot of each diluted sample was distributed over LLV-FOM plates and incubated in an aerobic environment at 37 °C for 96 h. LcS counts were determined from plates that developed 30-300 large, white, dome shaped colonies. A study beverage from the same lot of drinks consumed by participants was diluted at 10 −5 , 10 −6 and 10 −7 and cultured at each time point to serve as a positive control. Within each plate, up to eight randomly selected colonies were verified as LcS using a colony-direct PCR method with the forward primer LcS57F (5′-CTCAAAGCCGTGACGGTC -3′) and the reverse primer LcS-597R (5′-ACGTGGTGCTAATAATCCTAGTG-3′) (Fujimoto et al. 2008). PCR products were applied to an agarose gel electrophoresis to confirm presence and product size of the bands. The number of LcS per gram of faecal sample was calculated by the following formula: The lower limit of detection (LOD) was set to be 2.0 log 10 CFU/g faeces.

Compliance
Compliance with study product consumption was based on the number of returned bottles of the fermented milk beverage during the ingestion period. Additionally, a daily study diary was reviewed by clinic staff to determine intake of other fermented foods and concomitant medications that had the potential to influence study results.

Statistical analysis
Statistical analyses were performed using SAS version 9.4 (Cary, NC). LcS (log 10 CFU/g faeces) was evaluated with a repeated measures model with an unstructured covariance. The LcS number measured at each time point was included in the response vector. Counts below LOD were replaced with the value of the LOD (2.00) to allow for statistical analysis (Cox et al. 2019). The model derived absolute change in LcS from baseline (Day 14) to each of Days 21, 28, and 42 were estimated along with the corresponding 95% confidential interval (CI). Though the model suggested deviations from normality, no other transformation nor sub-setting to post consumption time points improved the model fit. Therefore, the repeated measures model included all time points and the log 10 CFU/g was used. Additionally, in the subset of subjects with elevated log 10 CFU/g while consuming study product (measured at Day 21 or 28), as compared to baseline, the proportion of subjects that returned to baseline by Day 42 were estimated along with a 95% CI.

Subject disposition
A total of 34 individuals were screened and 26 were enroled (Figure 2). One subject withdrew consent at baseline (Day 14) due to schedule concerns and did not provide any faecal samples, one subject terminated early (moved out of state) after Day 28 (end of ingestion period) but prior to Day 42 (end of no product ingestion follow up period) and therefore did not provide a faecal sample at Day 42, and one subject's Day 28 faecal sample was not usable due to competing bacterial growth that resulted in high background noise and LcS was not able to be counted. Therefore, in the intent-to-treat (ITT) analysis, data for faecal sample LcS counts were available for 25 participants at baseline (Day 14) and during study product consumption on Day 21, while data were available for 24 participants for the end of the study product consumption period (Day 28) and end of follow-up (Day 42). An additional per protocol (PP) population was also identified, defined as a subset of participants who completed the study in compliance. In addition to the two early termination subjects noted previously, an additional three subjects were deemed non-compliant and excluded from the PP population (n = 20) prior to statistical analysis of the data. The results were not materially different between the ITT and PP sample populations and did not meaningfully alter the interpretation of the overall findings. Therefore, the results for the ITT population (all available data) are presented herein.

Demographic characteristics
Twenty-six generally healthy adults (~2/3 female; age range 21 to 40 y; BMI range 18.8 to 29.5 kg/m 2 ) from the Chicago, US metropolitan area with a majority race/ethnicity indicated as white and non-hispanic/ latino were enroled. Descriptive statistics of participant demographic characteristics are shown in Table 1.

Fermented milk beverage compliance
Based on returned bottle counts during the ingestion period, only one participant had <100% compliance (85.7%) by missing 2 days of beverage intake during the 2 nd week of the ingestion period (Days 21 to 27). Two participants had >100% compliance as each consumed one extra bottle during the first week of product ingestion (Days 14 to 20), while another participant was >100% compliant by consuming one extra bottle during the 2 nd week of the ingestion period (Days 21 to 27). The remaining participants were 100% compliant during the entire ingestion period.

LcS recovery in faecal samples
Box and whisker plots showing the distribution of LcS recovery from faecal samples at each visit are shown in Figure 3 while the model derived mean estimates at each study visit are presented in Table 2. Consumption of the fermented dairy product with LcS resulted in a significant (p < 0.001) increase in LcS in faecal samples collected during the consumption period.
During the baseline period, there were no detectable LcS-like colonies in the faecal samples of 23 of the 25 subjects, while two subjects had detectable  concentrations of 3.00 and 5.22 log 10 CFU/g of faeces, respectively. Given the majority had non-detectable LcS levels at baseline, the lower LOD was set to 2.00 log 10 CFU/g faeces for change scores to be calculated over the remainder of the study. Using this approach, the change from baseline to end of ingestion period (primary outcome) was significant, with an increase of 3.10 log 10 CFU/g faeces (95% CI = 2.31, 3.89; p < 0.001). The change from baseline to the mid-point of the ingestion period was also significant, showing increases of 4.20 (95% CI = 3.68, 4.72; p < 0.001) log 10 CFU/g faeces. The mean number of viable LcS decreased after cessation of product ingestion, with most participants (87%; 95% CI = 67.6%, 97.3%) demonstrating a return to baseline at Day 42. LcS was only detected in 2 subjects at concentrations of 3.47 and 4.00 log 10 CFU/g faeces at Day 42.

Safety
With respect to safety outcomes, the fermented dairy product with LcS was well tolerated, with no product-specific adverse events during the 14-day ingestion period. One subject reported mild upper respiratory infection that was determined "Not Related" by the Clinical Investigator and resolved during the study. No other significant observations related to safety were reported during the study.

Discussion
In this study, the recovery of viable LcS from the faeces of generally healthy U.S. adults following consumption of a fermented milk beverage was investigated. After confirming no detectable LcS in 23 of 25 subjects at baseline prior to product consumption, average faecal LcS recovery reached approximately 6.4 log 10 CFU/g faeces after 7 days of daily beverage consumption that remained relatively stable after an additional 7 days of daily consumption, with most participants showing no detectable LcS after a subsequent 14 days of no beverage consumption follow-up period. Overall, these results add to the existing global body of evidence showing LcS consumption via a fermented milk beverage survives transit through the gastrointestinal tract of different human populations but may not persist following cessation of daily consumption. Notably, few probiotics have been as rigorously tested consistently demonstrating viable LcS recovery across diverse human races and ethnicities. The recovery of viable LcS following consumption of a fermented milk beverage is generally consistent with similar studies that have been conducted in China (Wang et al. 2015), Japan (Yuki et al. 1999), Vietnam (Mai et al. 2017), Thai (Tiengrim et al. 2012), Indonesia (Utami et al. 2015), Singapore (Khine et al. 2019), Europe (Tuohy et al. 2007;Sakai et al. 2010), and Australia (Cox et al. 2019). To the best of our knowledge, the present study results are the first to be shown specific to this study beverage in a representative sample of generally healthy adults in the United States. This is important because the previously studied populations reflect people of varying biological (e.g. race, health status) and environmental (e.g. habitual diet) factors that have been proposed to influence LcS survival through the gastrointestinal tract. Notably, our findings are most consistent with previous studies in populations, specifically Europe and Australia, which are more likely similar to the United States versus other previously studied Asian populations. For example, Tuohy and colleagues reported that adults in the United Kingdom had LcS concentrations of 7.1 ± 0.4 log 10 CFU/g faeces after 14 days of consuming fermented milk beverages providing 8.6 ± 0.1 log 10 LcS CFU/mL (Tuohy et al. 2007). In another European population, mean LcS counts in the faeces of Belgian adults was approximately 6.5 to 6.6 log 10 CFU/g faeces after 7 days of fermented milk consumption (Sakai et al. 2010). Meanwhile, Cox et al. showed a mean recovery of approximately 6.4 log 10 CFU/g faeces after 14 days of fermented milk consumption in Australian adults (Cox et al. 2019).
The interindividual variability in LcS recovery is noteworthy, although not drastically different from previous studies. While participants were closely observed and administered the same serving of fermented milk with documented high degrees of compliance, and the temperature of study beverages and faecal samples were monitored by temperature loggers to ensure a high probability of viable bacteria, the range of LcS counts varied from undetectable to >7.0 log 10 CFU/g faeces. Some possible explanations may include potential differences in background dietary composition between subjects that impacted LcS survival, although we did not collect detailed daily diet records to explore this hypothesis further. Additionally, as noted previously, individual differences in gastrointestinal tract acidity and motility may underly these variable responses, and future research is needed to better determine the extent to which these physiological parameters impact LcS survival. Such large variation between individuals is not uncommon in nutrition research, in general, and likely underscores the importance of future approaches to personalised nutrition (Kirk et al. 2021) to determine optimal serving/dose. Interestingly, after 14 days of stopping the tested fermented milk ingestion, LcS was not detected in 23 out of 25 subjects. This indicates that LcS may not have the ability to persist long-term in the gastrointestinal tracts of US adults. This study has some potential limitations. We did not characterise complete faecal microbiota of each participant to determine if LcS consumption impacted other bacterial populations, or vice versa. Indeed, there may be interactions between the existing gut microbiota, which itself is quite variable between individuals with differing species composition of the lactic acid and bifidobacterial microbiota, which may impact the ability of LcS to persist for an extended time after cessation of the fermented milk consumption (Shima et al. 2022). Such interactions may also play a role in the level of LcS survivability between individuals. We also did not observe any significant impact of race/ethnicity on the faecal LcS counts, although the study sample is likely too small to derive definitive conclusions as the majority of participants were non-Hispanic/Latino and white; however, part of the rationale of the study was to gather information on a generally representative sample of the U.S. population to compare to other unique populations. Future research should continue to address if sustained LcS survival following fermented milk consumption has health benefits in this population. That said, LcS is one of the most studied probiotics with clinical data suggesting it could support gastrointestinal health, in particular improvements in bowel habits, (Koebnick et al. 2003;Matsumoto et al. 2006;Sur et al. 2011) and immune system function (Gleeson et al. 2011).
In conclusion, this study shows that viable LcS probiotic bacteria are recovered in the faeces of a representative population of generally healthy adults in the United States following daily consumption of a fermented milk beverage for 14 days that was also well-tolerated. These findings demonstrate the survival of LcS in the gastrointestinal tract of this population, which are consistent with research conducted in Asia, Europe, and Australia involving LcS-containing fermented milk beverages.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.