Probiotic Supplementation Has a Limited Effect on Circulating Immune and Inflammatory Markers in Healthy Adults: A Systematic Review of Randomized Controlled Trials

RESEARCH

Review

Probiotic Supplementation Has a Limited Effect on Circulating Immune and Inflammatory Markers in Healthy Adults: A Systematic Review of Randomized Controlled Trials Alex E. Mohr, MS; Anthony J. Basile, MS; Meli’sa S. Crawford; Karen L. Sweazea, PhD; Katie C. Carpenter, PhD ARTICLE INFORMATION Article history: Submitted 15 March 2019 Accepted 12 August 2019

Keywords: Probiotic supplement Healthy adult Immunity Inflammation Systematic review 2212-2672/Copyright ª 2019 by the Academy of Nutrition and Dietetics. https://doi.org/10.1016/j.jand.2019.08.018

ABSTRACT Background A main mechanism of action proposed for oral probiotic supplementation is immunomodulation, which is expected to impart health benefits in the host by influencing circulating immune and inflammatory factors. To date, the effectiveness of probiotic supplementation for immunomodulation in healthy adults without disease has not been evaluated in a systematic review. Objective The objective of this systematic review was to evaluate the effect of probiotic supplementation on circulating immune and inflammatory markers of healthy adults compared to placebo. Methods PubMed, SCOPUS, ISI Web of Science, ProQuest, and Cochrane databases were searched for English articles up to May 15, 2019. Additional papers were identified by checking references of relevant papers. Only randomized controlled trials studying the administration of probiotic supplements compared to placebo on immune and inflammatory markers in healthy adults (aged 18 to 65 years), without acute or chronic disease, and in generally good health were examined. Independent extraction of articles was conducted by two authors using predefined search terms and restrictions/filters. The methodologic quality of each study was appraised using the Academy of Nutrition and Dietetics Evidence Analysis Library Quality Rating Worksheet and the body of evidence was assessed using the Academy of Nutrition and Dietetics Grade Definitions and Conclusion Grading Table. Results Eighteen articles, including 819 subjects, met eligibility criteria and were included in the present systematic review. Five articles were rated neutral in quality and 13 were rated high in quality. Eight articles reported a significant effect on immune and/ or inflammatory parameters including increases in natural killer cells, lymphocytes, and monocytes, and decreases in proinflammatory cytokine concentrations. Conclusions Based on the 18 articles extracted in this systemic review, probiotic supplementation was concluded to have a limited effect on immune and inflammatory markers in healthy adults. Overall, the evidence was heterogenous, precluding a metaanalysis, and difficult to aggregate and conclude on effect size. Systematic review registration number PROSPERO ref CRD42018110856. J Acad Nutr Diet. 2019;-(-):---.

D

ATA FROM THE US NATIONAL HEALTH INTERVIEW Survey show that probiotics are the third most commonly used dietary supplement among adults,1 other than vitamins and minerals, and are expected to exceed more than US$73 billion on the global market by 2024.2 Unique in comparison to other common dietary supplements, probiotic preparations comprise live microorganisms that, when administered in adequate amounts, confer a health benefit on the host.3 In the last several decades, research on the health effects of probiotics has progressed considerably and significant advances have been made in the selection and characterization of specific probiotic cultures.4

ª 2019 by the Academy of Nutrition and Dietetics.

Probiotics are now available commercially in a wide variety of products, including capsule or powder form, and in food products, such as fermented milk or yogurt.5 Research in humans has shown the beneficial effects of probiotics on several health outcomes, with the majority of studies conducted in clinical populations.6,7 In comparison, research supporting the use of probiotics in healthy adults has been more limited and inconsistent.7,8 The most studied target site for probiotic effects is the gastrointestinal tract, which throughout its length is increasingly colonized by microbes.3,6 A substantial portion of gut physiology, including the modulation of gut motility,

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

1

RESEARCH intestinal barrier homeostasis, nutrient absorption, and fat distribution, is influenced by the mutualistic relationship between intestinal microorganisms and their human host.9 Orally administered probiotics may interact with the approximately 200 m2 of gastrointestinal mucosa and gut associated lymphoid tissue where >70% of immune cells are localized.10 It is therefore not surprising that many of the recognized health benefits of probiotics are conferred mainly through stimulation/modulation of the immune system,11 affecting host immunity and inflammation.12 In relation, commercially available probiotic products are often promoted as capable of improving immune defenses13 and consumers are encouraged to try products shown to enhance immune function and reduce risk of common illness such as colds.14 A main mechanism of action proposed for probiotics is immunomodulation, which influences immune and inflammatory factors in the host, imparting positive health benefits, such as promoting pathogen resistance and anti-inflammation.4,15 Currently, no systematic review has been conducted on the effects of probiotics on circulating immune and inflammatory markers in healthy adults. Therefore, the objective of this systematic review was to examine whether probiotic supplements significantly influence immune and inflammatory markers in healthy adults compared with placebo. Research on this population is important for clinicians, consumers, industry and regulating bodies alike, who require information on whether these dietary supplements are indicated for healthy individuals.4

MATERIALS AND METHODS Protocol and Registration The available literature was identified and examined as a systematic review and not a meta-analysis due to the heterogeneity of probiotic strains and study outcomes. The results are reported in accordance with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement guidelines.16 Methods of the analysis and inclusion criteria were specified in advance and documented in a protocol that was published in a prospective register of systematic reviews, PROSPERO (www.crd.york.ac.uk/PROSPERO/; ref CRD42018110856).

Eligibility Criteria To be eligible for inclusion in this systematic review, studies had to meet the criteria of randomized controlled trials studying the administration of probiotic supplements on healthy adult humans, aged 18 to 65 years. No restriction of publication date or publication status was imposed, however, only articles published in English were included. Healthy was defined as individuals with no reported status of acute or chronic diseases, including but not limited to diabetes, cardiovascular disease, gastrointestinal diseases, autoimmune diseases, obesity (body mass index
30), cancer, and liver and kidney disease. In addition, pregnant women, individuals with mental disorders or reported drug use, and athletes or individuals involved in intense physical activity were excluded. Studies with interventions combining probiotics with other supplements (eg, prebiotics, antibiotics, and medications) were excluded. If studies had more than two arms, only the comparison of probiotics to placebo were 2

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

RESEARCH SNAPSHOT Research Question: Based on the current body of literature, does oral probiotic supplementation affect circulating immune and inflammatory markers compared to placebo in healthy adults? Key Findings: A systematic review of 18 randomized controlled trials that included 819 healthy subjects, found that there is limited evidence that oral probiotic supplementation affects circulating immune and inflammatory markers. Overall, this body of literature varied in quality and was limited in quantity, indicating the need for more appropriately powered randomized controlled trials in healthy adults. considered. Studies comparing healthy to unhealthy participants were also excluded. Only trials comparing the effects of orally ingested probiotic supplements (live cells) on circulating immune and inflammatory markers were included. For the purposes of this study, supplement was defined in accordance with the US Food and Drug Administration and as indicated in the Dietary Supplement Health and Education Act of 1994 as, “Intended for ingestion in a tablet, capsule, powder, softgel, gelcap, or liquid form.”17,18 Probiotic foods and beverages (such as fermented products), intense exercise, or vaccination tests were excluded due to the possible confounding effects on immune and inflammatory markers.4 Furthermore, this review sought to distinguish between probiotics as dietary supplements and functional foods. The primary outcome measures comprised circulating immune and inflammatory markers, including (but not limited to) monocytes, macrophages, immunoglobulins, lymphocytes, phagocyte activity, interferons (IFNs), interleukins (IL), Creactive protein (CRP), tumor necrosis factorea (TNF-a), and prostaglandins. Interventions and markers associated with the oral-pharyngeal cavity, respiratory tract, and genitalia were excluded.19-21

Search Strategy Articles were identified by applying the search to electronic databases PubMed, SCOPUS, ISI Web of Science, ProQuest, and Cochrane to capture as many relevant citations as possible. In addition, reference lists of included articles were searched for further resources. The search was conducted on May 14, 2019 for PubMed, ISI Web of Science, and ProQuest, and on May 15, 2019 for SCOPUS and Cochrane databases. The following search terms were used to search all databases; healthy volunteer OR healthy OR healthy subjects OR healthy participants OR healthy adults OR healthy persons OR healthy individuals OR healthy population OR healthy males OR healthy females AND probiotics OR probiotic supplementation OR probiotic supplement OR lactobacillus OR bifidobacterium OR streptococcus OR enterococcus OR bacillus OR saccharomyces OR lactobac* OR bifidobac* AND immunity OR immune OR lymphocyte OR interferon OR interleukin OR phagocyte OR immunoprotein OR leukocyte OR inflammat* OR cytokine OR prostaglandin OR monocyte OR macrophage OR immunoglobulin OR natural killer cells OR c-reactive protein OR tumor necrosis factor-alpha (asterisk is used in database search to --

2019 Volume

-

Number

-

RESEARCH capture multiple word endings). Figure 1 presents examples of the complete search strategies used in the electronic databases PubMed and SCOPUS. The search was limited to randomized controlled trials, human subject research, and articles published in the English language. No date restriction was applied. Duplicates were removed in the initial screening process, and titles and abstracts of the remaining studies were assessed for eligibility criteria by two investigators (A.E.M., K.C.C.). Full reports were then obtained and screened for all titles and abstracts appearing to meet the defined eligibility criteria according to the population, intervention, comparison (group or treatment), outcomes, and setting criteria as outlined in Figure 2. Eligibility was determined through agreement between two reviewers (A.E.M., K.C.C.). Disagreements between the two reviewers were resolved by consensus.

measure (including the inflammatory and/or immune marker, and the effect of the intervention). Differences in data extraction were discussed between reviewers (A.E.M., K.C.C.) until consensus was achieved. To ascertain the quality and validity of eligible randomized controlled trials, two investigators (A.E.M., A.J.B.) independently assessed each study according to the Academy of Nutrition and Dietetics Evidence Analysis Library Quality Criteria checklist for primary research.22 Discrepancies between reviewer’s quality ratings were discussed and resolved if possible.23 This checklist includes four relevance questions that address applicability to practice and 10 validity questions that address scientific soundness of included studies. Each article was assigned an overall quality rating of positive, negative, or neutral. Articles were not excluded based on risk of bias.

Data Extraction and Quality Evaluation

SUMMARY MEASURES

Based on the Academy of Nutrition and Dietetics Evidence Analysis Manual Evidence Worksheet, a data extraction form was developed (available from authors on request).22 Independent data extraction was performed by two investigators (A.E.M., K.C.C.). Information was extracted from each included trial on study details (including authors, year, and country of publication); characteristics of trial participants (including age, sex, race, physical activity), and inclusion and exclusion criteria; type of intervention (including type, dose, and frequency of the probiotic versus placebo); design (including study design and duration); and type of outcome

Statistically significant between-group differences were used to indicate intervention effects on circulating immune and inflammatory markers (significance level of P<0.05). An intention to treat approach was used where applicable. The Academy of Nutrition and Dietetics Grade Definitions and Conclusion Grading Table provided in the Academy of Nutrition and Dietetics Evidence Analysis Manual was used to provide an overall assessment of the body of evidence.22 The strength of evidence was determined by quality, consistency, quantity, clinical influence, and generalizability. Gradings range from I to V, where grade I¼good, grade II¼fair, grade

Database

Search termsa

PubMed (Search conducted up to May 14, 2019)

(((((((((((healthy volunteers) OR “healthy subjects”) OR “healthy participants”) OR “healthy adults”) OR “healthy persons”) OR “healthy individuals”) OR “healthy population”) OR “healthy males”) OR “healthy females”)) AND (((((((((((probiotics) OR “probiotic supplementation”) OR “probiotic supplement”) OR lactobacillus) OR bifidobacterium) OR streptococcus) OR enterococcus) OR bacillus) OR saccharomyces) OR lactobac*b) OR bifidobac*)) AND (((((((((((((((((immunity) OR “immune”) OR lymphocytes) OR interferons) OR interleukins) OR phagocytes) OR immunoproteins) OR leukocytes) OR monocytes) OR macrophages) OR immunoglobulins) OR natural killer cells) OR inflammat*) OR cytokines) OR prostaglandins) OR c-reactive protein) OR tumor necrosis factor-alpha)

SCOPUS (search conducted up to May 15, 2019)

(“healthy volunteers” OR {healthy subjects} OR {healthy participants} OR “healthy adults” OR {healthy persons} OR {healthy individuals} OR {healthy population} OR {healthy males} OR {healthy females}) AND (“probiotics” OR {probiotic supplementation} OR {probiotic supplement} OR lactobacillus OR bifidobacterium OR streptococcus OR enterococcus OR bacillus OR saccharomyces OR lactobac* OR bifidobac*) AND (immunity OR immune OR lymphocytes OR interferons OR interleukins OR phagocytes OR immunoproteins OR leukocytes OR inflammat* OR cytokines OR prostaglandins OR monocytes OR macrophages OR immunoglobulins OR {natural killer cells} OR {c-reactive protein} OR {tumor necrosis factor-alpha}) AND (LIMIT-TO (DOCTYPE, “ar”)) AND (LIMIT-TO (EXACTKEYWORD, “Human”)) AND (LIMITTO (LANGUAGE, “English”)) AND (LIMIT-TO (EXACTKEYWORD, “Randomized Controlled Trial”))

a

Searches were limited to randomized controlled trials, human subject research, and studies published in the English language using the appropriate filters and/or search terms depending on the database. b

Asterisk (*) used in database search to capture multiple word endings (eg, inflammat* will capture inflammation and inflammatory).

Figure 1. The effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults: Example of the database search strategy using PubMed and SCOPUS. --

2019 Volume

-

Number

-

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

3

RESEARCH Domain

Inclusion criteria

Population

Healthy adults (18 to 65 years of age) with no reported status of acute or chronic diseases, including diabetes, cardiovascular disease, gastrointestinal diseases, autoimmune diseases, obesity (body mass index
30), cancer, or liver and kidney disease. In addition, no pregnant women, nursing women, or any individuals with mental disorders or reported drug use.

Intervention

Oral probiotic (live cells) supplement consumption.

Comparator

Placebo group of randomized controlled trials studying oral probiotic supplementation on healthy adults (as described in “Eligibility Criteria”).

Outcome

Circulating immune and inflammatory markers, including (but not limited to): monocytes, macrophages, immunoglobulins, lymphocytes, phagocyte activity, interferons, interleukins, C-reactive protein, tumor necrosis factor-a, and prostaglandins.

Study design

Randomized controlled trials or randomized cross-over trials.

Figure 2. Summary of study eligibility criteria for the effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses framework (diagram adapted from Moher and colleagues16). III¼limited, grade IV¼expert opinion only, and grade V¼not assignable due to no available evidence.

RESULTS Study Selection The article selection process is illustrated in Figure 3. A total of 18 articles were identified for inclusion in the systematic review. The initial database search of PubMed, SCOPUS, ISI Web of Science, ProQuest, and Cochrane in May 2019 identified 3,401 publications. Four potentially eligible articles were identified from alternate methods and added to the pool of articles for consideration. After adjusting for duplicates, 3,039 unique articles remained. Of these, 2,912 articles were discarded after reviewing the titles and abstracts, as these papers did not meet the inclusion criteria. Reasons included not relevant outcome measures (n¼418), not adults between the ages of 18 to 65 years (n¼275), not a healthy study population (n¼547), not a randomized controlled trial (n¼231), animal study (n¼32), in vitro study (n¼61), not oral administration (n¼10), combined intervention (n¼101), pregnant or nursing (n¼13), obese subjects (n¼10), not 4

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

probiotic intervention (n¼1144), not in English (n¼68), and no control (n¼2). Next, the full texts of the remaining 127 articles were assessed to select articles for inclusion using the abovementioned criteria, resulting in the exclusion of 109 articles. After reviewing entire full-text articles, reasons for exclusion included not relevant outcome measures (n¼33), not healthy adults between the ages of 18 and 65 years (n¼3), not randomized controlled trial (n¼8), not a supplement (n¼45), combined intervention (n¼6), athletes/intense physical activity (n¼5), in vitro (n¼2), inactivated probiotics (n¼2), subgroup analysis (evaluation of treatment effects for a specific end point in a small subgroup of subjects)24 (n¼2), and conference abstract (n¼3). Several extracted publications presented information from the same research study and participant population but reported outcomes separately. In the first instance, two publications were generated from the same study participants, reporting different immune outcomes.25,26 In the second instance, two publications were generated from the same study with one reporting results on inflammatory markers,27 while the other reported on immune response.28 As this review is a qualitative assessment, these publications were presented separately to remain consistent with how the authors chose to present their findings.

Study Characteristics The characteristics of the 18 articles included in the review are outlined in Table 1. All articles were published in English between November 2003 and November 2018, ranging from 3 weeks to 5 months in duration. Studies were conducted in Australia,25,26 Belgium,29 Finland,30 Denmark,31 Korea,32,33 The Netherlands,34 Malaysia,27,28 Spain,35 New Zealand,36 United Kingdom,37 United States,38,39 India,40,41 and Ireland.42 All studies were randomized controlled trials, with one a doseeresponse31 and three cross-over designs (study design characteristics outlined in Table 2).29,37,42 Fifteen were double-blinded25-31,33-40 and one was single-blinded.41 Two articles reported their investigations were blinded, but did not report blinding methodology.32,42 Five articles reported sample size calculations,37,38,40-42 with three powered to immune or inflammatory markers.37,40,41 The included articles involved a total of 819 healthy participants with an age range of 18 to 65 years. Thirteen included female participants with a proportion that ranged from 50% to 72%.25,26,29-31,33-38,40,41 The interventions reported in the included articles were probiotics belonging to the genus Lactobacillus (n¼6)34,35,38,39,41,42; Bifidobacterium and Lactobacillus combined (n¼5)25-28,31; Bifidobacterium (n¼2)30,37; Bacillus (n¼1)32; Weissella (n¼1)33; Streptococcus (n¼1)36; Butyricicoccus (n¼1)29; and Bifidobacterium, Lactobacillus, and Streptococcus combined (n¼1).40 Supplements were provided as capsule (n¼9),29-31,33,35,39-42 sachet (n¼7),25-28,34,36,37 tablet (n¼1),32 or oil drops (n¼1)38 administered at a dose between 1  108 to 1  1011 colony-forming units. Where reported, compliance was assessed by return of unused product,29,36-38 daily record/checklist,27,28,34,39 detection of bacterial DNA from fecal samples,35 or an unspecified method.33 The control groups in all of the included studies received a placebo of similar size and appearance, except in one instance.30 Eleven studies prohibited the consumption of confounding dietary factors in order to avoid possible interferences, including fermented foods,32,35 --

2019 Volume

-

Number

-

RESEARCH

Identification

Records identified through database searching(n=3,401) - PubMed (n=968) - SCOPUS (n=1,179) - ISI Web of Science (n=730) - ProQuest (n=267) - Cochrane (n=257)

Additional records identified through other sources (n=4)a

Included

Eligibility

Screening

Records after duplicates removed (n=3,039)

Records screened (n=3,039)

Records excluded (n=2,912)

Full-text articles assessed for eligibility (n=127)

Full-text articles excluded due to:

Studies included in qualitative synthesis (n=18)

• • • • • • • • • •

Not relevant outcome Not healthy adult Not RCTb Not supplement Combined intervention Athletes/intense PAc In vitro Not live cells Abstract Sub-group analysis (n=109)

Figure 3. Flow diagram of literature selection process for the systematic review of randomized controlled trials investigating the effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults (diagram adapted from Moher and colleagues16). aIdentified by checking the references of the full-text articles assessed for eligibility. bRCT¼randomized controlled trial. cPA¼physical activity.

--

2019 Volume

-

Number

-

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

5

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS --

Author(s), year, country

Subject characteristicsa

Probiotic(s), delivery form

Dose

Duration

Design

Outcome

Mäkeläinen and colleagues, 2003,30 Finland

Healthy males and females; 19 to 60 y; n¼39

Bifidobacterium longum 2C and B longum 46 Capsule

2109 CFUb daily

3 wk

Double-blind RCT,c parallel-group

No effect

Christensen and colleagues, 2006,31 Denmark

Healthy males and females; 25.6 y (SDd not reported); n¼71

Bifidobacterium animalis subspecies lactis BB-12 and Lactobacillus paracasei subspecies paracasei CRL-431 Capsule

0; 1108, 1109, 11010, and 11011 CFU daily

3 wk

No effect Double-blind RCT, parallel-group, dose eresponse trial

Kim and colleagues, 2006,32 Korea

Healthy males; Trte: 25.575.04 y, Conf: 25.215.34 y; n¼25

Bacillus polyfermenticus SCDg 3.1108 CFU daily Tablet

8 wk

RCT, parallel-group

[Concentration of IgG,h %CD4þ T helper cells, %CD8þ cytotoxic T cells, %CD56þ NKi cells

Sierra and colleagues, 2010,35 Spain

Healthy male and females; 337.9 y; n¼40

Lactobacillus salivarius CECT5713 Capsule

2108 CFU daily

4 wk

Double-blind RCT, parallel-group

[IL-10j; YIL-12/IL-10, TNF-ak/IL-10

Wind and colleagues, 2010,34 The Netherlands

Healthy male and females; 4216 y; n¼34

L rhamnosus PRSF-L477 Sachet

11011 CFU daily

3 wk (3-wk washout)

Double-blind RCT, parallel-group

No effect

Burton and colleagues, 2011,36 New Zealand

Healthy males and females; Streptococcus salivarius K12 Trt: 39.7 y, Con: 37.2 y (SD Sachet not reported); n¼53

11010 CFU daily

4 wk

Double-blind RCT, parallel-group

No effect

Lactobacillus reuteri DSM 17938 Oil drops

5108 CFU daily

8 wk

Double-blind RCT, parallel-group

No effect

Mangalat and colleagues, Healthy males and females; 2012,38 United States Trt: 34.612.1 y, Con: 32.910.2 y; n¼40

2019 Volume -

Number

Childs and colleagues, 2014,37 United Kingdom

Healthy male and females; 4312 y; n¼44

B animalis subspecies lactis Bi-07 Sachet

1109 CFU daily

3 wk

Double-blind RCT, cross-over

YLPSl-stimulated IL-4 secretion; [IL-6 secretion

Cox and colleagues, 2014,25m Australia

Healthy physically active male and females; Trt 1: 42.216.2, Trt 2:

Trt 1: B animalis subspecies lactis Bl-04; Trt 2: Lactobacillus acidophilus

Trt 1: 2109 CFU; Trt 2: 11010 CFU

5 mo

Double-blind RCT, parallel-group

No effect

-

(continued on next page)

RESEARCH

6

Table 1. The effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults: Characteristics of included articles (by publication date)

--

2019 Volume

Table 1. The effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults: Characteristics of included articles (by publication date) (continued) Author(s), year, country

Subject characteristicsa

Duration

Design

Outcome

6 wk

Double-blind RCT, parallel-group

YCRPn

Trt 1: 2109 CFU; Trt 2: 11010 CFU (5109 CFU of each strain) daily

5 mo

Double-blind RCT, parallel-group

[MIP-1do (Trt 1 vs Con), YMMP-1p (Trt 2 vs Con)

2109 CFU daily

6 wk

Single-blind, RCT, parallel-group

YCRP, IL-6, IL-1b, TNF-

Kelly and colleagues, 2017,42 Ireland

Healthy males; 24.590.75 y; Lactobacillus rhamnosus JB-1 1109 CFU daily n¼29 Capsule

8 wk

RCT, cross-over

No effect

Marcial and colleagues, 2017,39 United States

Healthy males and females; 23.25.5 y; n¼42

Lactobacillus johnsonii N6.2 Capsule

5108 CFU daily

8 wk (4-wk washout)

Double-blind RCT, parallel-group

[Monocytes, NK cell (after 4-wk washout); [Thelper 1 cells, cytotoxic CD8þ T cells

Boesmans and colleagues, 2018,29 Belgium

Healthy males and females; 22 to 52 y; n¼30

Butyricicoccus pullicaecorum 25-3T Capsule

1108 CFU daily

4 wk (3-wk washout)

Double-blind RCT, cross-over

No effect

Ibrahim and colleagues, 2018,27q Malaysia

61010 CFU Healthy sedentary males; 19 Multistrain: L acidophilus daily to 26 y; n¼41 BCMC 12130, L casei BCMC 12313, L lactis BCMC

12 wk

Double-blind RCT, parallel-group

No effect

-

37.311.4, Con: 38.910.9 y; n¼129

Number -

Rajkumar and colleagues, Healthy overweight males 2014,40 India and females; 40 to 60 y; n¼60 (15 placebo; 15 probiotic)

West and colleagues, 2014,26m Australia

Probiotic(s), delivery form NCFM and B animalis subspecies lactis Bi-07 Sachet

9

(510 CFU of each strain) daily

11.251010 Multistrain: B longum, Bifidobacterium infantis, B. CFU daily breve, L acidophilus, L paracasei, L delbrueckii subspecies bulgaricus, L plantarum, and S salivarius subspecies thermophilus Capsule

Trt 1: B animalis subspecies Healthy physically active lactis Bl-04; Trt 2: L male and females; Trt 1: acidophilus NCFM and B 5013, Trt 2: 5111, Con: animalis subspecies lactis 4911 y; n¼144 Bi-07 Sachet L salivarius UBL S22 Capsule

a

7

(continued on next page)

RESEARCH

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

Rajkumar and colleagues, Healthy males and females; 2015,41 India 20 to 25 y; n¼45 (15 placebo; 15 probiotic)

Dose

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

Author(s), year, country

Subject characteristicsa

Probiotic(s), delivery form

Dose

Duration

Design

Outcome

12 wk

Double-blind RCT, parallel-group

No effect

8 wk

Double-blind RCT, parallel-group

[NK cell activity

12451, Bifidobacterium bifidum BCMC 02290, B infantis BCMC 02129, and B longum BCMC 02120 Sachet Ibrahim and colleagues, 2018,28q Malaysia

61010 CFU Healthy sedentary males; 19 Multistrain: L acidophilus daily to 26 y; n¼41 BCMC 12130, L casei BCMC 12313, L lactis BCMC 12451, B bifidum BCMC 02290, B infantis BCMC 02129, and B longum BCMC 02120 Sachet

Lee and colleagues, 2018,33 Korea

Healthy males and females; 43.22.13 y; n¼82

a

Weissella cibaria JW15 Capsule

Age reported as meanSD. When unavailable, age range is reported. CFU¼colony-forming unit. c RCT¼randomized controlled trial. d SD¼standard deviation. e Trt¼treatment group. f Con¼control group. g SCD denotes strain designation. h IgG¼Immunoglobulin G. i NK¼natural killer. j IL¼interleukin. k TNF-a¼tumor necrosis factorea. l LPS¼lipopolysaccharide. m Two publications were generated from the same study participants, reporting different immune outcomes.25,26 n CRP¼C-reactive protein. o MIP-1d¼macrophage inflammatory protein-1d. p MMP-1¼matrix metalloproteinase-1. q Two publications of the same study, separately reporting immune28 and inflammatory markers.27 b

11010 CFU daily

RESEARCH

8

Table 1. The effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults: Characteristics of included articles (by publication date) (continued)

--

2019 Volume -

Number -

--

2019 Volume

Table 2. The effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults: Research design characteristics of included articles (by publication date)

Supplement delivery

Appropriate use of placebo

Compliance assessment

Mäkeläinen and colleagues, 200330

Double

Capsule

No; appearance

NR

NR

NR

Yes

31

Christensen and colleagues, 2006

Double

Capsule

Yes

NR

Yes

NR

No

Kim and colleagues, 200632

NRa

Tablet

No; use of yeast tablets

NR

Yes

3-day dietary record

No

Sierra and colleagues, 201035

Double

Capsule

Yes

Presence of probiotic bacteriab

Yes

NR

No

Wind and colleagues, 201034

Double

Powder

Yes

Daily record

Yes

NR

Yes

Burton and colleagues, 201136

Double

Powder

Yes

Return of unused product

Yes

NR

Yes

Mangalat and colleagues, 201238

Double

Liquid

Yes

Return of unused product

Yes

NR

Yes

Childs and colleagues, 201437

Double

Powder

Yes

Return of unused product

Yes

4-day dietary record

No

Cox and colleagues, 201425

Double

Powder

Yes

NR

NR

NR

Yes

Rajkumar and colleagues, 201440

Double

Capsule

Yes

NR

Yes

NR

No

West and colleagues, 2014

Double

Powder

Yes

NR

NR

NR

No

Rajkumar and colleagues, 201541

Single

Capsule

Yes

NR

Yes

NR

No

NR

Capsule

Yes

NR

Yes

Food frequency questionnaire

No

Double

Capsule

Yes

Daily record

Yes

NR

Yes

Double

Capsule

Yes

Return of unused product

Yes

NR

Yes

Ibrahim and colleagues, 201827

Double

Powder

Yes

Daily record

Yes

NR

No

28

Double

Powder

Yes

Daily record

Yes

NR

No

Double

Capsule

Yes

Unspecified method

Yes

3-day dietary record

No

Author(s), year

-

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

26

Kelly and colleagues, 2017

42

Marcial and colleagues, 201739 Boesmans and colleagues, 2018

Ibrahim and colleagues, 2018

29

Lee and colleagues, 201833 a

NR¼not reported. Detection of bacterial DNA from fecal samples.

b

9

RESEARCH

Blinding type

-

Number

Dietary intake assessment

Safety/ tolerability as primary outcome

Restricted antibiotic use

RESEARCH probiotic-containing foods/products,29,30,33,36,37,39,41,42 and dietary supplements.27,28 Four studies analyzed dietary intake by such methods as dietary records32,33,37 and food frequency questionnaires.42 In the included studies, participants were required not to have taken antibiotics prior to and/or during the experimental period, except for a single study.30 One study had participants record physical activity during the experimental period33 and another had participants refrain from strenuous exercise before testing.42 Eight articles investigated inflammatory and immune markers as primary outcomes,26,28,31-33,35,40,41 whereas in three articles they were examined as secondary outcomes.27,37,42 Seven articles had safety and tolerability as the primary outcomes.25,29,30,34,36,38,39 The type of outcomes across the included articles were immune markers (n¼7),25,28-30,32,34,36 inflammatory markers (n¼3)27,40,41 or both (n¼8).26,31,33,35,37-39,42 Finally, only one article reported estimated effect sizes.38

Study Quality The quality assessment of included articles is presented in Table 3. Initially after reviewing the articles, the two investigators (AEM, AJB) had 78.2% agreement in scoring, which produced a .353 (fair agreement) Cohen’s k statistic.43 When a consensus was reached on scoring definitions and requirements, a 94.7% agreement was produced with a .872 (almost perfect agreement) Cohen’s k statistic. Ultimately, five articles were rated as neutral quality, indicating that the report was neither exceptionally strong nor exceptionally weak, and 13 articles were rated as high quality, indicating that the report clearly addressed issues of inclusion/exclusion, bias, generalizability, and data collection and analysis. Certain aspects were sometimes not reported, such as blinding,27,32,42 and number and reasons for withdrawals.25,28,30,32,40-42 Of the articles rated as neutral in quality, the method of randomization, selection biases, and study limitations were often not reported.25,30-32,38

Results of Individual Studies Although all articles reported measures of immune markers, inflammatory markers or both, there was considerable heterogeneity among studies with regard to the probiotic strain used and the specific outcomes reported. Therefore, focus was placed on describing the studies, their results, their applicability, and their limitations in a qualitative synthesis rather than a meta-analysis. Overall, 8 of the 18 included articles reported a significant effect on immune and/or inflammatory markers at the end of the study period with oral probiotic supplementation compared to placebo. Of the 12 articles investigating the effect of probiotic supplementation on immune markers, 3 reported significantly different treatment outcomes compared to the placebo group. Kim and colleagues32 reported supplementation with Bacillus polyfermenticus SCD (SCD denotes strain designation) had immunoenhancing effects, in part by modulation of innate, humoral and cell-mediated immune responses. Percentages of immunoglobulin G, CD4þ helper T cells, CD8þ cytotoxic T cells, and CD56þ natural killer (NK) cells in the probiotic-supplemented group were 12%, 32%, 28%, and 35% higher (all, P<0.05), respectively, compared with the control group after 8 weeks of supplementation. Additionally, the ratio of CD4þ/CD8þ T cells was greater in the experimental 10

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

group (P<0.05). Marcial and colleagues39 investigated the effects of Lactobacillus johnsonii N6.2 after 8 weeks of treatment and 4 weeks of washout on immune cell subsets of innate and adaptive immune systems. There was a significant increase in NK cells and monocytes (P<0.05) after the washout period, but not directly after treatment. In addition, there was a significant increase in circulating effector Th1 cells and cytotoxic CD8þ T-cell subsets. However, B-cell subsets, dendritic cells, and regulatory T-cell subsets did not show differences between groups during the treatment period or after washout. In a study by Lee and colleagues,33 supplementation with Weissella cibaria JW15 for 8 weeks resulted in significantly increased NK cell activities over placebo (P<0.05), but did not affect circulating immunoglobulin levels. Null findings included Mäkeläinen and colleagues,30 who did not find a significant effect on phagocytic activity from Bifidobacterium longum 2C and B longum 46 supplemented over 3 weeks. Similarly, Christensen and colleagues31 did not find a significant difference after supplementation with Bifidobacterium animalis subspecies (subsp) lactis BB-12 and Lactobacillus paracasei subsp paracasei CRL-431 in phagocytic activity in blood leukocytes compared to placebo. Wind and colleagues34 found no difference in leukocytes in those who supplemented with Lactobacillus rhamnosus PRSF-L477 for 3 weeks compared to placebo. Additionally, Burton and colleagues36 found no significant differences between the Streptococcus salivarius K12 and placebo-treated groups in white blood cell count, neutrophils, lymphocytes, monocytes, eosinophils, and basophils after 4 weeks. Mangalat and colleagues38 found no significant effect of Lactobacillus reuteri DSM 17938 on peripheral blood mononuclear cells Toll-like receptor expression on peripheral blood mononuclear cells after 8 weeks compared to placebo. Cox and colleagues25 did not detect any significant effect on white blood cell count, neutrophils, lymphocytes, monocytes, or eosinophils after 5 months (150 days) of supplementation in healthy physically active individuals with two arms of probiotic treatment, including B animalis subsp. lactis Bl-04 or Lactobacillus acidophilus NCFM and B animalis subsp lactis Bi-07 compared to placebo. Using participants from the same study, West and colleagues26 did not report a significant effect on NK-cell activity or phagocytosis capacity. Additionally, Boesmans and colleagues29 did not find a significant difference on white blood cell count from 4 weeks of Butyricicoccus pullicaecorum 25-3T supplementation compared to placebo. Finally, Ibrahim and colleagues28 did not report a significant effect on immune markers, including total leukocytes, total lymphocytes, T-lymphocytes, T-helper, T-cytotoxic, B-lymphocytes, or NK-cell counts after 12 weeks of treatment with a multi-strain probiotic containing L acidophilus BCMC 12130, Lactobacillus casei BCMC 12313, Lactobacillus lactis BCMC 12451, B bifidum BCMC 02290, Bifidobacterium infantis BCMC 02129, and B longum BCMC 02120. Of the 12 articles that investigated inflammatory markers, 5 reported significant differences in the probiotic treatment groups compared to placebo. In a 4-week study, Sierra and colleagues35 found a significant increase in the antiinflammatory cytokine IL-10 (P<0.05) and significant decreases in IL-12/IL-10 and TNF-a/IL-10 (P<0.05) ratios after supplementation with Lactobacillus salivarius CECT5713 compared to placebo. Childs and colleagues37 found a significant decrease in lipopolysaccharide-stimulated IL-4 secretion in whole-blood cultures (P¼0.035) and increased IL-6 secretion --

2019 Volume

-

Number

-

RESEARCH Table 3. Quality of included randomized controlled trials assessing the effect of oral probiotic supplementation on circulating immune and inflammatory markers in healthy adults using the Academy of Nutrition and Dietetics’ Evidence Analysis Library Quality Criteria Checklist22: Evaluation resultsa Validity Questionsc

Author(s), year

Relevance questionsb

1

2

3

4

5

6

7

8

9

10

Qualityd

Mäkeläinen and colleagues, 200330

Yes

Ye/Y

Uf/U

U/U

Ng/N

Y/Y

Y/Y

Y/Y

N/N

N/Y

U/U

Øh

Christensen and colleagues, 200631

Yes

Y/Y

U/U

U/U

N/N

Y/Y

Y/Y

Y/Y

Y/Y

U/U

U/U

Ø

Kim and colleagues, 200632

Yes

Y/Y

U/U

U/U

N/N

N/N

Y/Y

Y/Y

Y/Y

Y/Y

U/U

Ø

35

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

U/U

þi

34

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

Burton and colleagues, 2011

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

N/Y

N/N

þ

Mangalat and colleagues, 201238

Yes

Y/Y

N/N

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/N

Ø

Childs and colleagues, 201437

Yes

Y/Y

Y/Y

Y/Y

Y/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

N/N

þ

Cox and colleagues, 201425

Yes

Y/Y

U/U

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

U/U

N/N

Ø

Rajkumar and colleagues, 201440

Yes

Y/Y

Y/Y

Y/Y

N/N

N/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

West and colleagues, 201426

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

U/U

Y/Y

N/N

þ

Rajkumar and colleagues, 201541

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

Kelly and colleagues, 201742

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/U

Y/Y

Y/Y

Y/Y

Y/Y

Y/N

þ

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

U/U

U/N

þ

27

Ibrahim and colleagues, 2018

Yes

Y/Y

Y/Y

Y/Y

N/N

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

Ibrahim and colleagues, 201828

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

Yes

Y/Y

Y/Y

Y/Y

N/N

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

Y/Y

þ

Sierra and colleagues, 2010 Wind and colleagues, 2010

36

Marcial and colleagues, 2017

39

Boesmans and colleagues, 201829

Lee and colleagues, 2018

33

The k-statistic (a measure of interrater reliability score) was .872, indicating “almost perfect agreement” between the two raters. Results from both raters (A.E.M. and A.J.B.) are reported in the table. b Four relevance questions are included in the Quality Criteria Checklist (All studies were assigned the answer of “yes” to each of the following relevance questions): 1. Would implementing the studied intervention or procedure (if found successful) result in improved outcomes for the patients/clients/population group? 2. Did the authors study an outcome (dependent variable) or topic that the patients/clients/population group would care about? 3. Is the focus of the intervention or procedure (independent variable) or topic of study a common issue of concern to dietetics practice? 4. Is the intervention or procedure feasible? c The following 10 validity questions with sub-questions are included in the Quality Criteria Checklist and correspond to the validity question numbers in the table. The majority of responses to the sub-questions reflects the overall response to each validity question. For brevity, individual responses to each sub-question are not presented. Possible answers include; yes, no, unclear, and nonapplicable. Bold type indicates differences between raters. 1. Was the research question clearly stated? 1.1 Was the specific intervention(s) or procedure (independent variable[s]) identified? 1.2 Was the outcome(s) (dependent variable[s]) clearly indicated? 1.3 Were the target population and setting specified? 2. Was the selection of study subjects/patients free from bias? 2.1 Were inclusion/exclusion criteria specified (eg, risk, point in disease progression, diagnostic or prognosis criteria), and with sufficient detail and without omitting criteria critical to the study? 2.2 Were criteria applied equally to all study groups? 2.3 Were health, demographics, and other characteristics of subjects described? 2.4 Were the subjects/patients a representative sample of the relevant population? 3. Were study groups comparable? 3.1 Was the method of assigning subjects/patients to groups described and unbiased? (method of randomization identified if randomized controlled trial). 3.2 Was distribution of disease status, prognostic factors, and other factors (eg, demographics) similar across study groups at baseline? 3.3 Were concurrent controls used? (concurrent preferred over historical controls). 3.4 If cohort study or cross-sectional study, were groups comparable on important confounding factors and/or were pre-existing differences accounted for by using appropriate adjustments in statistical analysis? 3.5 If caseecontrol study, were potential confounding factors comparable for cases and controls? (If case series or trial with subjects serving as own control, this criterion is not applicable. Criterion may not be applicable in some cross-sectional studies.) 3.6 If diagnostic test, was there an independent blind comparison with an appropriate reference standard (eg, gold standard)? 4. Was method of handling withdrawals described? 4.1 Were follow-up methods described and the same for all groups? 4.2 Was the number, characteristics of withdrawals (ie, dropouts, lost to follow-up, and attrition rate), and/or response rate (cross-sectional studies) described for each group? (follow-up goal for a strong study is 80%). 4.3 Were all enrolled subjects/patients (in the original sample) accounted for? a

43

(continued on next page) --

2019 Volume

-

Number

-

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

11

RESEARCH 4.4 Were reasons for withdrawals similar across groups? 4.5 If diagnostic test, was decision to perform reference test not dependent on results of test under study? 5. Was blinding used to prevent introduction of bias? 5.1 In intervention study, were subjects, clinicians/practitioners, and investigators blinded to treatment group, as appropriate? 5.2 Were data collectors blinded for outcomes assessment? (if outcome is measured using an objective test, such as a laboratory value, this criterion is assumed to be met). 5.3 In cohort study or cross-sectional study, were measurements of outcomes and risk factors blinded? 5.4 In caseecontrol study, was case definition explicit and case ascertainment not influenced by exposure status? 5.5 In diagnostic study, were test results blinded to patient history and other test results? 6. Were intervention/therapeutic regimens/exposure factors or procedure and any comparison(s) described in detail? Were intervening factors described? 6.1 In randomized controlled trial or other intervention trial, were protocols described for all regimens studied? 6.2 In observational study, were interventions, study settings, and clinicians/provider described? 6.3 Was the intensity and duration of the intervention or exposure factor sufficient to produce a meaningful effect? 6.4 Was the amount of exposure and, if relevant, subject/patient compliance measured? 6.5 Were co-interventions (eg, ancillary treatments or other therapies) described? 6.6 Were extra or unplanned treatments described? 6.7 Was the information for 6.4, 6.5, and 6.6 assessed the same way for all groups? 6.8 In diagnostic study, were details of test administration and replication sufficient? 7. Were outcomes clearly defined and the measurements valid and reliable? 7.1 Were primary and secondary end points described and relevant to the question? 7.2 Were nutrition measures appropriate to question and outcomes of concern? 7.3 Was the period of follow-up long enough for important outcome(s) to occur? 7.4 Were the observations and measurements based on standard, valid, and reliable data collection instruments/tests/procedures? 7.5 Was the measurement of effect at an appropriate level of precision? 7.6 Were other factors accounted for (measured) that could affect outcomes? 7.7 Were the measurements conducted consistently across groups? 8. Was the statistical analysis appropriate for the study design and type of outcome indicators? 8.1 Were statistical analyses adequately described the results reported appropriately? 8.2 Were correct statistical tests used and assumptions of test not violated? 8.3 Were statistics reported with levels of significance and/or CIs? 8.4 Was intent to treat analysis of outcomes done (and as appropriate, was there an analysis of outcomes for those maximally exposed or a doseeresponse analysis)? 8.5 Were adequate adjustments made for effects of confounding factors that might have affected the outcomes (eg, multivariate analyses)? 8.6 Was clinical significance as well as statistical significance reported? 8.7 If negative findings, was a power calculation reported to address type 2 error? 9. Are conclusions supported by results with biases and limitations taken into consideration? 9.1 Is there a discussion of findings? 9.2 Are biases and study limitations identified and discussed? 10. Is bias due to study’s funding or sponsorship unlikely? 10.1 Were sources of funding and investigators’ affiliations described? 10.2 Was there no apparent conflict of interest? d Assignment of overall quality rating: Minus/negative (e): If most (6 or more) of the answers to the 10 validity questions are “no,” the report should be designated with a minus (e) symbol. Neutral (Ø): If the answers to validity criteria questions 2, 3, 6, and 7 do not indicate that the study is exceptionally strong, the report should be designated with a neutral (Ø) symbol. Plus/ positive (þ): If most of the answers to the above validity questions are “yes” (including criteria 2, 3, 6, 7 and at least one additional “yes”), the report should be designated with a plus symbol (þ). e Y¼yes. f U¼unclear. g N¼no. h ؼneutral. A neutral designation indicates that the report is neither exceptionally strong nor exceptionally weak. i þ¼Positive. A positive designation indicates that the report has clearly addressed issues of inclusion/exclusion, bias, generalizability, and data collection and analysis.

(P¼0.009) in comparison to placebo from supplementation with B animalis subsp lactis Bi-07.37 West and colleagues26 reported a significant 29% increase in the concentration of macrophage inflammatory protein-1d in those consuming B animalis subsp lactis Bl-04 and a 26% reduction in the concentration of matrix metalloproteinase-1 in the L acidophilus NCFM and B animalis subsp. lactis Bi-07 group compared with placebo supplementation in healthy, physically active male and females over five months.26 Rajkumar and colleagues40 reported a significant 24.5% reduction in CRP (P<0.01) after a 6-week treatment period with 112.5  109 colony-forming units daily of a multistrain probiotic compared to placebo. However, no difference in proinflammatory cytokines IL-1b, TNF-a or IL-6 were observed between groups. In a study with similar parameters using L salivarius UBL S22, Rajkumar and colleagues41 found 6 weeks of treatment significantly reduced plasma CRP, IL-1b, TNF-a, and IL-6 (P<0.05). Null findings included Christensen and colleagues,31 who did not find a difference after supplementation with B animalis 12

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

subsp lactis BB-12 and L paracasei subsp paracasei CRL-431 in production of IFN-g and IL-10 in blood cells compared to placebo. Mangalat and colleagues38 found no significant effect of L reuteri DSM 17938 on cytokine production by phorbol 12myristate 13-acetate and ionomycin stimulated peripheral blood mononuclear cell after 8 weeks compared to placebo. In healthy physically active males and females, Cox and colleagues25 did not report significance after 5 months of supplementation with B animalis subsp lactis Bl-04 or L acidophilus NCFM and B animalis subsp lactis Bi-07 on CRP. In a cross-over treatment, Kelly and colleagues42 found no effect on plasma IL10, IL-1b, IL-6, IL-8, TNF-a levels, or whole-blood Toll-like receptor-4 agonisteinduced cytokine release after an 8-week treatment with L rhamnosus JB-1 compared to placebo. Marcial and colleagues39 investigated the effects of L johnsonii N6.2 after 8 weeks of treatment and 4 weeks washout on serum cytokines. No statistical differences were obtained between the treatment groups or time points for IL-6, TNF-a, IFN-g, and IL-2 (P>0.1). In a 12-week study by Ibrahim and colleagues,27 --

2019 Volume

-

Number

-

RESEARCH supplementation with a multi-strain probiotic containing L acidophilus BCMC 12130, L casei BCMC 12313, L lactis BCMC 12451, B bifidum BCMC 02290, B infantis BCMC 02129, and B longum BCMC 02120 did not significantly affect inflammatory markers IL-10 and IL-6. Finally, in a study by Lee and colleagues,33 supplementation with Weissella cibaria JW15 for 8 weeks did not affect circulating cytokine levels, including TNF-a, IL-6, IL-b, IL-12, and IFN-g.

Syntheses of Results Based on the grading criteria for strength of evidence from Academy of Nutrition and Dietetics Evidence Analysis Manual,22 the current body of evidence was graded as grade IIIelimited. This grade was assigned due to the evidence that the quality of the extracted articles was of generally strong design, although there was uncertainty attached to this conclusion due to variation in probiotic strains used, dosages, outcomes measured, and findings across studies. Furthermore, there were doubts about the adequacy of sample sizes as only five articles reported sample size calculations,37,38,40-42 with three powered to immune or inflammatory markers.37,40,41 Therefore, there was some uncertainty about the statistical significance and error (type I and type II) due to power analyses not being reported. Finally, there were doubts about generalizability due to the presumed differences in subjects across the included articles.

DISCUSSION The aim of this systematic review was to examine the effect of probiotic supplementation on immune and inflammatory markers in healthy adults. As use of probiotics is widespread among healthy individuals, there is a need for robust studies on effects of probiotics in these individuals. Based on this systematic review, there is limited and varied evidence that oral probiotic supplementation in healthy adults modulates immune and inflammatory markers compared to placebo. While limited in quantity, the present review aligns with other reviews examining related factors in healthy adults, such as prevention of the common cold.7,44 However, the goal of this systematic review was to look at markers more proximal to the mechanisms of action of probiotics, examining the response in circulating immune and inflammatory markers. Overall, the effect of probiotics on inflammatory and immune markers showed a wide variance across the included studies. However, there were some similarities, including significant effects observed in immune (ie, NK-cell activity, monocytes, T cells, immnoglobulins)32,33,39 and inflammatory markers (ie, CRP, IL, TNF-a).35,37,40,41 Of the eight articles reporting significant effects from probiotic supplementation, four reported immune or inflammatory markers as the primary study outcome.26,33,40,41 Ten articles did not report an effect from probiotic supplementation in any of the targeted outcomes and did not investigate relevant parameters as the primary outcome. Considering the body of research, there is a limited strength of evidence for the targeted outcomes of the present systematic review. This is in agreement with a metaanalysis by Kang and colleagues44 showing a mild effect of probiotics on the prevention of the common cold in healthy adults, presumably mediated by the immune system. In addition, Khalesi and colleagues,7 after reviewing 16 studies --

2019 Volume

-

Number

-

looking primarily at upper respiratory infections, reported that probiotic consumption in healthy adults can improve immune function. Positive effects have also been reported in reviews looking at duration of illness in healthy children45 and immune markers in elderly adults.46 An important consideration of this systematic review, and probiotic research in general, is the vast range of strains used in clinical research. Of the included studies in this review, species and strains were the same in only a few instances. Despite the existence of shared, core mechanisms for probiotic functions, other mechanisms are likely narrowly distributed among probiotic species.4 Furthermore, strain specificity of probiotic benefits is presumed unless mechanistic and clinical evidence suggests otherwise.4 Therefore, the number of outcomes reported in the present review is not surprising, given the range of genera, species, and strains used in the included studies. It follows that various genera, species, and perhaps even strains, lumped together as “probiotics” will invariably lead to heterogeneity and perhaps should not be examined together but rather separately. Therefore, a meta-analysis is generally not appropriate for probiotics as a whole and may only be suitable at the species (or perhaps even the strain) level.47 In the studies included in this review, species from the genera Lactobacillus were most commonly administered (n¼12). Moreover, several studies had treatments composed of multiple strains. While more research is being published with multi-strain probiotics, it is currently unclear whether there is an advantage over singlestrain formulations. In addition, these formulations make it difficult to identify which strain is causing an effect, or whether an additive, synergistic, or antagonistic effect is occurring. This has implications for clinical recommendations and should be a topic of research for future studies. Another important consideration in probiotic clinical trials is whether the placebo used was appropriate to provide a more rigorous comparison between treatment and control. Given the tremendous resources invested in randomized controlled trials, development of consistent approaches to assess the effectiveness of probiotics, including the use of a placebo, is crucial. To reduce bias, a placebo should be as similar to the active intervention as possible, with the exception of the presence of the probiotic under investigation.48 In addition, it is imperative that the placebo is clearly described in the study protocol.48 Generally, the studies reviewed in the present review clearly described each placebo, which were identical or near identical to the treatment arm. However, there were three instances where the placebo was not clearly described, including the ingredient used27,28 and appearance.30 Common ingredients used in the placebo preparations included maltodextrin and dextrose, which are absorbed as glucose in the small intestine, reducing the likelihood of influence to the gut microbiota. The study participants across the included trials fit the inclusion criteria of healthy adults, however, there was heterogeneity. Participants in the studies by Rajkumar and colleagues40 and Lee and colleagues33 were middle-aged adults (approximately older than 40 years), who may respond differently to probiotics compared to younger individuals. There was also a large range of treatment dosages. The International Scientific Association for Probiotics and Prebiotics provides a list of dosages ranging from 1  108 to 1.8  1012 colony-forming units twice daily depending on strain and JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

13

RESEARCH disease,49 although currently there is no consensus regarding an ideal dosage or length of intervention. In addition, there was heterogeneity in the measurement of immune and inflammatory markers across studies. While there were similarities in some of the methodologies, no studies used the same approach. The measurement of these markers, as well as immune and inflammatory responses, are complex. The immunomodulatory effect of probiotics is attributed to the release of cytokines and chemokines from immune cells, which further regulate the innate and adaptive immune system.50 In terms of inflammation, indirect influence includes boosting the production of short-chain fatty acids with anti-inflammatory properties,51 increasing the synthesis of antimicrobial peptides that influence inflammation pathways in the mucosa,52 as well as protecting or repairing epithelial barriers.15,53 Direct effects include binding with the innate immune receptors53 and triggering pathways that cause the production of cytokines related to inflammation.54 Healthy participants generally have a well-functioning immune system, therefore, altering immune response and inflammation might be expected to be difficult to demonstrate. However, 3 of the 18 articles included in this systematic review reported a significant effect on immune markers. For instance, the activity of NK cells, a function of the innate immune system, appeared to be stimulated by the ingestion of probiotics in the current review.32,33,39 Innate immunity is activated very quickly after infection when acquired immunity has not yet been developed. Thus, the modulation of this response might improve the response against infections. In addition, Marcial and colleagues39 reported an increase in monocytes, involved in phagocytosis in the early stages of the immune response. Monocytes produce the cytokines that activate lymphocytes and consequently stimulate inflammation.55 Two studies also found an increase in lymphocyte T cells,32,39 which secrete antibodies and are a marker of adaptive immunity.56 It is important to note that it is unlikely that one probiotic exhibits all of these mechanisms of action and host conditions may have a profound influence on the effects of probiotics. Host conditions reported to modulate the gut microbiome include diet,57 medication use,58 age,59 environment,60 health state,9 geographical location,61 and race/ethnicity.62 Of the studies reviewed, only four accounted for diet. While the majority of studies limited the consumption of probiotics and/ or fermented foods, diet has a large impact on the gut microbiota. Another factor not accounted for in almost all studies was physical activity. Perhaps not as influential as diet on the gut microbiota, exercise does have a strong impact on immune and inflammatory markers.63,64 In addition, included studies were conducted in populations from a wide range of countries in Europe, Asia, and North America. Research has shown varying gut microbiota composition based on geography and race/ethnicity, which alone could contribute to variations in the outcome measures.61,62 This systematic review has several limitations. First, the search included probiotics of a common genus available commercially. It is possible the search strategy of this review missed some studies on novel strains that were not yet classified as probiotics. Second, the quality of the studies varied. Assessment of the included articles found most to be of positive quality, yet five were rated as neutral, indicating the study was not exceptionally strong and missed 14

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

important validity criteria. These articles were primarily published earlier (2011 or before), when research on probiotic supplementation was not as prevalent.7 In future research, reporting could be improved, such as blinding, representativeness of the population, and number and reasons for withdrawals. Furthermore, only five articles reported sample size calculation. This calls into question whether the other studies were appropriately powered. Another limitation was the heterogeneity of outcomes. Immunity and inflammation are inherently complex, although related processes, with a large array of markers. Furthermore, the included studies examined different features of immune and/or inflammatory responses. To pool enough resources for this systematic review, a wider range of these markers were included. A fourth limitation was the exclusion of studies that used immunostimulants, such as vaccination models,65 to test immune system response. These studies were excluded due to the use of a combined intervention, heterogeneity of treatment, and the existence of previous systematic reviews on this topic.66-68 Also excluded were studies with athletes and study participants who underwent intense physical activity. While athletes are generally considered healthy, and moderate exercise beneficially influences the immune system,69 a heavy schedule of training and competition can lead to immune impairment and inflammatory response.70,71 To reduce confounding effects, studies with these participants were therefore excluded. Finally, the search was limited to studies published in English, therefore, language bias cannot be ruled out.

PRACTICE IMPLICATIONS What Is the Current Knowledge on this Topic? Information on oral probiotic supplementation for potential health benefit is not sufficiently summarized for healthy adults to allow practical and consistent recommendations to be made by nutrition professionals and food and nutrition practitioners.

How Does this Research Add to Knowledge on this Topic? This research provides a review on the health effects of oral probiotic supplementation in healthy adults, specifically showing a limited and varied effect on circulating immune and inflammatory markers.

How Might this Knowledge Influence Current Dietetics Practice? Practitioners should be cautious when making recommendations for oral probiotic use in healthy individuals to affect circulating immune and inflammatory markers, as the current literature on this topic provides a limited level of evidence.

--

2019 Volume

-

Number

-

RESEARCH CONCLUSIONS Based on the 18 articles extracted in this systematic review, oral probiotic supplementation was concluded to have limited and varied effects on immune and inflammatory markers in healthy adults. While eight of included articles did report significant effects, caution is warranted in making general recommendations for probiotic use due to the general quality, methodology, and heterogeneity of outcomes in this body of literature. Given the potential differences in mechanisms of action between probiotic strains to elicit different immune/inflammatory effects, it was not unexpected to find multiple reported outcomes. This feature, along with the vast number of probiotic strains, precluded a meta-analysis, which might only be appropriate for examining more focused criteria, such as probiotic species, or perhaps even strains. Because a large segment of probiotic consumers are healthy individuals, further research examining whether probiotic supplementation benefits this population is warranted.

References 1.

Clarke T, Black L, Stussman B, et al. Trends in the use of complementary health approaches among adults: United States, 2002-2012. Natl Health Stat Report. 2015;(79):1-16.

2.

Market Research Engine. Probiotics Market By Ingredient Analysis (Bacteria, Yeast); by Application Analysis (Food & Beverages, Dietary Supplements, Animal Feed); by End User Analysis (Human Probiotics, Animal Probiotics) and by Regional Analysis—Global Forecast by 2018- 2024. Market Research Engine website. https://www. marketresearchengine.com/probiotics-market-report. Published November 2018. Accessed January 12, 2019.

Dietary Supplements website. https://ods.od.nih.gov/About/DSHEA_ Wording.aspx. Approved October 25, 1994. Accessed November 15, 2018. 18.

US Food and Drug Administration. Dietary Supplements Guidance Documents & Regulatory Information. Food and Drug Administration website. https://www.fda.gov/Food/GuidanceRegulation/ GuidanceDocumentsRegulatoryInformation/DietarySupplements/ default.htm. Published 2018. Accessed December 3, 2018.

19.

Plaza-Díaz J, Gomez-Llorente C, Fontana L, et al. Modulation of immunity and inflammatory gene expression in the gut, in inflammatory diseases of the gut and in the liver by probiotics. World J Gastroenterol. 2014;20(42):15632-15649.

20.

Smolinska S, Groeger D, O’Mahony L. Biology of the microbiome 1: Interactions with the host immune response. Gastroenterol Clin North Am. 2017;46(1):19-35.

21.

La Fata G, Weber P, Mohajeri MH. Probiotics and the gut immune system: Indirect Regulation. Probiotics Antimicrob Proteins. 2018;10(1):11-21.

22.

Academy of Nutrition and Dietetics. Evidence Analysis Manual. Academy of Nutrition and Dietetics website. https://www.andeal. org/evidence-analysis-manual. Published April 2016. Accessed January 1, 2019.

23.

Handu D, Moloney L, Wolfram T, et al. Academy of Nutrition and Dietetics methodology for conducting systematic reviews for the Evidence Analysis Library. J Acad Nutr Diet. 2016;116(2):311-318.

24.

Wang R, Lagakos SW, Ware JH, et al. Statistics in medicine— Reporting of subgroup analyses in clinical trials. N Engl J Med. 2007;357(21):2189-2194.

25.

Cox AJ, West NP, Horn PL, et al. Effects of probiotic supplementation over 5 months on routine haematology and clinical chemistry measures in healthy active adults. Eur J Clin Nutr. 2014;68(11):12551257.

26.

West NP, Horn PL, Barrett S, et al. Supplementation with a single and double strain probiotic on the innate immune system for respiratory illness. ESPEN J. 2014;9(5):e178-e184.

3.

Hill C, Guarner F, Reid G, et al. Expert consensus document: The international scientific association for probiotics and prebiotics consensus statement on the scope and appropriate use of the term probiotic. Nat Rev Gastroenterol Hepatol. 2014;11(8):506-514.

27.

Ibrahim NS, Muhamad AS, Ooi FK, et al. The effects of combined probiotic ingestion and circuit training on muscular strength and power and cytokine responses in young males. Appl Physiol Nutr Metab. 2018;43(2):180-186.

4.

Sanders ME, Merenstein D, Merrifield CA, et al. Probiotics for human use. Nutr Bull. 2018;43(3):212-225.

28.

5.

Di Cerbo A, Palmieri B. Review: The market of probiotics. Pak J Pharm Sci. 2015;28(6):2199-2206.  Markowiak P, Slizewska K. Effects of probiotics, prebiotics, and synbiotics on human health. Nutrients. 2017;9(9).

Ibrahim NS, Ooi FK, Chen CK, et al. Effects of probiotics supplementation and circuit training on immune responses among sedentary young males. J Sports Med Phys Fitness. 2018;58(7-8): 1102-1109.

29.

Boesmans L, Valles-Colomer M, Wang J, et al. Butyrate producers as potential next-generation probiotics: Safety assessment of the administration of Butyricicoccus pullicaecorum to healthy volunteers. mSystems. 2018;3(6).

30.

Kristensen NB, Bryrup T, Allin KH, et al. Alterations in fecal microbiota composition by probiotic supplementation in healthy adults: A systematic review of randomized controlled trials. Genome Med. 2016;8(1):52.

Mäkeläinen H, Tahvonen R, Salminen S, et al. In vivo safety assessment of two Bifidobacterium longum strains. Microbiol Immunol. 2003;47(12):911-914.

31.

9.

Shreiner AB, Kao JY, Young VB. The gut microbiome in health and in disease. Curr Opin Gastroenterol. 2015;31(1):69-75.

Christensen HR, Larsen CN, Kaestel P, et al. Immunomodulating potential of supplementation with probiotics: A dose-response study in healthy young adults. FEMS Immunol Med Microbiol. 2006;47(3):380-390.

32.

10.

Lebeer S, Vanderleyden J, De Keersmaecker SC. Host interactions of probiotic bacterial surface molecules: Comparison with commensals and pathogens. Nat Rev Microbiol. 2010;8(3):171-184.

Kim HS, Park H, Cho IY, et al. Dietary Supplementation of probiotic Bacillus polyfermenticus, bispan strain, modulates natural killer cell and T cell subset populations and immunoglobulin G levels in human subjects. J Med Food. 2006;9(3):321-327.

11.

Ouwehand AC, Salminen S, Isolauri E. Probiotics: An overview of beneficial effects. Antonie van Leeuwenhoek. 2002;82(1-4):279-289.

33.

Lee YJ, Lee A, Yoo HJ, Kim M, Noh GM, Lee JH. Supplementation with the probiotic strain Weissella cibaria JW15 enhances natural killer cell activity in nondiabetic subjects. J Funct Foods. 2018;48:153-158.

12.

Oelschlaeger TA. Mechanisms of probiotic actions—A review. Int J Med Microbiol. 2010;300(1):57-62.

34.

13.

Elmadfa I, Klein P, Meyer AL. Immune-stimulating effects of lactic acid bacteria in vivo and in vitro. Proc Nutr Soc. 2010;69(3):416-420.

Wind RD, Tolboom H, Klare I, et al. Tolerance and safety of the potentially probiotic strain Lactobacillus rhamnosus PRSF-L477: A randomised, double-blind placebo-controlled trial in healthy volunteers. Br J Nutr. 2010;104(12):1806-1816.

14.

Douglas LC, Sanders ME. Probiotics and prebiotics in dietetics practice. J Am Diet Assoc. 2008;108(3):510-521.

35.

15.

Bermudez-Brito M, Plaza-Díaz J, Muñoz-Quezada S, et al. Probiotic mechanisms of action. Ann Nutr Metab. 2012;61(2):160-174.

Sierra S, Lara-Villoslada F, Sempere L, et al. Intestinal and immunological effects of daily oral administration of Lactobacillus salivarius CECT5713 to healthy adults. Anaerobe. 2010;16(3):195-200.

36.

16.

Moher D, Liberati A, Tetzlaff J, et al. Preferred reporting items for systematic reviews and meta-analyses: The PRISMA statement. BMJ. 2009;339:b2535.

Burton JP, Cowley S, Simon RR, et al. Evaluation of safety and human tolerance of the oral probiotic Streptococcus salivarius K12: A randomized, placebo-controlled, double-blind study. Food Chem Toxicol. 2011;49(9):2356-2364.

17.

103rd Congress. Dietary Supplement Health and Education Act of 1994; Public Law 103-417. National Institutes of Health, Office of

37.

Childs CE, Röytiö H, Alhoniemi E, et al. Xylo-oligosaccharides alone or in synbiotic combination with Bifidobacterium animalis subsp.

6. 7.

8.

--

Khalesi S, Bellissimo N, Vandelanotte C, et al. A review of probiotic supplementation in healthy adults: Helpful or hype? Eur J Clin Nutr. 2019;73(1):24-37.

2019 Volume

-

Number

-

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

15

RESEARCH lactis induce bifidogenesis and modulate markers of immune function in healthy adults: A double-blind, placebo-controlled, randomised, factorial cross-over study. Br J Nutr. 2014;111(11):1945-1956.

53.

Thomas CM, Versalovic J. Probiotics-host communication modulation of signaling pathways in the intestine. Gut Microbes. 2010;1(3): 148-163.

38.

Mangalat N, Liu Y, Fatheree NY, et al. Safety and tolerability of Lactobacillus reuteri DSM 17938 and effects on biomarkers in healthy adults: Results from a randomized masked trial. PLoS One. 2012;7(9): 2012;e43910.

54.

Djaldetti M, Bessler H. Probiotic strains modulate cytokine production and the immune interplay between human peripheral blood mononucear cells and colon cancer cells. FEMS Microbiol Lett. 2017;364(3):fnx014.

39.

Marcial GE, Ford AL, Haller MJ, et al. Lactobacillus johnsonii N6.2 modulates the host immune responses: A double-blind, randomized trial in healthy adults. Front Immunol. 2017;8:655.

55.

Arango Duque G, Descoteaux A. Macrophage cytokines: Involvement in immunity and infectious diseases. Front Immunol. 2014;5:491.

56.

40.

Rajkumar H, Mahmood N, Kumar M, et al. Effect of probiotic (VSL#3) and omega-3 on lipid profile, insulin sensitivity, inflammatory markers, and gut colonization in overweight adults: A randomized, controlled trial. Mediators Inflamm. 2014;2014:348959.

Alberts B, Johnson A, Lewis J, et al. Lymphocytes and the cellular basis of adaptive immunity. Mol Biol Cell. 2002;4552:4-8.

57.

David LA, Maurice CF, Carmody RN, et al. Diet rapidly and reproducibly alters the human gut microbiome. Nature. 2014;505(7484): 559-563.

58.

Kim DH. Gut microbiota-mediated drug-antibiotic interactions. Drug Metab Dispos. 2015;43(10):1581-1589.

59.

Buford TW, Carter CS, VanDerPol WJ, et al. Composition and richness of the serum microbiome differ by age and link to systemic inflammation. GeroScience. 2018;40(3):257-268.

60.

Segata N. Gut Microbiome: Westernization and the disappearance of intestinal diversity. Curr Biol. 2015;25(14):R611-613.

61.

Gupta VK, Paul S, Dutta C. Geography, ethnicity or subsistencespecific variations in human microbiome composition and diversity. Front Microbiol. 2017;8(1162):1162.

62.

Deschasaux M, Bouter KE, Prodan A, et al. Depicting the composition of gut microbiota in a population with varied ethnic origins but shared geography. Nat Med. 2018;24(10): 1526-1531.

63.

Walsh NP, Gleeson M, Pyne DB, et al. Position statement part two: Maintaining immune health. Exerc Immunol Rev. 2011;17:64-103.

64.

Allen J, Sun Y, Woods JA. Exercise and the regulation of inflammatory responses. Prog Mol Biol Transl Sci. 2015;135:337-354.

65.

West NP, Cripps AW. Are vaccination models suitable to determine whether probiotics have beneficial health effects in the general population? Hum Vaccin Immunother. 2013;9(3):621-624.

66.

Yeh TL, Shih PC, Liu SJ, et al. The influence of prebiotic or probiotic supplementation on antibody titers after influenza vaccination: A systematic review and meta-analysis of randomized controlled trials. Drug Des Devel Ther. 2018;12:217-230.

67.

Lei WT, Shih PC, Liu SJ, et al. Effect of probiotics and prebiotics on immune response to influenza vaccination in adults: A systematic review and meta-analysis of randomized controlled trials. Nutrients. 2017;9(11).

68.

Zimmermann P, Curtis N. The influence of probiotics on vaccine responses—A systematic review. Vaccine. 2018;36(2):207-213.

41.

Rajkumar H, Kumar M, Das N, et al. Effect of probiotic Lactobacillus salivarius UBL S22 and prebiotic fructo-oligosaccharide on serum lipids, inflammatory markers, insulin sensitivity, and gut bacteria in healthy young volunteers: A randomized controlled single-blind pilot study. J Cardiovasc Pharmacol Ther. 2015;20(3):289-298.

42.

Kelly JR, Allen AP, Temko A, et al. Lost in translation? The potential psychobiotic Lactobacillus rhamnosus (JB-1) fails to modulate stress or cognitive performance in healthy male subjects. Brain Behav Immun. 2017;61:50-59.

43.

McHugh ML. Interrater reliability: The kappa statistic. Biochem Medica (Zagreb). 2012;22(3):276-282.

44.

Kang EJ, Kim SY, Hwang IH, et al. The effect of probiotics on prevention of common cold: A meta-analysis of randomized controlled trial studies. Korean J Fam Med. 2013;34(1):2-10.

45.

King S, Glanville J, Sanders ME, et al. Effectiveness of probiotics on the duration of illness in healthy children and adults who develop common acute respiratory infectious conditions: A systematic review and meta-analysis. Br J Nutr. 2014;112(1):41-54.

46.

Miller LE, Lehtoranta L, Lehtinen MJ. The effect of Bifidobacterium animalis ssp. lactis HN019 on cellular immune function in healthy elderly subjects: Systematic review and meta-analysis. Nutrients. 2017;9(3).

47.

McFarland LV, Evans CT, Goldstein EJC. Strain-specificity and disease-specificity of probiotic efficacy: A systematic review and meta-analysis. Front Med. 2018;5:124.

48.

Shane AL, Cabana MD, Vidry S, et al. Guide to designing, conducting, publishing and communicating results of clinical studies involving probiotic applications in human participants. Gut Microbes. 2010;1(4):243-253.

49.

Guarner F, Khan AG, Garisch J, et al. World gastroenterology organisation global guidelines: Probiotics and prebiotics October 2011. J Clin Gastroenterol. 2012;46(6):468-481.

50.

Azad MAK, Sarker M, Wan D. Immunomodulatory effects of probiotics on cytokine profiles. Biomed Res Int. 2018;2018: 8063647.

69.

Nieman DC, Henson DA, Austin MD, et al. Upper respiratory tract infection is reduced in physically fit and active adults. Br J Sports Med. 2011;45(12):987-992.

51.

Vinolo MA, Rodrigues HG, Nachbar RT, et al. Regulation of inflammation by short chain fatty acids. Nutrients. 2011;3(10):858-876.

70.

Walsh NP, Gleeson M, Shephard RJ, et al. Position statement part one: Immune function and exercise. Exerc Immunol Rev. 2011;17:6-63.

52.

Schlee M, Harder J, Köten B, et al. Probiotic lactobacilli and VSL#3 induce enterocyte beta-defensin 2. Clin Exp Immunol. 2008;151(3): 528-535.

71.

Gleeson M, Bishop NC. URI in athletes: Are mucosal immunity and cytokine responses key risk factors? Exerc Sport Sci Rev. 2013;41(3): 148-153.

16

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

--

2019 Volume

-

Number

-

RESEARCH AUTHOR INFORMATION A. E. Mohr is a PhD student, College of Health Solutions, Arizona State University, Phoenix, and a research and development scientist, Isagenix International LLC, Gilbert, AZ. A. J. Basile and M. S. Crawford are PhD students, School of Life Sciences, Arizona State University, Tempe. K. L. Sweazea is an associate professor, College of Health Solutions, Arizona State University, Phoenix, and an associate professor, School of Life Sciences, Arizona State University, Tempe. K. C. Carpenter is senior scientific content specialist, Isagenix International LLC, Gilbert, AZ. Address correspondence to: Katie C. Carpenter, PhD, Isagenix International LLC, 155 East Rivulon Blvd, Gilbert, AZ 85297. E-mail: [email protected]

STATEMENT OF POTENTIAL CONFLICT OF INTEREST A. E. Mohr and K. C. Carpenter are employees of Isagenix International LLC. Isagenix International LLC was not involved in the design, implementation, data collection, or analyses of this research. The remaining authors declare no conflict of interest.

FUNDING/SUPPORT There is no funding to disclose.

ACKNOWLEDGEMENTS The authors would like to thank Mary Ellen Sanders, PhD, from the International Scientific Association for Probiotics and Prebiotics for her valuable guidance and recommendations during the preparation of this manuscript.

AUTHOR CONTRIBUTIONS A. E. Mohr conceptualized and designed this systematic review with the aid of K. L. Sweazea and M. S. Crawford. A. E. Mohr and K. C. Carpenter (senior author) conducted the literature search and performed the study extraction. A. E. Mohr and A. J. Basile evaluated the quality of included studies. A. E. Mohr prepared and compiled the draft for review and editing by coauthors. All authors reviewed, edited, and approved the draft, and the final manuscript.

--

2019 Volume

-

Number

-

JOURNAL OF THE ACADEMY OF NUTRITION AND DIETETICS

17