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Cited by 123Gut microbiota and healthProbiotics and Fermented FoodsGastrointestinal motility and disorders

Distinct actions of the fermented beverage kefir on host behaviour, immunity and microbiome gut-brain modules in the mouse

Open access
van de Wouw, Marcel, Walsh, Aaron M., Crispie, Fiona, van Leuven, Lucas, Lyte, Joshua M., Boehme, Marcus, Clarke, Gerard, Dinan, Timothy G. +2 more·2020·Microbiome
DOI 10.1186/s40168-020-00846-5PMC PMC7236220

Published version of a preprint

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Abstract

Background: Mounting evidence suggests a role for the gut microbiota in modulating brain physiology and behaviour, through bi-directional communication, along the gut-brain axis. As such, the gut microbiota represents a potential therapeutic target for influencing centrally mediated events and host behaviour. It is thus notable that the fermented milk beverage kefir has recently been shown to modulate the composition of the gut microbiota in mice. It is unclear whether kefirs have differential effects on microbiota-gut-brain axis and whether they can modulate host behaviour per se. Methods: To address this, two distinct kefirs (Fr1 and UK4), or unfermented milk control, were administered to mice that underwent a battery of tests to characterise their behavioural phenotype. In addition, shotgun metagenomic sequencing of ileal, caecal and faecal matter was performed, as was faecal metabolome analysis. Finally, systemic immunity measures and gut serotonin levels were assessed. Statistical analyses were performed by ANOVA followed by Dunnett's post hoc test or Kruskal-Wallis test followed by Mann-Whitney U test. Results: Fr1 ameliorated the stress-induced decrease in serotonergic signalling in the colon and reward-seeking behaviour in the saccharin preference test. On the other hand, UK4 decreased repetitive behaviour and ameliorated stress-induced deficits in reward-seeking behaviour. Furthermore, UK4 increased fear-dependent contextual memory, yet decreased milk gavage-induced improvements in long-term spatial learning. In the peripheral immune system, UK4 increased the prevalence of Treg cells and interleukin 10 levels, whereas Fr1 ameliorated the milk gavage stress-induced elevation in neutrophil levels and CXCL1 levels. Analysis of the gut microbiota revealed that both kefirs significantly changed the composition and functional capacity of the host microbiota, where specific bacterial species were changed in a kefir-dependent manner. Furthermore, both kefirs increased the capacity of the gut microbiota to produce GABA, which was linked to an increased prevalence in Lactobacillus reuteri . Conclusions: Altogether, these data show that kefir can signal through the microbiota-gut-immune-brain axis and modulate host behaviour. In addition, different kefirs may direct the microbiota toward distinct immunological and behavioural modulatory effects. These results indicate that kefir can positively modulate specific aspects of the microbiota-gut-brain axis and support the broadening of the definition of psychobiotic to include kefir fermented foods.

AI snapshot

Written from the paper’s full text.

TL;DR

Two distinct kefirs (Fr1 and UK4) were administered to mice and produced partly divergent effects on behaviour, peripheral immunity, gut serotonin, and microbiota function. Both kefirs altered the gut microbiota and increased predicted GABA-producing capacity linked to *Lactobacillus reuteri*, supporting kefir as a potential psychobiotic fermented food.

Key findings

  • UK4 decreased repetitive behaviour in the marble burying test (F(2,35) = 5.464, p = 0.009), increased fear-dependent contextual memory (trend: F(2,34) = 3.181, p = 0.055), but worsened reversal learning in the appetitive Y-maze (treatment effect: F(2,33) = 3.870, p = 0.031).
  • Fr1 increased saccharin preference (χ2(2) = 12.826, p = 0.002) and ameliorated stress-induced changes in colonic serotonergic signalling (5-HT: F(2,35) = 6.387, p = 0.005; 5HIAA/5-HT ratio: F(2,35) = 9.026, p < 0.001).
  • UK4 increased mesenteric lymph node Treg cells (F(2,34) = 8.709, p < 0.001), circulating Treg cells (F(2,31) = 3.420, p = 0.046), and plasma IL-10 (F(2,32) = 6.205, p = 0.006); Fr1 reduced milk gavage-induced neutrophil levels (F(2,34) = 5.412, p = 0.009) and CXCL1 (F(2,32) = 7.006, p = 0.003).
  • Both kefirs changed microbiota composition and function: 15 bacterial species were differentially abundant, 59 level-4 EC categories differed, and faecal “GABA synthesis III” was higher with Fr1 (LDA = 4.39) and UK4 (LDA = 4.21), attributed to increased *L. reuteri*.

Methods

  • Male C57BL/6j mice (8 weeks old; n = 12/group, n = 48 total) received milk control, kefir Fr1, kefir UK4, or no gavage for 3 weeks before a behavioural battery, with tissues collected postmortem.
  • Shotgun metagenomic sequencing was performed on ileal, caecal, and faecal matter; faecal metabolome was analysed by GC-MS; immunity was assessed by flow cytometry and cytokine quantification; gut serotonin was measured by HPLC.
  • Behavioural/physiological data used ANOVA with Dunnett’s post hoc or Kruskal-Wallis with Mann-Whitney U tests; microbiota analyses used PERMANOVA, LEfSe, HAllA, StrainPhlAn/PanPhlAn, HUMAnN2, and flux variability analysis.

Limitations

  • The paper states that it remains unclear whether gut-derived GABA can cross the blood-brain barrier or whether increasing gut-derived GABA improves mood, and that more research is warranted to validate whether the proposed microbiota-gut-brain pathways are involved.
  • The paper notes weak serotonin-related effects, no corresponding serotonin-related changes in the microbiome/metabolome, no changes in other depressive-like behaviour measures, and no significant faecal metabolite differences after p-value adjustment.
  • Evident from the text: findings are from male mice only, kefir-derived strains did not colonise to high levels, many microbiota-immunity/behaviour links are correlational, and faecal metabolome analysis used a subset of animals (n = 6 per group).

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1186/s40168-020-00846-5

Cites

Statistics

22 tests recomputed · 0 with the reported and recomputed p on opposite sides of .05

Claims the paper makes

0 of 40 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    UK4 modestly increased reward-seeking behaviour in the female urine sniffing test.

    “and modestly increased reward-seeking behaviour in the female urine sniffing test, also often used as a measure of depression-related anhedonia.”
    Quote in the paperNot a substantial claim
  • Findingown data

    L. reuteri encodes enzymes and antiporters associated with GABA production.

    “Our data additionally reveals that L. reuteri encodes enzymes and antiporters associated with GABA production.”
    Quote in the paperSubstantial claim
  • Findingown data

    Kefir-induced increases in L. reuteri levels were linked to an increased capacity to produce GABA.

    “Importantly, kefir-induced increases in L. reuteri levels were linked to an increased capacity to produce GABA.”
    Quote in the paperSubstantial claim
  • Findingown data

    Fr1 ameliorated stress-induced deficits in colonic serotonergic signalling.

    “Our results also reveal that Fr1 ameliorated stress-induced deficits in colonic serotonergic signalling”
    Quote in the paperSubstantial claim
  • Findingown data

    UK4 increased predicted GBM inositol synthesis.

    “while UK4 increased “inositol synthesis”.”
    Quote in the paperNot a substantial claim
  • May combine two claims. 13
  • Classified as probably not a substantial claim. 26

  • May combine two claims.

    Findingnone given

    Fr1 increased circulating neutrophil and CXCL1 levels.

    “Furthermore, Fr1 increased circulating neutrophil and CXCL1 levels.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    UK4 increased circulating Treg cells and IL-10 levels.

    “Finally, UK4 increased circulating Treg cells and IL-10 levels.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Beta diversity showed a trend towards significant separation induced by both Fr1 and UK4, which resulted in a significant separation induced by kefir overall.

    “Analysis of beta diversity revealed a trend towards significant separation induced by both Fr1 and UK4, which resulted in a significant separation induced by kefir overall.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both kefirs significantly modulated the functional capacity of the gut microbiota and altered the levels of GBMs, which was related to the prevalence of specific bacterial strains.

    “Our data also reveals that both kefirs significantly modulated the functional capacity of the gut microbiota and altered the levels of GBMs, which was related to the prevalence of specific bacterial strains.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fr1 increased abundances of P. goldsteinii, B. intestinalis, and Anaerotruncus unclassified in the gut microbiota.

    “We observed some changes in the gut microbiota that were specific to kefir Fr1, such as an increase in P. goldsteinii, B. intestinalis, Anaerotruncus unclassified and P. goldsteinii.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Fr1 decreased predicted GBM p-Cresol biosynthesis and inositol synthesis.

    “Analysis of GBMs revealed that Fr1 decreased the GBM “p-Cresol biosynthesis” and “inositol synthesis””
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Increase in predicted S-adenosylmethionine synthesis could indicate increased levels of S-adenosylmethionine, which might have contributed to the Fr1-induced increase in reward-seeking behaviour.

    “As such, the increase in predicted S-adenosylmethionine synthesis could indicate increased levels of S-adenosylmethionine, which in turn might have contributed to the Fr1-induced increase in reward-seeking behaviour.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Specific bacterial species increased by kefir administration, such as B. pseudolongum and L. reuteri, have previously been associated with increased levels of anti-inflammatory cytokine IL-10 in mice and Treg cells in mice and humans, respectively.

    “In addition, specific bacterial species that were increased by kefir administration, such as B. pseudolongum and L. reuteri, have previously been associated with increased levels of the anti-inflammatory cytokine IL-10 in mice and Treg cells in mice and humans, respectively [53, 65].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [53] Mu Q, Tavella VJ, Luo XM. Role of Lactobacillus reuteri in human health and diseases. Front Microbiol. 2018;9:757. source

      Not checked in the free run.

    • cites [65] Sasajima N, Ogasawara T, Takemura N, Fujiwara R, Watanabe J, Sonoyama K. Role of intestinal Bifidobacterium pseudolongum in dietary fructo-oligosaccharide inhibition of 2,4-dinitrofluorobenzene-induced contact hypersensitivity in mice. Br J Nutr. 2009;103(4):539–48. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    S-adenosylmethionine supplementation has been studied in numerous randomised controlled trials involving depressed adults.

    “S-adenosylmethionine supplementation has been studied in numerous randomised, controlled trials involving depressed adults [36, 70].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [36] Joffe H, Partridge A, Giobbie-Hurder A, Li X, Habin K, Goss P, Winer E, Garber J. Augmentation of venlafaxine and selective serotonin reuptake inhibitors with zolpidem improves sleep and quality of life in breast cancer patients with hot flashes: a randomized, double-blind, placebo-controlled trial. Menopause. 2010;17(5):908–16. source

      Not checked in the free run.

    • cites [70] Sharma A, Gerbarg P, Bottiglieri T, Massoumi L, Carpenter LL, Lavretsky H, Muskin PR, Brown RP, Mischoulon D, R. as Work Group of the American Psychiatric Association Council on. S-Adenosylmethionine (SAMe) for Neuropsychiatric Disorders: A Clinician-Oriented Review of Research. J Clin Psychiatry. 2017;78(6):e656–67. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Two GABA-producing Lactobacillus strains have recently been shown to reduce depressive-like behaviour in high fat diet-induced obese mice.

    “Indeed, two GABA-producing Lactobacillus strains have recently been shown to reduce depressive-like behaviour in high fat diet-induced obese mice [58].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [58] Patterson E, Ryan PM, Wiley N, Carafa I, Sherwin E, Moloney G, Franciosi E, Mandal R, Wishart DS, Tuohy K, Ross RP, Cryan JF, Dinan TG, Stanton C. Gamma-aminobutyric acid-producing lactobacilli positively affect metabolism and depressive-like behaviour in a mouse model of metabolic syndrome. Sci Rep. 2019;9(1):16323. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Probiotics most frequently do not colonise the gut.

    “This has parallels with the fact that probiotics most frequently do not colonise the gut [21].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [21] Derrien M, van Hylckama Vlieg JE. Fate, activity, and impact of ingested bacteria within the human gut microbiota. Trends Microbiol. 2015;23(6):354–66. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    GABA is the primary inhibitory neurotransmitter in the brain.

    “GABA is the primary inhibitory neurotransmitter in the brain and central GABA levels have been linked to anxiety and depression [7]”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Bienenstock J, Forsythe P, Karimi K, Kunze W. Neuroimmune aspects of food intake. Int Dairy J. 2010;20(4):253–8. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Previous reports have demonstrated an increased prevalence of Lactobacillus species in response to kefir supplementation, as well as L. kefiri CIDCA 8348 administration.

    “Notably, previous reports have also demonstrated an increased prevalence of Lactobacillus species in response to kefir supplementation [40], as well as L. kefiri CIDCA 8348 administration, a species frequently found in kefir [15].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [40] Kim DH, Kim H, Jeong D, Kang IB, Chon JW, Kim HS, Song KY, Seo KH. Kefir alleviates obesity and hepatic steatosis in high-fat diet-fed mice by modulation of gut microbiota and mycobiota: targeted and untargeted community analysis with correlation of biomarkers. J Nutr Biochem. 2017;44:35–43. source

      Not checked in the free run.

    • cites [15] Carasi P, Racedo SM, Jacquot C, Romanin DE, Serradell MA, Urdaci MC. Impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota. J Immunol Res. 2015;2015:361604. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Various Lactobacillus strains, or Lactobacillus-containing supplements, have been shown to reduce depressive-like behaviour in rodents and improve measures of depression in humans.

    “Various Lactobacillus strains, or Lactobacillus-containing supplements, have been shown to reduce depressive-like behaviour in rodents [1, 13, 23, 44, 45] and improve measures of depression in humans [3, 5, 39, 76].”
    Quote in the paperNot a substantial claim0 of 9 sources checked
    • cites [1] Abildgaard A, Elfving B, Hokland M, Wegener G, Lund S. Probiotic treatment reduces depressive-like behaviour in rats independently of diet. Psychoneuroendocrinology. 2017;79:40–8. source

      Not checked in the free run.

    • cites [13] Bravo JA, Forsythe P, Chew MV, Escaravage E, Savignac HM, Dinan TG, Bienenstock J, Cryan JF. Ingestion of Lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve. Proc Natl Acad Sci U S A. 2011;108(38):16050–5. source

      Not checked in the free run.

    • cites [23] Dhaliwal J, Singh DP, Singh S, Pinnaka AK, Boparai RK, Bishnoi M, Kondepudi KK, Chopra K. Lactobacillus plantarum MTCC 9510 supplementation protects from chronic unpredictable and sleep deprivation-induced behaviour, biochemical and selected gut microbial aberrations in mice. J Appl Microbiol. 2018;125(1):257–69. source

      Not checked in the free run.

    • cites [44] Liang L, Zhou H, Zhang S, Yuan J, Wu H. Effects of gut microbiota disturbance induced in early life on the expression of extrasynaptic GABA-A receptor alpha5 and delta subunits in the hippocampus of adult rats. Brain Res Bull. 2017;135:113–9. source

      Not checked in the free run.

    • cites [45] Liang S, Wang T, Hu X, Luo J, Li W, Wu X, Duan Y, Jin F. Administration of Lactobacillus helveticus NS8 improves behavioral, cognitive, and biochemical aberrations caused by chronic restraint stress. Neuroscience. 2015;310:561–77. source

      Not checked in the free run.

    • cites [3] Akkasheh G, Kashani-Poor Z, Tajabadi-Ebrahimi M, Jafari P, Akbari H, Taghizadeh M, Memarzadeh MR, Asemi Z, Esmaillzadeh A. Clinical and metabolic response to probiotic administration in patients with major depressive disorder: A randomized, double-blind, placebo-controlled trial. Nutrition. 2016;32(3):315–20. source

      Not checked in the free run.

    • cites [5] Bagga D, Reichert JL, Koschutnig K, Aigner CS, Holzer P, Koskinen K, Moissl-Eichinger C, Schopf V. Probiotics drive gut microbiome triggering emotional brain signatures. Gut Microbes. 2018:1–11. source

      Not checked in the free run.

    • cites [39] Kazemi A, Noorbala AA, Azam K, Eskandari MH, Djafarian K. Effect of probiotic and prebiotic vs placebo on psychological outcomes in patients with major depressive disorder: a randomized clinical trial. Clin Nutr. 2019;38(2):522–8. source

      Not checked in the free run.

    • cites [76] Steenbergen L, Sellaro R, van Hemert S, Bosch JA, Colzato LS. A randomized controlled trial to test the effect of multispecies probiotics on cognitive reactivity to sad mood. Brain Behav Immun. 2015;48:258–64. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    L. reuteri is a bacterial strain that has been ascribed numerous beneficial effects on host immunity and metabolism.

    “Notably, L. reuteri is a bacterial strain that has been ascribed numerous beneficial effects on host immunity and metabolism [52, 53].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [52] Mobini R, Tremaroli V, Stahlman M, Karlsson F, Levin M, Ljungberg M, Sohlin M, Berteus Forslund H, Perkins R, Backhed F, Jansson PA. Metabolic effects of Lactobacillus reuteri DSM 17938 in people with type 2 diabetes: a randomized controlled trial. Diabetes Obes Metab. 2017;19(4):579–89. source

      Not checked in the free run.

    • cites [53] Mu Q, Tavella VJ, Luo XM. Role of Lactobacillus reuteri in human health and diseases. Front Microbiol. 2018;9:757. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    Kefir can signal through the microbiota-gut-immune-brain axis and modulate host behaviour.

    “Altogether, these data show that kefir can signal through the microbiota-gut-immune-brain axis and modulate host behaviour.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Premisecitation

    Central GABA levels have been linked to anxiety and depression.

    “GABA is the primary inhibitory neurotransmitter in the brain and central GABA levels have been linked to anxiety and depression [7]”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [7] Bienenstock J, Forsythe P, Karimi K, Kunze W. Neuroimmune aspects of food intake. Int Dairy J. 2010;20(4):253–8. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    p-Cresol alters neutrophil function in dogs.

    “Notably, p-Cresol, of which the predicted biosynthesis was decreased by Fr1, alters neutrophil function in dogs [10].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Bosco AM, Pereira PP, Almeida BF, Narciso LG, Dos Santos DB, Santos-Neto AJ, Ferreira WL, Ciarlini PC. Free p-Cresol Alters Neutrophil Function in Dogs. Artif Organs. 2016;40(5):480–8. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Changes in the overall composition of the gut microbiota induced by kefir, or kefir-associated bacterial strains, have been reported previously.

    “Changes in the overall composition of the gut microbiota induced by kefir, or kefir-associated bacterial strains have been reported previously [15, 30, 34, 40, 90].”
    Quote in the paperNot a substantial claim0 of 5 sources checked
    • cites [15] Carasi P, Racedo SM, Jacquot C, Romanin DE, Serradell MA, Urdaci MC. Impact of kefir derived Lactobacillus kefiri on the mucosal immune response and gut microbiota. J Immunol Res. 2015;2015:361604. source

      Not checked in the free run.

    • cites [30] Gao J, Ding G, Li Q, Gong L, Huang J, Sang Y. Tibet kefir milk decreases fat deposition by regulating the gut microbiota and gene expression of Lpl and Angptl4 in high fat diet-fed rats. Food Res Int. 2019;121:278–87. source

      Not checked in the free run.

    • cites [34] Hsu YJ, Huang WC, Lin JS, Chen YM, Ho ST, Huang CC, Tung YT. Kefir supplementation modifies gut microbiota composition, reduces physical fatigue, and improves exercise performance in mice. Nutrients. 2018;10(7). source

      Not checked in the free run.

    • cites [40] Kim DH, Kim H, Jeong D, Kang IB, Chon JW, Kim HS, Song KY, Seo KH. Kefir alleviates obesity and hepatic steatosis in high-fat diet-fed mice by modulation of gut microbiota and mycobiota: targeted and untargeted community analysis with correlation of biomarkers. J Nutr Biochem. 2017;44:35–43. source

      Not checked in the free run.

    • cites [90] Yilmaz I, Dolar ME, Ozpinar H. Effect of administering kefir on the changes in fecal microbiota and symptoms of inflammatory bowel disease: a randomized controlled trial. Turk J Gastroenterol. 2019;30(3):242–53. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Lactobacillus strains have previously been reported to produce GABA.

    “Indeed, Lactobacillus strains have previously been reported to produce GABA [6, 7, 46].”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [6] Barrett E, Ross RP, O'Toole PW, Fitzgerald GF, Stanton C. gamma-Aminobutyric acid production by culturable bacteria from the human intestine. J Appl Microbiol. 2012;113(2):411–7. source

      Not checked in the free run.

    • cites [7] Bienenstock J, Forsythe P, Karimi K, Kunze W. Neuroimmune aspects of food intake. Int Dairy J. 2010;20(4):253–8. source

      Not checked in the free run.

    • cites [46] Lin Q. Submerged fermentation of Lactobacillus rhamnosus YS9 for gamma-aminobutyric acid (GABA) production. Braz J Microbiol. 2013;44(1):183–7. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The order of faecal pellet collection was counterbalanced between groups to minimise the effect of circadian rhythm.

    “The order of faecal pellet collection was counterbalanced between groups to minimise the effect of circadian rhythm.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The sequence did not contain more than three consecutive starts in the same position to avoid temporary position preferences.

    “This sequence did not contain more than three consecutive starts in the same position to avoid temporary position preferences.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductreasoning

    Food wells were not cleaned between mice so that a slight odour of milk reward remained, ensuring mice found the goal arm based on spatial cues rather than olfactory cues.

    “Between mice, the food wells were not cleaned so that a slight odour of milk reward remained at all times, ensuring mice found the goal arm based on spatial cues, rather the olfactory cues.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    An undisturbed control group was added to control for potential effects of chronic oral gavage or milk administration on behaviour or physiology.

    “The last group was added to control for the fact that chronic oral gavage or milk administration could affect behaviour or physiology [87].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [87] Walker MK, Boberg JR, Walsh MT, Wolf V, Trujillo A, Duke MS, Palme R, Felton LA. A less stressful alternative to oral gavage for pharmacological and toxicological studies in mice. Toxicol Appl Pharmacol. 2012;260(1):65–9. source

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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    There was a minimum of 36 h between behavioural tests.

    “In addition, there was a minimum of 36 h between tests.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The time of testing was counterbalanced between groups and rotated each day to reduce the effect of testing during a specific time of the day.

    “The time of testing was counterbalanced between groups and rotated each day to reduce the effect of testing during a specific time of the day.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Behavioural tests were ordered from least stressful to most stressful to reduce the likelihood of prior behavioural tests influencing subsequent ones.

    “which were formed in order of least stressful to most stressful to reduce the likelihood of prior behavioural tests influencing subsequent ones (Fig. 9).”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Study design and conductown data

    Behavioural experiments were videotaped and scored blinded.

    “All experiments were videotaped using a ceiling-mounted camera and were scored blinded for the time interacted with the wire cup-like cages.”
    • Also stated in: § Elevated plus maze · plain
      “Experiments were videotaped using a ceiling-mounted camera and videos were scored blinded for time spent in the open arms, which was defined as all paws in the open arm.”
    • Also stated in: § Tail-suspension test · plain
      “Experiments were videotaped using a numeric tripod-fixed camera and videos were scored blinded for the time mice spent immobile.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The starting position for each trial in the appetitive Y-maze was determined by a pseudorandomised computer sequence.

    “The starting position for each trial was determined by a pseudorandomised computer sequence, which was different for each mouse but was the same across treatment groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductreasoning

    Mice were habituated to the testing room 40 min before the 3-chamber test to reduce potential anxiogenic factors.

    “To reduce potential anxiogenic factors, all mice were habituated to the testing room 40 min before the test”
    Quote in the paperNot a substantial claim

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    Study design and conductnone given

    Animals were sacrificed by decapitation in a random fashion regarding test groups.

    “Animals were sacrificed by decapitation in a random fashion regarding test groups between 9.00 am and 2.00 pm.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The side on which the regular drinking water bottle and the saccharin solution bottle were placed was randomised and counterbalanced between groups.

    “The side on which the regular drinking water bottle and the one containing saccharine solution were randomised and counterbalanced between groups.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Abundances of faecal GABA-producers, such as Bacteroides, correlate negatively with brain signatures associated with depression.

    “Furthermore, abundances of faecal GABA-producers, such as Bacteroides, correlate negatively with brain signatures associated with depression [77].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [77] Strandwitz P, Kim KH, Terekhova D, Liu JK, Sharma A, Levering J, McDonald D, Dietrich D, Ramadhar TR, Lekbua A, Mroue N, Liston C, Stewart EJ, Dubin MJ, Zengler K, Knight R, Gilbert JA, Clardy J, Lewis K. GABA-modulating bacteria of the human gut microbiota. Nat Microbiol. 2019;4(3):396–403. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The location of conspecific mice and object were systematically altered between test mice.

    “The location of the conspecific mice and object were systemically altered in-between test mice.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Study design and conductnone given

    A trial where the mouse did not walk into any arm was excluded from analysis because it indicates the mouse was anxious.

    “A trial where the mouse did not walk into any arm was excluded from the analysis, as this indicates that the mouse was anxious.”
    Quote in the paperNot a substantial claim
35 of 35

  • The paper relies on this without support.

    It is unclear whether kefirs have differential effects on the microbiota-gut-brain axis and whether they can modulate host behaviour per se.

    “It is unclear whether kefirs have differential effects on microbiota-gut-brain axis and whether they can modulate host behaviour per se.”

  • The paper relies on this without support.

    It is not clear if kefirs exert differential influence across the microbiota-gut-brain axis due to their compositional differences.

    “It is not clear if kefir’s exert differential influence across the microbiota-gut-brain axis due to their compositional differences [12].”

  • The paper relies on this without support.

    Decreased reward-seeking behaviour (anhedonia) is a key feature of depression.

    “Notably, one of the key features of depression is decreased reward-seeking behaviour (i.e. anhedonia)”
    “Notably, one of the key features of depression is decreased reward-seeking behaviour (i.e. anhedonia) and that both kefirs increased reward-seeking behaviour.”
3 of 3

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • In mice, kefir administration increased the abundance of L. reuteri, Eubacterium plexicaudatum, and Bifidobacterium pseudolongum, decreased Lachnospiraceae bacterium 3_1_46FAA, Propionibacterium acnes, and Bacillus amyloliquefaciens, and shifted the functional potential of the gut microbiota toward production of neuroactive metabolites.

    “In a recent mouse study, kefir administration increased the abundance of L. reuteri, Eubacterium plexicaudatum, and Bifidobacterium pseudolongum, and decreased Lachnospiraceae bacterium 3_1_46FAA, Propionibacterium acnes, and Bacillus amyloliquefaciens, and shifted the functional potential of the gut microbiota toward the production of neuroactive metabolites (58).”

    From Diet and the Microbiota–Gut–Brain Axis: Sowing the Seeds of Good Mental Health (2021) · cites this paper as [58]

Statistics that don’t add up

Each reported p-value recomputed from its test statistic and degrees of freedom.

  • Inconsistent p-value: t(22) = 2.650, p < .001

    Minor

    The paper reports p < .001 here; recomputing from the test statistic and its degrees of freedom gives p = .015 (two-tailed). The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p < .001 · recomputed p = .015

  • Inconsistent p-value: χ2(2) = 13.238, p < .001

    Minor

    The paper reports p < .001 here; recomputing from the test statistic and its degrees of freedom gives p = .001. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p < .001 · recomputed p = .001

  • Inconsistent p-value: F(2,35) = 6.387, p = .005

    Minor

    The paper reports p = .005 here; recomputing from the test statistic and its degrees of freedom gives p = .004. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .005 · recomputed p = .004

  • Inconsistent p-value: F(2,32) = 6.205, p = .006

    Minor

    The paper reports p = .006 here; recomputing from the test statistic and its degrees of freedom gives p = .005. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .006 · recomputed p = .005

  • Inconsistent p-value: F(2,34) = 3.181, p = .055

    Minor

    The paper reports p = .055 here; recomputing from the test statistic and its degrees of freedom gives p = .054. The check cannot tell whether the statistic or the p-value is the misprint.

    Reported p = .055 · recomputed p = .054

5 of 5

Statistics recomputed

Every reported test result, recomputed from its test statistic and degrees of freedom. A result shows as not adding up only once an independent check has confirmed it.

  • Doesn’t add upχ²(2) = 13.238reported p < .001 · recomputed p = .001

    “χ2(2) = 13.238, p < .001”

  • Doesn’t add upF(2, 34) = 3.181reported p = .055 · recomputed p = .054

    “F(2,34) = 3.181, p = .055”

  • Doesn’t add upF(2, 32) = 6.205reported p = .006 · recomputed p = .005

    “F(2,32) = 6.205, p = .006”

  • Doesn’t add upt(22) = 2.65reported p < .001 · recomputed p = .015

    “t(22) = 2.650, p < .001”

  • Doesn’t add upF(2, 35) = 6.387reported p = .005 · recomputed p = .004

    “F(2,35) = 6.387, p = .005”

  • ConsistentF(2, 35) = 5.464reported p = .009 · recomputed p = .009

    “F(2,35) = 5.464, p = .009”

  • Consistentχ²(1) = 6.367reported p = .012 · recomputed p = .012

    “χ2(1) = 6.367, p = .012”

  • Consistentχ²(2) = 6.28reported p = .043 · recomputed p = .043

    “χ2(2) = 6.280, p = .043”

  • Consistentχ²(2) = 12.826reported p = .002 · recomputed p = .002

    “χ2(2) = 12.826, p = .002”

  • ConsistentF(2, 33) = 3.87reported p = .031 · recomputed p = .031

    “F(2,33) = 3.870, p = .031”

  • ConsistentF(2, 33) = 3.387reported p = .046 · recomputed p = .046

    “F(2,33) = 3.387, p = .046”

  • Consistentt(22) = -2.303reported p = .031 · recomputed p = .031

    “t(22) = − 2.303, p = .031”

  • Consistentt(22) = -3.583reported p = .002 · recomputed p = .002

    “t(22) = − 3.583, p = .002”

  • ConsistentF(2, 34) = 5.412reported p = .009 · recomputed p = .009

    “F(2,34) = 5.412, p = .009”

  • Consistentt(21) = -2.589reported p = .017 · recomputed p = .017

    “t(21) = − 2.589, p = .017”

  • ConsistentF(2, 32) = 7.006reported p = .003 · recomputed p = .003

    “F(2,32) = 7.006, p = .003”

  • ConsistentF(2, 34) = 8.709reported p < .001 · recomputed p < .001

    “F(2,34) = 8.709, p < .001”

  • ConsistentF(2, 34) = 7.548reported p = .002 · recomputed p = .002

    “F(2,34) = 7.548, p = .002”

  • ConsistentF(2, 31) = 3.42reported p = .046 · recomputed p = .045

    “F(2,31) = 3.420, p = .046”

  • Consistentt(21) = 2.65reported p = .015 · recomputed p = .015

    “t(21) = 2.650, p = .015”

  • Consistentt(22) = -1.937reported p = .066 · recomputed p = .066

    “t(22) = − 1.937, p = .066”

  • Consistentt(22) = 2.907reported p = .008 · recomputed p = .008

    “t(22) = 2.907, p = .008”

22 of 22

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 3 caption and results assign different outcomes to panels

    Minor

    The caption assigns panels e and f to neutrophils and CXCL1, while the results cite those panels for circulating Treg cells and IL-10.

  • Ileal functional separation is called significant at p = 0.052

    Minor

    The results call the ileal Fr1 separation significant, although its reported p value is above the paper's stated significance threshold.

  • Saccharin-preference formula uses sucrose intake

    Minor

    The test administers saccharin solution, but its stated preference formula uses sucrose intake as the numerator.

  • Discussion calls beta-diversity separation significant despite reported p-value range

    Minor

    The discussion says kefir overall produced significant beta-diversity separation, but the results give a p-value range entirely above 0.05 for all regions.

  • Discussion reverses Fr1's reported effect on neutrophils and CXCL1

    Minor

    The results report that Fr1 ameliorated elevations in neutrophils and CXCL1, but the discussion says Fr1 increased both.

5 of 5

The citation lists haven’t been fetched yet

References 90 · Cited by 123

Research works printed but not linked: 1

  1. Oksanen J, Kindt R, Legendre P, O’Hara B, Stevens MHH, Oksanen MJ, Suggests M. The vegan package. Comm Ecol Package. 2007;10:631–7.

Authors
van de Wouw, M., Walsh, A. M., Crispie, F., van Leuven, L., Lyte, J. M., Boehme, M., Clarke, G., Dinan, T. G., Cotter, P. D., & Cryan, J. F.
Affiliations
  • Teagasc - The Irish Agriculture and Food Development Authority (IE)
  • University College Cork (IE)
Corresponding author
  • Cotter, Paul D.
  • Cryan, John F.
ORCID
Venue
Microbiome
Publisher
Springer Science and Business Media LLC
Published
2020-05-18
Volume
8
Issue
1
Pages
67-67
ISSN
2049-2618 (ISSN-L)
DOI
10.1186/s40168-020-00846-5
PMC
PMC7236220
OpenAlex
W3026259629
Semantic Scholar
bce58de2464961f3f5f687be5d25fae25a50e632
Cited by
123
References
90
Citations by year
2026: 19 · 2025: 22 · 2024: 30 · 2023: 18 · 2022: 13 · 2021: 16 · 2020: 3 · 2019: 1 · 2017: 1
FWCI
4.05
Type
journal article
Language
en
Open access
Licence
Locations
  • Microbiome (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Cork Open Research Archive (University College Cork, Ireland) (submittedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • T-Stór (Teagasc) (submittedVersion, cc-by-nc-sa)
Funders
Science Foundation Ireland (SFI/12/RC/2273, 15/JP‐HDHL/3270)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
  • Health Sciences › Medicine › Gastroenterology › Gastrointestinal motility and disorders
Keywords
kefir, fermented milk beverages, gut microbiota, gut-brain axis, psychobiotics, host behavior, stress, reward-seeking behavior, repetitive behavior, contextual fear memory, spatial learning, serotonin, regulatory T cells, IL-10, neutrophils, CXCL1, GABA production, Lactobacillus reuteri
MeSH terms
Gastrointestinal Microbiome, Kefir, Host Microbial Interactions, Animals, Behavior, Animal, Male, Mice, Inbred C57BL, Mice, Microbial Interactions, Microbiota

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-09.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-09.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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