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A probiotic amylase blend reduces gastrointestinal symptoms in a randomised clinical study

In: Beneficial Microbes
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M.B. La Monica The Center for Applied Health Sciences, 6570 Seville Dr., Canfield, OH 44406, USA

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B. Raub The Center for Applied Health Sciences, 6570 Seville Dr., Canfield, OH 44406, USA

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H.L. Lopez The Center for Applied Health Sciences, 6570 Seville Dr., Canfield, OH 44406, USA

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T.N. Ziegenfuss The Center for Applied Health Sciences, 6570 Seville Dr., Canfield, OH 44406, USA

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Abstract

A randomised, placebo-controlled, double-blind, parallel clinical study was performed to examine the effects of a probiotic- amylase (PRO) blend on gastrointestinal (GI) symptoms. Sixty men and women (44.4 ± 8.9 yr; 82.0 ± 18.4 kg; 170.3 ± 11.5 cm; 28.1 ± 4.6 kg/m2) were randomised into PRO (n = 29) or placebo (PLA: n = 31) groups. Participants exhibited mild to moderate GI symptoms and severity [via Gastrointestinal Symptom Rating Scale (GSRS)] to be eligible for participation. Participants were tested before (Baseline) and after (POST) 6 weeks of supplementation on various gastrointestinal indices, the GSRS (to assess GI symptoms, frequency, and severity), an anxiety questionnaire (GAD-7), and an overall well-being questionnaire (SF-36). Two (PRO vs PLA) × 2 (Baseline vs POST) mixed factorial ANOVAs were completed to assess group, time, and (group × time) interaction effects. Fifty-two subjects who completed the entire study were analysed (PRO: n = 25, PLA: n = 27). There were statistically significant (P0.05) interactions for bloating, GSRS score, and abdominal discomfort but time effects for flatulence, constipation, stool regularity, and GAD-7 total score. PRO significantly reduced GSRS score (∼60 vs 25%, d = 0.72), bloating (∼49% vs 25%, d = −0.63) and abdominal discomfort (59% vs 32%, d = −0.66) to a greater degree than PLA. PRO significantly reduced subjective feelings of irritability, pain, and overall health interference. Oral supplementation of the probiotic-amylase blend was very well tolerated. Our study showed that the probiotic-amylase blend reduced the GSRS score and other GI symptoms to a greater degree than PLA.

Clinical trial registration: clinicaltrials.gov #NCT05614726

1 Introduction

Digestive health has emerged as an important topic for many consumers (Forssten et al., 2011) and the use of microbes as medicine has been steadily gaining traction as increased understanding of the role of the microbiome in health and disease provided by improved genomics, metagenomics, and metabolomics platforms has emerged (Zmora et al., 2019). In 2013, the International Scientific Association for Probiotics and Prebiotics (ISAPP) defined Probiotics as: ‘Live microorganisms that, when administered in adequate amounts, confer a health benefit on the host’ (Hill et al., 2014). In the time since probiotics were officially defined, they have become a popular dietary supplement (Hill et al., 2014). Probiotics have been used for over a century (Zmora et al., 2019) and are found in supplement form as well as in specific foods (e.g. yoghurt, kefir, sauerkraut, tempeh, and kimchi).

The microbial specificity of each bodily site is dependent upon the habitat provided by the host and the commensal organisms that thrive in each habitat. The gastrointestinal tract (gut) represents the richest niche for microorganisms where inter-kingdom interactions between bacterial (bacteriome) and fungal (mycobiome) communities occur (Mukherjee et al., 2015). The gut represents a very diverse area within the human host. However, the diversity represented within the gut can be altered by exogenous and endogenous factors including diet, lifestyle, generalised health, therapeutics, or other environmental factors.

Probiotics may beneficially modulate the gut microbiota in several ways including expansion of beneficial bacteria and yeast, increasing the mucus layer to improve the physiological barrier function within gut mucosal epithelial cells, keeping pathogens under control, and stimulating immune cells (Mukherjee et al., 2015). Beyond the gut, probiotics may also have beneficial effects on anxiety, stress, and improving mood through the gut-brain axis (Butel, 2014). Since beneficial attributes of a given microbial species is strain specific, testing strain specific effects and formulating a mixture of strains to provide an optimal benefit for the host at an effective dose is necessary (Wallace and Milev, 2017).

Dysbiosis occurs when the gut microbial communities (e.g. bacterial and fungal) are imbalanced and may lead to gastrointestinal (GI) issues as well as numerous beyond the gut diseases (Bubnov et al., 2018), although probiotics can help provide balance (symbiosis) within the gut (Gebrayel et al., 2022) addressing the GI issues. Dysbiosis of the gut microbiota has been associated with adverse conditions, such as Clostridioides difficile infection (CDI) (Santiago et al., 2019), metabolic syndrome (Scheithauer et al., 2020), inflammatory bowel disease (IBD) (Zuo and Ng, 2018), and colorectal cancer (Wong and Yu, 2019). Previously, it had been shown that the abundance of pathogenic Candida tropicalis (a fungus), Escherichia coli and Serratia marcescens (bacteria) were elevated in Crohn’s disease (CD) patients compared to their non-affected relatives (Hoarau et al., 2016). Additionally, a previous investigation demonstrated that these pathogens formed thick polymicrobial biofilms (PMB) (Hoarau et al., 2016). Recently, it was discovered that a novel formulation of probiotic strains (Bifidobacterium breve 19bx, Lactobacillus acidophilus 16axg, Lacticaseibacillus rhamnosus 18fx, and Saccharomyces boulardii 16 mxg) plus amylase was able to prevent and treat the formation of PMB (Hager et al., 2019).

Recent evidence indicates that an overgrowth of fungus in the small intestine of non-immunocompromised subjects may contribute to unexplained GI symptoms. Furthermore, two recent studies showed that 26 and 25.3% of patients with unexplained GI symptoms had small intestinal fungal overgrowth (SIFO) which is characterised by the presence of an excessive number of fungal organisms in the small intestine, associated with GI symptoms (Erdoğan, 2014; Jacobs et al., 2013). The most common symptoms observed in these patients were gas, bloating, intestinal cramps, altered bowel function, indigestion, nausea, diarrhea, belching, and rectal itching (Erdogan and Rao, 2015). While several different bacteria are known to occur in cases of small intestinal bacterial overgrowth (SIBO), Candida spp. are implicated in nearly all cases of SIFO (Martins et al., 2014).

Recently, it was demonstrated that the probiotic formulation used in the current study was able to prevent and treat biofilm formation in vitro (Hager et al., 2019). In the same in vitro study, it was shown that Candida albicans or C. tropicalis, compared to either Trichosporon inkin or Saccharomyces fibuligera formed significantly thicker polymicrobial biofilms (PMB) in combination with E. coli and S. marcescens, indicating that this interaction is Candida specific. Furthermore, it was shown that the probiotic could prevent or treat mature biofilms, and that C. tropicalis PMB exposed to this probiotic filtrate had reduced biofilm matrix, decreased thickness, and inhibited hyphal formation (Hager et al., 2019).

In addition to in vitro work, an in vivo preclinical study evaluating the effect of the probiotic formulation using a spontaneous chronic CD like-ileitis animal model (SAMP1/YitFc) (Reuter et al., 2011) was conducted. Three groups of 7-week-old SAMP mice were compared using (1) the current probiotic formulation (4 probiotic strains + amylase), (2) the probiotic supplement without amylase, and (3) control animals administered sterile phosphate-buffered saline alone. After treatment, mice were euthanised, and ilea were collected for histologic scoring of ileitis. Stool samples were evaluated by 16S ribosomal RNA and gas chromatography/mass spectrometry analyses. Histology scores showed that mice treated with the complete formulation (4 probiotic strains + amylase) had a significant decrease of ileitis severity compared to the other 2 groups. 16S ribosomal RNA and gas chromatography/ mass spectrometry analyses showed that the abundance of species belonging to genus Lachnoclostridium and the species Mucispirillum schaedleri were significantly increased compared to the other 2 groups, and this increase was associated with augmented production of short chain fatty acids (SCFAs) (Di Martino et al., 2023). These findings suggest that administration of this novel probiotic formulation leads to functional changes that ameliorate the severity of CD-like ileitis. In addition, the hydrolytic activity of amylase appears to be essential for the anti-inflammatory effects of beneficial bacteria in the intestine (Di Martino et al., 2023). Previous studies have reported that amylase causes a disruption in the biofilm matrix which supplies probiotic strains with the ability to inhibit pathogenic fungi and bacteria (Di Martino et al., 2023; Gowen et al., 2023; Hager et al., 2019). This disruption in the biofilm matrix protects the intestinal barrier and causes marked alterations in faecal microbial population providing the murine model with greater resistance to colonization of microorganisms, which was dependent on the presence of amylase and the probiotic strains (Di Martino et al., 2023). A follow-up pilot clinical trial showed that consumption of this novel formulation results in positively modifying both bacterial and fungal abundance in the gut within 4 weeks of daily consumption (Ghannoum et al., 2021).

Given the demonstration that the probiotic plus amylase formulation was capable of altering biofilm formation in vitro and in vivo, combined with reports demonstrating the ability to modulate the gut microbiota structure, including significantly decreasing the pathogenic genus Candida, altering biofilm formation, and positively impacting the microbiota in an ileitis model, provided the rationale for performing further placebo-controlled clinical studies. We designed the current study to address the potential for the combined probiotic and amylase treatment to ameliorate or prevent common gastrointestinal symptoms often reported with IBD or gut dysbiosis. We initiated these studies in a cohort of individuals that reported mild to moderate GI symptoms, while excluding any individuals that reported chronic health issues. Thus, the cohort of interest was individuals that demonstrated only mild-moderate GI symptoms, and underlying chronic health issues would not confound the observations.

Therefore, this study examined the effects of oral supplementation with this novel formulation on gastrointestinal symptoms, stress response, and overall well-being, as assessed by quality-of-life metrics. We hypothesised that consumption of the designated probiotic + amylase formulation would decrease symptoms associated with GI discomfort and stress, and therefore enhance overall quality of life.

2 Materials and methods

2.1 Experimental design

The current study was a randomised, parallel, placebo-controlled, double-blind investigation consisting of three study visits. This study was conducted according to the Declaration of Helsinki guidelines and all procedures involving human subjects were approved by Pearl IRB on 6/18/21 (#21-CAHS-102). The study was registered on clinicaltrials.gov (‘A Probiotic Amylase Blend Reduces Gastrointestinal Symptoms and Positively Impacts Gut Microbiota Modulation in a Randomized Study’, #NCT05614726). Written informed consent was obtained from all subjects prior to enrolment. This study was conducted at a contract research organisation (CRO) in Northeast Ohio. Participants were recruited from the Northeast Ohio area and within the CRO’s subject database via advertisements, phone calls, and word of mouth. During the study screening visit, each participant’s medical history and routine blood work [Complete Blood Count (CBC), Comprehensive Metabolic Panel (CMP), and Lipid Panel] were collected, and a 24-h dietary recall was performed. At the next two visits (baseline and post- supplementation), body weight, baseline measurements of vital signs, visual analog scales (VAS) for flatulence, bloating, abdominal discomfort, stool consistency/regularity & constipation, and questionnaires that assess the participants’ GI health [Gastrointestinal Symptom Rating Scale (GSRS)], overall well-being (e.g. SF-36), physical activity (e.g. Framingham), and general anxiety (i.e. GAD-7) were performed. On the third visit (post-supplementation) routine blood work (CBC, CMP, and Lipid Panel) was again collected. After completion of visit 2 (baseline testing), participants were randomised in a parallel, double-blind, placebo-controlled fashion to ingest either; a daily dose of a 575 mg [30 billion cfu probiotic + amylase blend (PRO (see Table 1))] consisting of B. breve 19bx, L. acidophilus 16axg, L. rhamnosus 18fx, S. boulardii 16 mxg, and alpha amylase 500 SKB (Alpha-amylase-Dextrinizing Units) (provided by BIOHM Health, LLC, Cleveland, OH, USA), or a placebo (PLA) consisting of rice oligodextrin for 6 weeks. Compliance was monitored and assessed with a daily supplement log in which subjects checked off each day they took the investigational product. Investigators reviewed the supplement log with the subjects at visit 3, and subjects returned their supplement bottles to the lab during their last visit to confirm that the correct dose was taken. Supplement bottles, from the manufacturer, were plain, labelled with a study code and instructions to take 1 capsule with their largest meal of the day, and marked as ‘A’ or ‘B’ for each respective group as they arrived to the research lab. The researchers were unblinded only after statistical analyses were finalised. Compliance to the supplementation regimen was >90% for all participants.

Table 1
Table 1

The composition of probiotic blend (PRO) BIOHM 30B, consisting of 575 mg [30 billion cfu] of Bifidobacterium breve, Lactobacillus acidophilus, Ligilactobacillus rhamnosus, and Saccharomyces boulardii plus 500 SKB of amylase.1

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

Table 2
Table 2

Anthropometrics and vitals on study participants total and in the active probiotic (PRO) and placebo (PLA) groups

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

Prior to all study visits, participants were asked to replicate their initial dietary intake for the 24 h prior to their visit, refrain from caffeine and exercise for 24 h, and fast for 10 h. In addition to the clinical endpoints, a concurrent study (targeted for an independent publication on microbiome changes) was performed to assess the gut microbiota following supplementation, therefore individuals were requested to refrain from caffeine due to the potential impact of caffeine on the composition of the gut microbiota (i.e. association with greater alpha diversity) (Barandouzi et al., 2021). The first participant was enrolled on 7/7/21, and data collection concluded on 2/15/22. Comprehensive side effect profile/adverse event monitoring took place throughout the study.

2.2 Study participants

Fifty-two (14 men and 38 women) participants were randomised (allocation ratio 1:1 via research randomizer, https://www.randomizer.org/) into the study. Twenty-seven individuals were assigned to PLA (44.7 ± 7.9 yr, 169.2 ± 12.7 cm, 78.7 ± 18.0 kg, 27.4 ± 4.5 kg/m2), and 25 were assigned to PRO (44.0 ± 10.0 yr, 171.4 ± 10.3 cm, 85.5 ± 18.6 kg, 28.9 ± 4.6 kg/m2) by the researchers. Table 2 presents the baseline demographics of the study cohort, showing that the two groups were similar at screening. Review of health/medical history documents and a physical exam showed that all study participants were free of chronic health issues. Inclusion criteria were established so that all participants were required to be between 30-60 years old, have a GSRS score ≥ 12 (corresponding to mild to moderate GI symptoms), have a minimum body mass of 120 pounds (54.5 kg), and body mass index (BMI) between 20.0-34.99 kg/m2. Inclusion also required participants to be normotensive (<140/<90 mm Hg) with a normal resting heart rate (<90 beats/min). Female participants were not eligible if they were determined to be pregnant, nursing, or trying to become pregnant. Exclusion criteria included any history of: unstable or new-onset cardiovascular or cardiorespiratory disease; stroke, diabetes, or other endocrine disorder; use of any nutritional supplement known to alter the gut microbiome/microflora, probiotic supplements, use of prebiotic supplements in the previous 4 weeks and for the duration of the study; use of any antibiotics, antifungals, antivirals, or antiparasitic within 8 weeks of the start of the study or throughout the study; any changes in diet within 4 weeks of study start date or throughout study duration; if the subject was unwilling to abstain from gut altering supplements for the duration of the study; malignancy in the previous five years except for non-melanoma skin cancer (basal cell cancer or squamous cell cancer of the skin); prior gastrointestinal bypass surgery (i.e. Lapband); any known gastrointestinal or metabolic diseases that might impact nutrient absorption or metabolism [e.g. short bowel syndrome, diarrheal illnesses, history of colon resection, gastroparesis, Inborn-Errors-of-Metabolism (such as PKU)]; any chronic inflammatory condition or disease (e.g. rheumatoid arthritis, Crohn’s disease, ulcerative colitis, Lupus, HIV/AIDS, etc.); known sensitivity to any ingredient in the test formulations as listed in the certificates of analysis. Participants were excluded if they were currently participating in another research study with an investigational product or had participated in another research study in the past 30 days, or if they had any other diseases or conditions that, in the opinion of the medical staff, could confound the primary endpoints or place the subject at increased risk of harm if they were to participate. Figure 1 displays a Consort flow diagram.

Figure 1
Figure 1

Consort diagram.

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

2.3 Procedures

Standing height was determined using a stadiometer with participants in socks or bare feet with heels together. Body mass was measured using a Seca 767™ Medical Scale (Hamburg, Germany). Resting heart rate and blood pressure were measured using an automated blood pressure cuff (Omron HEM-780; Osaka, Japan) after participants had remained seated for a minimum of 5 min.

Health-associated questionnaires including Framingham Global Risk Assessment, Short Form Health Survey questionnaire (SF-36), a modified GSRS, a Generalised Anxiety Disorder Assessment (GAD-7), and VAS, to rate flatulence, bloating, abdominal discomfort, stool consistency/regularity and constipation were completed by each participant before and after 6 weeks of supplementation. VAS questions were constructed using a 10 cm line anchored by ‘Lowest Possible’ and ‘Highest Possible’ except for stool consistency which was anchored by ‘Liquid’ and ‘Hard’ and stool regularity which was anchored by ‘Irregular’ and ‘Regular’. The validity and reliability of VAS to assess fatigue and energy have been previously established (Lee et al., 1991) and reported (Lopez et al., 2020; Ziegenfuss et al., 2018). The Framingham physical activity questionnaire was used to assess physical activity habits throughout the study and to ensure participants complied with their instructions to maintain their physical activity habits. The SF-36 Health Survey was used to assess quality-of-life (McHorney, 1993; McHorney et al., 1994; Orrell et al., 2017; Wayne et al., 2015). The modified GSRS was used to assess symptoms of gastrointestinal health (Dimenas, 1995). The GSRS is a self-administered questionnaire designed to subjectively evaluate the intensity, frequency, duration, and impact on daily living for commonly reported GI symptoms (Svedlund et al., 1988). The questionnaire includes 15 symptoms and uses a graded Likert scale to assess the severity of symptoms and includes items that are frequently reported by patients with GI diseases (Dimenäs et al., 1993). A zero on any one item within the questionnaire corresponds to the absence of a symptom whereas a higher overall total score on the GSRS indicates greater severity/frequency of symptoms and/or the existence of more GI symptoms. The total score on the GSRS can range from 0-45 and has previously demonstrated good construct validity and interrater reliability (Dimenäs et al., 1993; Svedlund et al., 1988). The GAD-7 was used to assess general anxiety (Spitzer, 2006). The GAD-7 assessed the frequency of 7 items over the previous two weeks (‘Feeling nervous, anxious, or on edge’, ‘Not being able to stop or control worrying’, ‘Worrying too much about different things’, ‘Trouble relaxing’, ‘Being so restless that it’s hard to sit still’, ‘Becoming easily annoyed or irritable’, ‘Feeling afraid as if something awful might happen’). A higher score on the GAD-7 indicates a greater degree of anxiety.

2.4 Statistical analyses

Primary outcome measures included subjective changes in flatulence, bloating, and abdominal discomfort, and GSRS score. Secondary outcome measures included subjective changes in stool consistency, stool regularity, and constipation. Tertiary outcome measures included changes in anxiety/stress (GAD-7), changes in general health (SF-36), vital signs, bloodwork, side effect profile/adverse events monitoring and adverse events. Quaternary outcome measures included changes in physical activity (Framingham score) and body weight. A priori power analysis was conducted via G*Power (https://www.psychologie.hhu.de/arbeitsgruppen/allgemeine-psychologie-und-arbeitspsychologie/gpower) for a mixed factorial ANOVA with repeated measures, a within-between interaction and a small effect size of 0.25 (as a conservative approach based on several of our primary outcome measures). With two groups and two time points a sample size of 34 was needed to achieve 80% power. Normality of each variable was assessed using the Shapiro-Wilk test. Two (PRO vs PLA) × 2 (week 0-Baseline, week 6-POST) mixed factorial ANOVAs were completed to assess group, time, and (group × time) interaction effects. When sphericity was violated, Greenhouse-Geisser corrected P-values were used for main effects/interactions. In the event of missing data, a mixed-effects model was utilised in GraphPad Prism. Sidak post-hoc procedures were used to assess individual comparisons and adjust for multiple comparisons providing tighter bounds than Bonferroni between time points and/or groups. A significance level of ≤ 0.05 was accepted as statistical significance. Significant interactions were followed up with dependent t-tests to identify potential differences within groups or independent t-tests to identify potential differences between groups. For between-group changes over time, independent t tests (for data displaying normal distribution) and Mann-Whitney tests (for data not displaying normal distribution) were used to assess the change score (deltas) on all variables. Delta values were computed by the differences in time points relative to baseline (i.e. Post-Baseline). All data points less than −3SD or greater than +3SD were deemed outliers and removed before analyses. Effect sizes are expressed as Cohen’s d with 95% confidence intervals and interpreted as ≥ 0.2 (small), ≥ 0.5 (moderate), and ≥ 0.8 (large). All analyses were completed with GraphPad Prism version 9.2.0 (GraphPad Software, San Diego, CA, USA).

3 Results

Fifty-two subjects who received the intervention, complied, and adhered to the intervention, and completed the clinical trial in-full were analysed (PRO: n = 25, PLA: n = 27).

3.1 Visual analog scales and gastrointestinal symptom rating scale

Table 3 presents VAS for (flatulence, bloating, abdominal discomfort, stool consistency & regularity, and constipation), and questionnaires that assess the participants’ gastrointestinal health (GSRS) in comparison of baseline versus Post (Time) for treatment with PRO versus PLA. Significant time × group interactions and main effects of time were observed for bloating (P=0.016 and P<0.001, respectively), abdominal discomfort (P=0.027 and P<0.001, respectively), and GSRS score (P<0.001 and P<0.001, respectively). There were also significant main effects of time for flatulence (P=0.002), stool regularity (P<0.001), and constipation (P=0.010).

Table 3
Table 3

Visual analog scales (VAS) for (flatulence, bloating, abdominal discomfort, stool consistency/regularity & constipation), and questionnaires that assess the participants’ gastrointestinal health [Gastrointestinal Symptom Rating Scale (GSRS)] are summarised before (Pre) and after (Post) active probiotic (PRO) and placebo (PLA) consumption.1

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

Post-hoc testing for bloating indicated that Post was significantly lower than baseline in the PRO treatment group by an average of −2.8 cm (∼49% reduction relative to baseline) (95% CI: −4.0 to −1.6) (P<0.001, d = 1.14) and in PLA by an average of −1.3 cm (∼25% reduction relative to baseline) (95% CI: −2.3 to −0.3) (P=0.012, d = 0.58). In addition, the delta between groups for bloating indicated significant differences (P=0.027, d = −0.63). On average PRO had a larger delta (i.e. reduction) in bloating than PLA (−2.8 ± 3.4 vs −1.3 ± 2.3 cm, respectively). Post-hoc testing for abdominal discomfort indicated that Post was significantly lower than baseline in the PRO group by an average of −3.2 cm (∼59% reduction relative to baseline) (95% CI: −4.5 to −1.9) (P<0.001, d = 1.14) and in the PLA group by an average of −1.5 cm (∼32% reduction relative to baseline) (95% CI: −2.5 to −0.4) (P=0.012, d = 0.65). The delta between groups for abdominal discomfort indicated significant differences (P=0.021, d = −0.66). PRO had a larger average delta (i.e. reduction) in abdominal discomfort vs PLA (−3.2 ± 2.8 vs −1.5 ± 2.3 cm, respectively). Post-hoc testing for GSRS indicated that Post was significantly lower than baseline in PRO by an average of −9.1 au (∼60% reduction relative to baseline) (95% CI: −11.0 to −7.2) (P<0.001, d = 2.31) and in PLA by an average of −3.2 au (∼25% reduction relative to baseline) (95% CI: −5.2 to −1.1) (P=0.002, d = 0.72). The GSRS score for PLA was significantly greater than PRO at Post-treatment with a mean difference of 3.7 au (95% CI: 0.6 to 6.9) (P=0.014, d = −0.89) indicating a more positive effect within the PRO-treated cohort. In addition, the delta between groups for GSRS indicated significant differences (P<0.001, d = −1.46). PRO had a larger delta (i.e. reduction) in their GSRS score than PLA (−9.1 ± 3.9 au vs −3.0 ± 4.5 au, respectively). There were no significant differences noted for stool consistency (time: P=0.45; group: P=0.58; time × group: P=0.48).

Individual scores (box and whisker plots) for flatulence, constipation, bloating, abdominal discomfort, GSRS total score, and changes in GSRS score before and after the intervention are shown in Figure 2.

Figure 2
Figure 2

Box and whisker plots for flatulence (A), constipation (B), bloating (C), abdominal discomfort (D), GSRS scores (E), and GSRS change (delta) scores (F). *** P<0.001, ** P<0.01, * P<0.05.

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

3.2 Quality of life measurements (GAD-7, SF-36, Framingham)

Data from the generalised anxiety disorder (GAD-7) questionnaire is shown in Table 4. Within the GAD-7 questionnaire there was a significant time x group interaction (P=0.048) for the question ‘Becoming easily annoyed or irritable’. Post hoc testing indicated that post was significantly less than baseline for the PRO group (P=0.025). There was a significant main effect of time (P=0.024) on the GAD-7 total score. The delta between groups for total score on the GAD-7 indicated significant differences (P=0.041). On average PRO had a larger delta (i.e. reduction) relative to baseline in their GAD-7 total score than PLA [−3.1 ± 4.8 au (∼44%) vs −0.4 ± 2.3 au (∼10%), respectively]. There was a significant main effect of time (P=0.016) and group (P=0.006) on ‘Feeling nervous, anxious, or on edge’. There was a significant main effect for group (P=0.034) for ‘Not being able to stop or control worrying’. There was a significant main effect of time (P=0.049) for ‘Trouble relaxing’. There were no significant differences (P>0.05) in ‘Worrying too much about different things’, ‘Being so restless that it’s hard to sit still’, or ‘Feeling afraid as if something awful might happen’.

Table 4
Table 4

General anxiety disorder (GAD-7) assessments before (Pre) and after (Post) active probiotic (PRO) and placebo (PLA) consumption.1

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

Data from the quality-of-life health survey questionnaire (SF-36) are summarised in Table 5. Specifically for the items within the SF-36, there was a significant interaction (P=0.016) for the question ‘Didn’t do work/activities carefully due to emotional problems’, however there were no post-hoc differences. The delta between groups for ‘Didn’t do work/activities carefully due to emotional problems’ indicated a significant difference (P=0.019) with PRO showing an improvement (by ∼20%) while PLA had a poorer score (by ∼4%) from baseline to post (16.7 ± 38.1 au vs −3.9 ± 19.6 au, respectively). There was a significant interaction (P=0.013) for ‘Health interfered with normal activities’. Post hoc testing indicated that post was greater than baseline within PRO (P=0.020) showing an improvement after the intervention. In addition, the change score/delta between groups for ‘Health interfered with normal social activities’ indicated a significant difference (P=0.012). On average PRO had an improvement while PLA had a worse score from baseline to post (13.5 ± 24.4 vs −1.9 ± 17.2 au, respectively). There was a significant interaction (P=0.048) for Social Functioning, however there were no post hoc differences. There was a significant interaction (P=0.047) for Pain. Post hoc testing indicated that post was significantly greater than baseline for PRO (P=0.019) showing an improvement in Pain after the intervention. There was a significant main effect of time (P=0.032) for the composite score of physical functioning. There were no significant main effect differences (P>0.05) or interaction for several questions including; ‘Cut down time on work/activities due to emotional problems’, ‘Accomplish less than would like due to emotional problems’, ‘Have you been nervous’, ‘Calm and peaceful’, ‘Role limitations due to physical health’, ‘Role limitations due to emotional health’, ‘Energy/Fatigue’, ‘Emotional well-being’, ‘General health’, or ‘Health change’.

Table 5
Table 5

36-Item Short Form Health Survey (SF-36) assessments before (Pre) and after (Post) active probiotic (PRO) and placebo (PLA) consumption to address health-related quality of life responses.1

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

Table 5
Table 5

(Continued)

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

No differences were noted in Framingham score at baseline or post intervention (PRO: 35.6 ± 5.2 au to 35.7 ± 5.2 au vs PLA: 35.6 ± 6.5 au to 36.6 ± 7.0 au, respectively).

3.3 Adverse events

Participants in both groups tolerated PRO and PLA well with ∼10% of participants in both groups reporting possible grade 1 adverse events to either test article including: mild abdominal pain, vivid dreams, a maculo-papular rash, vertigo, and headache (Table 6).

Table 6
Table 6

Adverse events (AE) summary

Citation: Beneficial Microbes 14, 5 (2023) ; 10.1163/18762891-20230043

4 Discussion

This study examined the effects of a novel probiotic + amylase formulation on quantified digestive symptoms. Previous preliminary data testing the probiotic blend on in vitro biofilm formation, and preclinical studies indicated that the probiotic amylase formulation should reduce biofilm formation. In particular, we hypothesized that biofilms formed from bacterial interactions with fungal organisms (as observed in Crohn’s Disease) would improve, based on our preliminary studies (Hager et al., 2019). Thus, our rationale for the current study was to investigate the effect of the probiotic blend in a cohort of subjects that previously reported levels of mild-moderate GI symptoms. The current study showed that the probiotic and enzyme blend reduced the severity and frequency of overall GI symptoms and positively modulated specific clinical features (i.e. flatulence, bloating, stool regularity, constipation, and abdominal discomfort) to a greater degree than placebo. The changes in the GSRS, bloating and abdominal discomfort appear clinically relevant based on minimal important differences (Khanna et al., 2017). In addition, PRO improved irritability, mitigated health interferences with normal social activities, reduced subjective feelings of pain, and improved the change in emotional problems that interfered with working or carrying out activities carefully. Importantly the probiotic + amylase formulation was very well tolerated in individuals in comparison to PLA.

Other studies investigating probiotics have demonstrated similar modulation of GI-associated endpoints. For example, Diop et al. (2008) reported reductions in abdominal pain and nausea/vomiting and decreased levels of flatulence and gas production during probiotic (L. acidophilus Rosell-52 and Bifidobacterium longum Rosell-175) supplementation compared to placebo. While the current investigation observed improvements in bloating, Verdenelli et al. (2011), reported that probiotic enhanced foods failed to alleviate constipation and flatulence, but enhanced stool regularity after 12 weeks. In support of the current results, Bonfrate et al. (2020) reported that a double strain probiotic (B. longum BB536 and L. rhamnosus HN001) containing vitamin B6 was able to reduce abdominal pain (∼49%), bloating (∼36%), and GI-related interference with quality of life compared to placebo in IBS patients. Additionally, Kajander et al. (2005), showed a significantly greater reduction in GI symptoms (76% vs 43%) in the second half of a 6-month controlled trial with a 8-9 billion cfus blend of probiotics (L. rhamnosus LC705, B. breve Bb99, Propionibacterium freudenreuchii ssp. Shermanii JS) versus a placebo in IBS patients. The mixture of strains + amylase in the current probiotic accounted for greater changes in GI symptoms from baseline to Post-treatment time points compared to PLA, demonstrating that, similar to the work of Bonfrate and colleagues, combinations of Bifidobacterium and Lactobacillus can be beneficial to gut health (Bonfrate et al., 2020).

The gut-microbiota and the brain communicate through a variety of different pathways that offer crosstalk to multiple organs and influences the behaviour of the host (Cryan et al., 2019). Probiotic strains may modulate the microbiome structure including the hypothalamic-pituitary-adrenal (HPA) axis (leading to potential benefits supporting mental health) and the amygdala influencing social behaviour (Cryan et al., 2019). Species such as the ones found in the tested probiotic (L. acidophilus and L. rhamnosus) have been shown to lower depression and anxiety (Cryan et al., 2019). In the current investigation PRO was beneficial in mitigating irritability and overall health and emotional interference compared to PLA. The underlying mechanism behind probiotics enhancing mood and reducing anxiety is thought to be due to improving the integrity of the gastrointestinal lining, reducing leaky gut, and decreasing global inflammation which can improve neurotransmitter activity through the gut-brain axis and/or improve production of free tryptophan and subsequently improve serotonin secretion (Wallace and Milev, 2021). This may mirror preclinical studies that demonstrated a reduction in intestinal inflammation exhibited in the CD-like ileitis model SAMP1/YitFc mice following probiotic feeding (Di Martino et al., 2023).

Although speculative, these neurological effects may be why the current investigation demonstrated improvements in items within the stress/anxiety questionnaire (GAD-7) and the health questionnaire (SF-36). Wallace and Milev (2017) reported a reduction in GAD-7 scores after a 4-week daily consumption of 2 probiotic strains (3 billion cfus of Lactobacillus helveticus R0052 and Bifidobacterium longum R0175) in men and women with moderate depression (Wallace and Milev, 2021). On the other hand, a randomised controlled trial using L. helveticus R0052 and B. longum R0175 at 3 billion cfu for 8 weeks did not appear to improve depressive symptoms versus a placebo in mildly depressed subjects (Romijn et al., 2017). Altogether, these results show that there may be strain specific effects for mental health that have not been fully elucidated and thus, more specific studies focused on anxiety and probiotic use should be undertaken.

In conjunction with depression and anxiety, probiotics may also have indirect implications on overall health and associated feelings of well-being. Given the link between the gut and the brain, particularly within the HPA axis (Cryan et al., 2019), certain probiotic strains (i.e. B. longum NCC3001) may have an impact on brain activity which has been shown to coincide with less depression and improve quality-of-life in a 6-week intervention versus a placebo (Pinto-Sanchez et al., 2017). In the current investigation PRO demonstrated potential improvements in a variety of health outcomes (i.e. mitigated irritability, pain, and health interference), whereas PLA did not. Several studies targeting a variety of clinical populations have also seen increased quality of life (e.g. vitality, social functioning, mental health, pain, physical functioning) following probiotic consumption (Jalali et al., 2019; Mazzawi et al., 2013; Ohigashi et al., 2011; Pellino et al., 2013; Preston et al., 2018). Although probiotics alone did not achieve beneficial results, Kim et al. (2006), reported significant improvements in physical functioning and ‘role-physical’ domains within the SF-36 after 12 weeks with probiotics + fermented plant nutrients. This may suggest why the probiotic + amylase formulation is important, as a key interaction is the mechanism(s) of amylase, combined with the probiotic, as demonstrated in the preclinical model (Di Martino et al., 2023), as well as in vitro work demonstrating an effect on reducing biofilm formation (Hager et al., 2019), although more definitive research needs to be executed on the impact probiotics play on general health outcomes.

This study is not without limitations. The responses to the survey instruments are self-reported interpretation with no clinical verification regarding the response. The makeup of microbial constituents of the gut may be affected by diet (Singh et al., 2017), which was not controlled in this study other than the 24 h diet repeat prior to each study visit. Although the study design was a randomised, placebo-controlled, double-blind parallel study, the potential for placebo effects in any clinical study is well documented (Gupta and Verma, 2013). Lastly, future studies on this novel blend should consider including a comparator arm of amylase alone.

Our data demonstrate that consumption of the probiotic-amylase (PRO) formulation was well-tolerated with very mild side-effects. Ingestion reduced the severity and frequency of overall GI symptoms and positively modulated clinical features associated with gut discomfort (e.g. bloating, and abdominal discomfort) to a greater degree than PLA. In addition to the improved clinical features modulated by the probiotic + amylase formulation, there was also improvement in behavioural (irritability) symptoms and overall health (i.e. improved pain and lessened emotional and overall health interference) as measured by the validated SF-36 instrument.

Clinical implications regarding probiotic consumption and alteration of GI symptoms provide face validity that modulating microbiota gut constituents via probiotic supplementation can result in symptom improvement. Modulation of the gut microbiota suggests that it may be possible one day to tailor probiotics blends that would augment host microbial composition and may show efficacy as primary or adjuvant therapies for the treatment of diseases such as IBS, CD, or obesity (Fysekidis et al., 2012; Pascal et al., 2017). Indeed, the ability to modulate the microbiota through the rational design of probiotics would be useful in any number of clinical outcomes influenced by the gut microbiota.

Thus, we demonstrated herein that subjects consuming the complete probiotic + amylase formulation who completed the entire study were characterised by a significant (P0.05) reduction in GSRS score and other GI symptoms to a greater degree than individuals who consume the placebo formulation. In addition, oral supplementation of the probiotic + amylase blend was very well tolerated, suggesting incorporation of a probiotic formulation into the diet of individuals with gut-associated dysbiosis may improve overall outcomes.

*

Corresponding author; e-mail: ml@appliedhealthsciences.org

Acknowledgements

We would like to thank the dedicated group of subjects who participated in this study.

Author contributions

M.B.L. contributed to data collection, data analysis, interpretation of the findings, and manuscript preparation, B.R. contributed to data collection, H.L.L. contributed to the study design, interpretation of the findings, and helped review the manuscript, T.N.Z. contributed to the study design, interpretation of the findings, and helped prepare the manuscript. All authors read and approved the final version of the manuscript.

Conflicts of interest

This research was sponsored in part by a grant from BIOHM Health, LLC. This study was conducted at a consulting research organisation (CRO) in which all the authors are affiliated.

Financial support

This research was sponsored in part by a grant from BIOHM Health, LLC. BIOHM contributed to the design of this research study. The sponsor did not contribute to the data collection, data analysis, or writing of the manuscript. The presentation of results of this study does not constitute endorsement by any of the researchers or their affiliation.

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