Bifidobacterium animalis subsp. lactis HN019 Effects on Gut Health: A Review

Optimal gut motility is central to bowel function and gut health. The link between the gut dysmotility related disorders and dysfunctional-intestinal barriers has led to a hypothesis that certain probiotics could help in normalizing gut motility and maintain gut health. This review investigates the roles of Bifidobacterium animalis subsp. lactis HN019 (B. lactis HN019™) on gut health, and its mechanisms of action in various pre-clinical and clinical studies. Research supports the hypothesis that B. lactis HN019™ has a beneficial role in maintaining intestinal barrier function during gastrointestinal infections by competing and excluding potential pathogens via different mechanisms; maintaining normal tight junction function in vitro; and regulating host immune defense toward pathogens in both in vitro and human studies. This has been observed to lead to reduced incidence of diarrhea. Interestingly, B. lactis HN019™ also supports normal physiological function in immunosenescent elderly and competes and excludes potential pathogens. Furthermore, B. lactis HN019™ reduced intestinal transit time and increased bowel movement frequency in functional constipation, potentially by modulating gut–brain–microbiota axis, mainly via serotonin signaling pathway, through short chain fatty acids derived from microbial fermentation. B. lactis HN019™ is thus a probiotic that can contribute to relieving gut dysmotility related disorders.


INTRODUCTION
Bowel function plays a central role in gut health and overall well-being. A healthy gut involves many factors, including intact epithelial barrier function, homeostatic intestinal microbiota, optimal functioning digestive organs (stomach, liver/gallbladder, pancreas), and definitely optimal gut motility. The interactions of those systems are in homeostasis in healthy subjects with normal bowel function and balanced immune function. However, this can be perturbed by antibiotic usage, unbalanced diet, and other life-style factors, infections, and other disease conditions. This may lead to changes in bowel habits and stool consistency, diarrhea, constipation, or a spectrum of both them, such as manifested in different subtypes of irritable bowel syndrome (IBS) (1)(2)(3).
The human gastrointestinal tract (GIT) represents an extremely complex ecosystem, comprising of interactions within the digestive system as well as cross-functioning with various immune cells, endocrine cells, and nerve cells, and organ systems beyond the GIT (4)(5)(6). Evidence also showed that the interactions between GIT and other organs often involve the residential intestinal microbiota, which is a dense community (estimated to 4 × 10 13 microbial cells), contributing to various metabolic functions, and immunological defenses in the maintenance of normal health (7)(8)(9). Therefore, gut health has traditionally been one of the most important targets for the majority of probiotic applications, and central for connections to other health benefits via the intestinal microbiota.
Probiotics are defined as live microorganisms that, when administered in adequate amounts, confer a health benefit on the host (10). Studies have shown that probiotics are beneficial for supporting the human body's natural functions in both health and disease; either by interacting with the host directly or indirectly by optimizing the composition and/or activity of the intestinal microbiota. Although common probiotic benefits have been proposed, the core benefits are strainspecific (10).
Bifidobacterium animalis subsp. lactis HN019 TM (B. lactis HN019 TM ) is a strain originally isolated from commercial yogurt, and commercialized as an ingredient for dietary supplements, fermented and non-fermented foods, and beverages for decades (11). Its complete genome sequence was published in 2018 (12), allowing for stringent control of product quality, safety, purity, and consistency by strain identity confirmation at industrial scale. B. lactis HN019 TM is available in various finished formats, granting it profound application potential in food and beverage, dietary supplement, and pharmaceutical industries.
The objective of this review is to discuss the properties of B. lactis HN019 TM in the context of both preclinical and clinical studies on the following aspects of gut health; survival through the gut, modulation of intestinal microbiota, maintain intestinal barrier functions during gastrointestinal infections, regulate gut motility, and improve symptoms in constipation, assist digestion, and utilization of macronutrients, and their plausible mechanisms of action. Further, the safety of the strain will be discussed. Although the same strain has been investigated for other health benefits (13), these will not be discussed here.

SAFETY
Bifidobacterium animalis subsp. lactis has been documented to be present in human food since 1980 but most likely has been consumed before that. The species is listed in Inventory of Microbial Food Cultures with Safety Demonstration in Fermented Food Products (14). The European Food Safety Authority (EFSA) has included the subspecies in the Qualified Presumption of Safety list (15). In China, B. lactis HN019 TM has been specifically permitted for use in infant and toddler (>1 year) food since 2011 (16), and it was accepted to be Generally Recognized as Safe (GRAS, GRN445) in US in 2012 (17).
By December 2020, there were 42 studies/clinical trials published for 27 investigated cohorts, including both healthy and compromised subjects in all age groups from newborns to elderly ( Table 1). The investigational products contain B. lactis HN019 TM as a single strain or combined with other probiotics and/or prebiotics. The daily dosage of B. lactis HN019 TM in the products ranged from 10 7 to 10 11 colony forming units (CFU) per day and consumption lasted for 7 days to 2 years. None of these trials have reported any safety concerns related to B. lactis HN019 TM consumption, and it may be concluded that infants, children, adults, and seniors can safely consume B. lactis HN019 TM at doses up to 3 × 10 11 CFU/day.
To date, most of the published human studies for B. lactis HN019 TM focused on gut health of which six studies contained B. lactis HN019 TM as a single strain product in at least one arm ( Table 2).

SURVIVAL THROUGH THE GUT
Although not stipulated in the definition, it is often assumed that probiotic strains should be able to survive passage through the digestive system, transiently colonize in the GIT, and potentially modulate host factors, such as immune responses, digestion, or the intestinal microbiota composition and/or activity. These probiotic attributes may be observed more pronounced in subjects with suboptimal physiological status but are often not observed in the healthy subjects. These functional characterizations can be investigated in various in vitro and/or animal models, while ultimately, health efficacy can only be confirmed by human clinical studies (64). Although adhesion is not a pre-requisite for a strain to elicit probiotic properties, interaction with intestinal epithelial cells (IECs) and intestinal mucosa is considered important for colonization and modulation of host factors. The excellent adhesion property of B. lactis HN019 TM to IEC lines, such as HT29, Caco-2, and HT29-MTX, were documented in vitro in comparison with two other probiotic lactobacilli strains and a negative control (non-probiotic Lactobacillus bulgaricus) (65).
B. lactis HN019 TM demonstrates high tolerance to low pH and varying resistance to bile salts in vitro, which are two important markers for assessing survival during intestinal passage (66 (24,25,35,58,59). Regardless of the variations caused by different quantitative techniques, given the observations that B. lactis/bifidobacteria was/were not detected in the control group (24) or counts decreased after the cessation of the supplementation (35,58), it is highly likely that B. lactis/bifidobacteria detected in the probiotic group in these studies was B. lactis HN019 TM . These studies suggest that B. lactis HN019 TM could survive and transiently persist in intestinal transit in both short-term (2-4 weeks) and long-term (>6 months) dietary interventions in almost all age groups, including infants, toddlers, pre-school children, adults, and elderly. This, notwithstanding a high inter-individual variability (0.1-68.8%) of B. lactis HN019 TM colonies quantified by strainspecific probes from total fecal bifidobacteria was reported   in healthy adults (59). Fecal recovery has also been assessed with products containing B. lactis HN019 TM in combination with other active components, such as prebiotics where increases in B. lactis or B. lactis HN019 TM were observed (31,34). However, Horvath et al. (67) did not observe such increases with a multi-strain probiotic containing B. lactis HN019 TM (67).

MODULATION OF INTESTINAL MICROBIOTA
Bifidobacterium and Lactobacillus sensu lato have long been used in processing and preserving food and are considered as beneficial (68). Although they are not the most abundant, they are relatively stable in the adult intestinal microbiota, maintaining essential metabolic functions, such as fermentation of undigested carbohydrates into short-chain fatty acids (SCFAs), lipid metabolism, and vitamin synthesis throughout the entire lifespan (69,70). This balance could be breached during aging, where the gut microbiota become less diverse and total bifidobacterial counts decrease (71). Therefore, sustaining these two genera at a stable level may be beneficial for maintaining a balanced healthy gut microbiota, particularly for elderly. B. lactis HN019 TM can become a significant component of the normal fecal bifidobacterial population, and can increase total Lactobacillus sensu lato and Bifidobacterium spp. counts in feces, with daily consumption of 6.5 × 10 7 CFU to 3 × 10 10 CFU (58,59). There were no significant differences between the responses of the different dose groups, indicating that even the lowest dose (6.5 × 10 7 CFU/day) was able to confer desired changes with regards to these two beneficial bacterial groups in the intestinal microbiota (58).
Furthermore, in these two studies fecal bifidobacterial and Lactobacilli counts decreased to baseline levels after cessation of the supplementation, remaining at 10 8 -10 9 CFU/g for bifidobacteria and 10 7 -10 8 CFU/g for lactobacilli. Interestingly, Ahmed et al. (58) reported significant increases in enterococci in the placebo group during the intervention with healthy elderly subjects. Although enterococci are normal members of the colonic microbiota, they are also opportunistic pathogens and may increase during aging (72). Moreover, in the same study, enterococci were reduced significantly in the same group during B. lactis HN019 TM consumption, indicating that B. lactis HN019 TM can reduce levels of fecal enterococci in elderly (58). B. lactis HN019 TM was shown to support maintaining the healthy/normal intestinal microbiota against aging process and by competing and excluding harmful pathogens, both at taxonomical and functional levels (25,58,59). In addition, further evidence for intestinal microbiota support can be found from combination products containing B. lactis HN019 TM . For example, both B. lactis HN019 TM (10 9 CFU/day) alone or with polydextrose (6.25 g/day) for 2 weeks increased the proportion of fecal Bacteroides in healthy young Japanese females, while the synbiotic format had better synergistic outcomes than probiotic alone, in terms of greater proportion of this genus among the entire microbial community (23). In a recent pilot study in Chinese neonates, after 28 days supplementation with a B. lactis HN019 TM synbiotic since birth, the complexity and similarity of gut microbiota in cesarean-born neonates across time and across individuals were similar to those in the vaginallyborn infants (52). Furthermore, both Bifidobacterium and Lactobacillus increased in their abundances after the third day of the intervention.
These findings from B. lactis HN019 TM alone or in combination with other ingredients indicate that the abovementioned modulations of the intestinal microbiota are ubiquitous, across age and geography.

MAINTAIN INTESTINAL BARRIER FUNCTIONS DURING GASTROINTESTINAL INFECTIONS
Gastrointestinal infections often lead to diarrhea in humans, which was estimated as a leading cause of death among all ages across the world (73). Rotavirus, Shigella spp., and Salmonella spp. are the top three etiologies for diarrhea mortality, whereas the first two are typical for children under the age of five (73). The leading risk factors for diarrhea were unchanged from 2005 to 2015, which are poor hygiene in water supply and food chain, where bacterial pathogens are food poisoning organisms (73).
The gut forms the border between the inside (body) and the outside (lumen), an intestinal barrier is therefore indispensable and includes both a physical and an immunological barrier, preventing pathogenic bacteria and other harmful substances from entering the body while at the same time allowing nutrients and water to be absorbed. Intestinal barrier integrity is a prerequisite for homeostasis of mucosal function, which is balanced to maximize absorptive capacity, while maintaining efficient defensive reactions against chemical and microbial challenges.

B. lactis HN019 TM Maintains Epithelium Integrity
The inner surface of the intestine consists of a layer of cells (epithelium), which are covered by a mucus layer (a viscoelastic layer consisting mainly of protein-linked carbohydrates) which plays a key role in the barrier effect mechanism.
Tight junctions (TJ) are cell-cell junctional complexes that form a continuous intercellular barrier between epithelial cells, that the major components are claudin and occludin proteins (74). These structures control and maintain balanced intestinal permeability. Increased permeability is associated with disease conditions, which are often characterized with intestinal mucosal inflammation (74). Therefore, a proper regulation of the function of TJ is important in health maintenance and disease prevention. Putaala et al. (75) showed that cell-free supernatant (CFS) of B. lactis HN019 TM may increase the strength of TJ, measured as trans-epithelial electrical resistance (TEER), although not to an extent that is statistically significant, while CFS of enterohemorrhagic Escherichia coli (EHEC) reduced the TEER significantly (75).
Cyclooxygenases (COX) play a role in normal physiological function as well as fight against pathogenic bacteria. There are two isoforms of COX in humans: COX-1, contributing to the maintenance of the physiological functions, whereas COX-2 as an inducible enzyme mediates pathogenic inflammatory responses (75). In Putaala et al. (75), CFS of both B. lactis HN019 TM and EHEC exhibited significant increase in the ratio of COX-2 to COX-1, but the underlying mechanisms are different. EHEC CFS induced increase of COX-2 and decrease of COX-1, whereas B. lactis HN019 TM CFS induced a slightly increase of COX-2 and retained level of COX-1, confirming the immunostimulatory properties of B. lactis HN019 TM , which lead to the activation and maintenance of normal physiological function in elderly under immunosenescence, but not pathological inflammation (47,76).

B. lactis HN019 TM Shows Competitive Advantages Against Entero-Pathogens
Pre-treatment of EHEC with CFS of B. lactis HN019 TM reduced the culturable E. coli numbers along with their invasive ability and cell association characteristics of the pathogenic strain (65). Both B. lactis HN019 TM and Salmonella typhimurium adhere effectively to INT-407 cells, and the adhesive capacity of B. lactis HN019 TM was similar to S. typhimurium. Nevertheless, the adhesion values obtained in co-treated assays showed that B. lactis HN019 TM could significantly decrease the number of attached S. typhimurium while the number of attached B. lactis HN019 TM was not affected by S. typhimurium (77).

B. lactis HN019 TM Regulates Host Immune Defense Toward Pathogens
B. lactis HN019 TM showed potential to protect enterocytes from inflammatory responses induced by S. typhimurium and lipopolysaccharide (LPS) in vitro. A robust interleukin (IL)-8 expression was induced when INT-407 cells were exposed to S. typhimurium. However, live and heat-killed B. lactis HN019 TM not only functionally modulated the epithelium by inhibiting the constitutive mRNA level of IL-8 and attenuating S. typhimuriuminduced IL-8 gene expression, but also protect the INT-407 cells from IL-8 protein production activated by LPS. Similar results were discovered on tumor necrosis factor (TNF)-α and IL-1β gene expression. TNF-α expression was up-regulated by live B. lactis HN019 TM , but the expression levels were much lower than with S. typhimurium (77).
Both S. typhimurium and EHEC are important causes of food poisoning under poor hygienic conditions, whereas E. coli and rotavirus are common causes of diarrhea in infants and young animals. The latter two infectious agents also commonly cause diarrhea in piglets during weaning, making piglets an ideal model for studying this type of gastrointestinal infections. The administration of B. lactis HN019 TM to weaning piglets resulted in a significantly lower incidence of diarrhea during the first 2 days after weaning. The fecal levels of E. coli and rotavirus were also lower in the treatment group, which exhibited a significantly higher titer of specific antibodies in the feces (78). In addition, in mice models, B. lactis HN019 TM was found to protect the animals against S. typhimurium and EHEC with lower rate of morbidity (79,80). The probiotic-fed mice also showed significantly higher numbers of phagocytic active leukocytes (80). These findings indicated a mechanism of enhanced immunemediated protection by B. lactis HN019 TM against gastrointestinal infections. Moreover, in a large randomized trial in India, B. lactis HN019 TM showed efficacy in reducing the incidence of diarrhea and fever during the rainy season (months August and September), when incidence of diarrhea is the highest (24). Fecal immunoglobulin (Ig)A and serum inflammatory marker IL-8 were also significantly decreased in the B. lactis HN019 TM arm compared to placebo (24). Similar observations were made with B. lactis HN019 TM in combination with galacto-oligosaccharides (GOS); reducing the incidence of dysentery and showing a trend (p = 0.08) for reduced incidence of diarrhea (28).

REGULATE GUT MOTILITY AND IMPROVE SYMPTOMS IN CONSTIPATION
Constipation without identifiable organic causes, may refer to slow-transit constipation (STC) or obstruction constipation, which are related to poor lifestyles, such as high fat/protein diet and lack of fiber and liquid intake and exercise. The typical clinical symptom of STC is the prolonged gut transit time (GTT), which may be resulted from the dysfunction of colonic smooth muscles (81).
B. lactis HN019 TM showed prokinetic effects on subjects suffered functional constipation (FC). For example, daily intake of B. lactis HN019 TM at both high (1.72 × 10 10 CFU) and low (1.8 × 10 9 CFU) doses for 2 weeks decreased colonic transit time (CTT) in 100 constipated adults, with no change observed in the placebo group (32). This finding of shorten CTT was not observed in Ibarra et al. (22), who investigated the effects of B. lactis HN019 TM using a similar intervention regime but longer duration (4 weeks) in 228 constipated adults diagnosed with the same ROME III criteria as in Waller et al. (32). However, a subgroup of subjects (N = 65) with bowel movement frequency (BMF) of less than three bowel movements per week (i.e., those who were constipated) benefitted from the consumption of the strain (22). After 4 weeks, daily doses of 10 10 CFU (high does) and 10 9 CFU (low dose) increased 2 and 1.7 bowel movements/week, respectively. The stool consistency outcomes indicated that the participants in Ibarra et al. (22) were mildconstipated subjects (Bristol Stool Scale: 3.3) without changing their level of constipation after the intervention. However, Aoe et al. (23) did not observe an effect of B. lactis HN019 TM alone on BMF in constipated women after 2-week supplementation. The study population was, however, under-powered; with only seven subjects per treatment group (23). In the same study, Aoe et al. (23) explored the outcomes on stool characteristics in a synbiotic intervention arm, where polydextrose (6.25 g/day) and B. lactis HN019 TM (10 9 CFU/day) were combined. This synbiotic showed increased stool amounts which positively correlated with the relative abundance of Bacteroides. It is important to note that PDX has been reported to positively influence stool consistency and bowel function (82). Other studies with B. lactis HN019 TM in combination with other probiotics or prebiotics observed improvements after intervention in bowel functions, such as shortened CTT (26), increased stool frequency and consistency (18,27), reduced flatulence symptoms (21). However, although Botelho et al. observed a change in fecal microbiota composition, the study failed to observe a positive effect on bowel function as compared to the placebo group (19) ( Table 1).
These observations on bowel habits led to the hypothesis that B. lactis HN019 TM affects colonic motility patterns, which most probably via direct interactions with host factors rather than alteration of water and electrolyte secretion, even though improvements for stool amount were observed in combination with polydextrose, which is a soluble fiber. This hypothesis was investigated in an ex vivo model, where B. lactis HN019 TM extract markedly increased the contractile amplitude of synchronous contractions spanning the proximal colon to the rectum. Interestingly, this effect occurred post-treatment (83). Even though the exact components of B. lactis HN019 TM extract are not clear, several potential mechanistic pathways including potential active molecules will be reviewed below (Figure 1).

Modulate Microbiota-Fermentation-Gut-Brain Signaling
The primary pathophysiological mechanism for constipation is gut dysmotility, presenting dysregulated or deficient colonic propulsive motor patterns (6,84). So far, molecules reported to impact on gut motility are SCFAs, formylated peptides, and serotonin (5-hydroxytryptamine, 5-HT), which might be derived from bacterial surface structures or bacterial fermentation of nutrients (6,85). Short-chain fatty acid concentrations can directly influence motility through the G protein-coupled receptor (GPR) 41 and GPR43, where the underlying mechanisms have been mostly addressed in animal studies (86)(87)(88). For example, intra-luminal administration of a blend of acetate, propionate and butyrate in rat was shown to lead to an increased 5-HT concentration and subsequently decreased CTT (86). B. lactis HN019 TM has been shown to produce lactate and acetate, where acetate was more selective potent agonist for GPR43 (48,89). However, there are a few studies confirming the effects of SCFAs on motility in humans (90,91).
In addition, B. lactis HN019 TM may also influence gut motility via gut commensals, which are known to be involved in serotonin biosynthesis and bile acid metabolism (92,93). Bile salts are known to stimulate colonic contractility and transit in humans (94)(95)(96), where probiotic strains may deconjugate bile salts, leading to the formation of secondary bile salts with laxative effects (6). However, to date, no data is available on the ability of B. lactis HN019 TM to deconjugate bile salts.

Improve Gastrointestinal Symptoms in Constipation
Except bowel habits, B. lactis HN019 TM has been reported to reduce the frequency of functional gastrointestinal symptoms in adults, including vomiting, regurgitation, abdominal pain, nausea, gurgling, constipation, diarrhea, and flatulence (32). Most symptoms improved in the high dose (eight of nine symptoms) and low dose (seven of nine symptoms) groups, respectively, while only two of nine symptoms showed a statistically significant improvement with placebo. In Ibarra et al. (22), when comparing the measured gastrointestinal symptoms between subjects taken B. lactis HN019 TM vs. placebo, only a reduction observed in the degree of straining in the subgroup of subjects (BMF<3). Improvements of gastrointestinal symptoms were also observed in studies with B. lactis HN019 TM in combination with other probiotics or prebiotics (21, 27) ( Table 1).

ASSIST DIGESTION AND UTILIZATION OF MACRONUTRIENTS
Some of the constipation related symptoms are associated with inadequate digestion of fibrous components from the diet. Plantbased diets, rich in fermentable residues, could be a solution to constipation but may be accompanied with complaints of gas produced during endogenous microbial fermentation. Aiding their digestion may thus help reduce these symptoms. In vitro, B. lactis HN019 TM was shown to utilize commercial oligosaccharides: FOS, GOS, and xylo-oligosaccharide (XOS) (46,50). Further, the analysis of complete genomes from several commercial B. lactis strains, including B. lactis HN019 TM supports this, as well as the identification of several putative carbohydrate-modifying enzymes in the genome of B. lactis HN019 TM for a wide range of complex carbohydrates (97,98). This is supported by the utilization of oligosaccharides by B. lactis HN019 TM seems best for mono-and di-saccharides, such as stachyose, raffinose, but not able to utilize oligosaccharides with a degree of polymerization of more than 7 (99,100). Such oligosaccharide utilization may contribute to a reduced fermentation by the endogenous microbiota and improve tolerance to such fibers. Bifidobacterium spp. are usually non-gas producers, since in general, they metabolize monosaccharides via the fructose-6-phosphate pathway eventually to SCFAs without gas as a by-product (98,101). Therefore, endogenous and consumed bifidobacteria, including B. lactis HN019 TM , could divert the fermentation in the colon toward non-gaseous endproducts. This property of B. lactis HN019 TM may support tolerance for the fermentation of oligosaccharides in vivo, which is in line with clinical observations of B. lactis HN019 TM on reduced flatulence (21,32). In addition, lactate and acetate are produced during B. lactis HN019 TM fermentation, indicating potential roles in digestion of dietary components without differentiation of the origin; carbohydrate or protein (89,102).
Weak indications were observed for the involvement of B. lactis HN019 TM in digestion of specific dietary component(s) in human (44). In this study, a combination of L. rhamnosus HN001 and B. lactis HN019 TM was examined on established atopic dermatitis (AD) in children SCORAD (SCORing Atopic Dermatitis), a measure of the extent and severity of AD, was assessed at baseline, 2 and 12 weeks after starting treatment and 4 weeks after treatment was discontinued. The supplement alleviated AD symptoms, but only in a sub-group of foodsensitized children, whereas no effect on children sensitized to environmental allergens, suggesting the beneficial effect of the probiotic may only relate to local effects on GIT, mainly toward food challenges (44). However, since the results came from sub-group analysis in children only tested for several common food allergens, the results should be interpreted with caution, and more confirmatory human studies should be carried out in larger population.
Gut microbial bile salt hydrolase (BSH) enzymes promote deconjugation, dehydrogenation, and dihydroxylation of primary bile acids, increasing the chemical diversity of bile acids, which can in turn have an impact on host lipid metabolism (93). Unconjugated bile acids are less efficient emulsifiers of dietary fats and may positively influence blood lipid profiles. Even though no data showed B. lactis HN019 TM could deconjugate bile salts, as discussed earlier, B. lactis HN019 TM may have a role in bile acid metabolism via affecting gut commensals, which in turn can change the amount of fat that the body is able to absorb. In addition, several studies have shown the potential of B. lactis HN019 TM alone or in combination with other probiotics in improving blood lipid profiles (61,63).

DISCUSSION
Different aspects of digestive health are the most common health benefits addressed by probiotics (103). The above review has shown that also B. lactis HN019 TM has several health benefits in this area, in particular in the area of bowel function and intestinal motility. Bifidobacterium animalis subsp. lactis is one of the most common lactic acid producing probiotics in North-America and Europe (104) B. lactis DN-173010/B. lactis CNCM I-2494 in fermented milk at a dosage of 1.25 × 10 10 CFU two to three times/day, has shown beneficial effects on acceleration of gastrointestinal transit in both a healthy and constipation dominant irritable bowel syndrome (IBS-C) population after 10 days to 4 weeks intervention (105)(106)(107). Moreover, this probiotic fermented milk (PFM) has been shown to improve general gastrointestinal well-being, reduce symptomatology including abdominal distention and discomfort in individuals from the general population with minor digestive symptoms but not functional gastrointestinal disorders (107)(108)(109), as well as in IBS-C population (106). These studies were all performed with fermented milk which also contained the classic yogurt cultures: S. thermophilus and L. bulgaricus. Noteworthy, both Guyonnet et al. (107) and Marteau et al. (109) recruited healthy women without any diagnosis of digestive disease, including FBD, such as IBS with normal BMF (3-21/week). This hypothesis was further confirmed, in rat model, where B. lactis CNCM I-2494 reduced stress-induced visceral hypersensitivity by restoring intestinal barrier function (110), and in a proofof-concept study where healthy women consumed this PFM and modulation of brain activities related to mood was reported (111).
B. lactis BB-12 is another well-documented probiotic strain that has been investigated for its digestive health benefits (112).
In several studies the strain has been evaluated for the effects it has on BMF. In a large study testing two doses; 10 9 and 10 10 CFU/day for four weeks, a beneficial effect was observed especially in the lower tested dose (113). Similarly, a fermented oat product with B. lactis BB-12 was observed to normalize bowel function in elderly. In combination with Lacticaseibacillus paracasei CRL-341 softer stools were observed (114). This could suggest a potential mechanism of action. A recent study was, however, not able to replicate the effect on intestinal transit time (115); most likely because of the small number of subjects and that subjects were not selected to have long transit times or be constipated. Consumption of yogurt or a capsule with the strain was not found to change the intestinal microbiota, this is not unusual; the resilience of a healthy colonic microbiota is likely to resist change from one (probiotic) strain (116). However, consumption of the probiotic yogurt did lead to fecal recovery of the strain (115).
In summary, all three B. lactis strains discussed above do seem to have quite similar effects on bowel movements. This is confirmed by a recent meta-analysis which concluded that B. lactis may be more effective in increasing stool frequency and in improving stool consistency (117). It is therefore tempting to speculate that this relates to the high genome similarity and almost complete synteny of the genomes between various strains within the subspecies B. lactis (97,118). The small genetic difference between strains of B. lactis could be investigated further and correlated with phenotypic traits to help understand mechanisms of action.
As manifested by the current research, serotonin signaling pathway seems to be a major effector pathway underlining the efficacy of B. lactis HN019 TM on gut motility, where SCFAs derived from gut microbial fermentation seem to be promising candidate effector molecules. The contribution from other bacterial derived molecules, such as formylated peptides acting on FPR1 cannot be ruled out with the current knowledge. Although probiotics in healthy subject generally cause no or minor changes in the gut microbiota (113); it nevertheless seems to be involved in the complex interactions of B. lactis HN019 TM with host metabolisms and immune defenses, including endocrine cells, immune cytokines, and neurotransmitters, which are yet to be fully understood. Therefore, future investigations on the exact role of B. lactis HN019 TM on gut motility should focus on identifying key molecules and signaling pathways in the gut-brain-microbiota interactions in humans, which could enhance our understanding. To this end, focus of microbiota analysis should shift or expand to the analysis of the colonic mucosal microbiota and metabolomics of the intestinal microbiota (119). Recent scientific advances suggest great potential for bacterial-derived microvesicles (MVs) on gut health. Microvesicles from L. reuteri DSM 17938 were shown in an ex vivo mouse colon to recapitulate the effects of the whole bacteria on increasing colonic propagating contraction frequency (120). Application of L. rhamnosus JB-1 and its MVs separately to the apical side of the gut epithelium, could reduce the amplitude of propagating neural contractions in ex vivo mouse colon via interactions with ENS (121). These findings suggest a role for MVs from probiotics on signaling and their significance in the mechanisms of action within the gut-brain-microbiota axis, would therefore be potential mode of action to be investigated in the future for a better understanding of mechanisms of B. lactis HN019 TM .
In conclusion, B. lactis HN019 TM has been observed to have benefits on different aspects of digestive health; not unlike some other commercial probiotic B. lactis strains. What is needed, however, is a better understanding of the mechanisms of action on how B. lactis HN019 TM and probiotics in general function (122). Further, clinical trials should be better designed; taking into account the heterogeneity across different populations (123,124) with sufficiently sized studies and clearly defined endpoints.

AUTHOR CONTRIBUTIONS
JC, AL, and AO: conceptualization, review, and editing. JC and AO: original draft preparation. All authors have read and agreed to the published version of the manuscript. Publisher's Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.
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