Antimicrobial effect of gastric acid secretion

High-Ca diets (30 mmol Ca/l⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 )} and 180 mmol Ca/kg diet⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )}) are associated with reduced number of viable bacteria in the stomach. A high intraluminal concentration of Ca²⁺ stimulates the release of gastrin and, consequently, increases acid secretion⁽ Reference Kopic and Geibel ^{28 )}. Several bacterial species (e.g. Salmonella) are destroyed by gastric acid⁽ Reference Álvarez-Ordóñez, Begley and Prieto ^{29 )}. However, some factors, such as the buffering effect of food, gastric emptying rate and mechanisms of bacterial resistance, interfere with the interaction of gastric acid and ingested bacteria⁽ Reference Sarker and Gyr ^{30 )}. Wistar rats (n 9 per group) were fed ad libitum for 12 d a low-Ca diet (control: lactose-free low-Ca milk, 3·8 % fat, 6 mmol Ca/l), regular lactose-free milk (3·7 % fat, 30 mmol Ca/l), acidified milk or yoghurt (both prepared with regular lactose-free milk)⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 )}. These rats were orally infected with Salmonella enteritidis just after food consumption. Animals fed yoghurt had lower faecal excretion of bacteria than those in the other groups. The authors suggest that the lower gastric emptying rate after yoghurt consumption could have prolonged the exposure to gastric acid and, thus, reduced the effectiveness of the inoculation. Thereafter, faecal Salmonella excretion declined rapidly in all high-Ca groups compared with the control group⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 )}. Another study conducted by the same authors showed similar results⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )}. Mice infected with S. enteritidis were fed high-, medium- or low-Ca diets (n 8 per group; Ca in the form of CaHPO₄: 180, 60 and 20 mmol/kg diet, respectively). Compared with the low-Ca diet, the medium- and high-Ca diets favoured bacterial colonisation⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )}. High-Ca intake (30 mmol Ca/l⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{2 6)} and 180 mmol Ca/kg diet⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )}) probably increased the gastric acidity and, hence, reduced viable counts of S. enteritidis ⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )}. Other studies also showed the antimicrobial effect of gastric acid⁽ Reference Álvarez-Ordóñez, Begley and Prieto ^{29 ,} Reference Zhu, Hart and Sales ^{31 )}, which potentially modified the endogenous microbiota by reducing viable bacteria in the gut. In general, few microorganisms, such as Helicobacter pylori, some streptococci, lactobacilli and probiotics, can survive extremely acidic conditions within the stomach⁽ Reference Berg ^{32 ,} Reference Dicksved, Lindberg and Rosenquist ^{33 )}. However, in some studies⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )}, gastrin release and/or acid secretion were not measured. Therefore, we cannot conclude that the results of those studies⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 )} were mediated by changes in gastric secretion. High protein content and the liquid state of the diets facilitated bacterial survival within the stomach⁽ Reference Álvarez-Ordóñez, Begley and Prieto ^{29 )}. Furthermore, some dairy product components, such as Ig, peptides, lactoferrin, lactoperoxidase and lysozyme, have antimicrobial effects. Other dairy components such as lactose, peptides and probiotics stimulate potentially beneficial bacteria that compete with pathogens for nutrients and attachment, and enhance the mucosal immune response to pathogens⁽ Reference Mills, Ross and Hill ^{34 ,} Reference Venema ^{35 )}.

Bile acid and fatty acid precipitation: reducing luminal cytotoxicity

Because of the low pH, dietary Ca in the stomach exists in dissociated form, whereas in the small intestine there is an equilibrium between its dissociated and nondissociated forms. In the distal ileum and the colon, where pH>6, Ca interacts with dietary phosphate, forming an insoluble complex that precipitates intestinal BA and FA. Hence, Ca increases their faecal excretion in animals⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 ,} Reference Lapré, De Vries and Koeman ^{36 –} Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 )} and humans⁽ Reference Van der Meer, Welberg and Kuipers ^{39 –} Reference Zheng, Yde and Clausen ^{44 )}. BA precipitation increases the de novo synthesis of BA from cholesterol in the liver and, hence, reduces serum cholesterol. FA precipitation reduces fat absorption, increasing faecal energy loss⁽ Reference Jacobsen, Lorenzen and Toubro ^{5 )}.

Primary BA (cholic acid and chenodeoxycholic acid) are synthesised in the liver from cholesterol and then conjugated with either glycine or taurine, often called bile salts. About 5 % of BA are deconjugated and dehydroxylated by bacterial enzymes in the intestine to form secondary BA (deoxycholic acid and lithocholic acid), which are more cytotoxic. The dehydroxylation process involves the removal of the OH group at the 7-position of the steroid nucleus (also termed 7-dehydroxylation). Deconjugation results in amino acid side chain cleavage. Among intestinal bacteria, 7-dehydroxylase was detected in the Eubacterium and Clostridium genera but not in lactobacilli and bifidobacteria⁽ Reference Begley, Gahan and Hill ^{45 ,} Reference Floch ^{46 )}. BA hydrolysis is mediated by several gut microbiota genera, including Clostridium, Bacteroides, Lactobacillus, Bifidobacterium and Enterococcus ⁽ Reference Begley, Gahan and Hill ^{45 )}. Approximately 95 % of BA are reabsorbed in the distal ileum and return to the liver (enterohepatic circulation of BA). About 400–800 mg BA/d elude enterohepatic circulation and are subjected to extensive modifications by the endogenous colonic microbiota. Secondary BA formed by colonic bacteria can be absorbed passively and, thus, may contribute to the BA pool⁽ Reference Kopic and Geibel ^{28 )}.

A small amount of NEFA and ionised secondary BA that reaches the colon can damage the intestinal epithelium and thus increase colonic permeability⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 )}. Therefore, BA and FA precipitation caused by dietary Ca promotes cytoprotective effects by reducing the bacteria’s formation of cytotoxic surfactants. BA and FA precipitation ultimately maintains the integrity of the colonic epithelium.

Measuring the BA and FA concentrations in the soluble portion of the faeces (so-called faecal water (FW)) is more reflective of luminal cytotoxicity than measuring the total faecal BA and FA concentration⁽ Reference Roberton ^{48 )}. FW refers to the supernatant obtained after intense centrifugation of the faeces. FW contains aqueous soluble BA and FA that are not linked to other faecal compounds⁽ Reference Lapré, Termont and Groen ^{49 )}. In some studies, a high-Ca diet reduced the BA and FA concentration in FW⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 ,} Reference Lapré, De Vries and Koeman ^{36 –} Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Govers and Van der Meer ^{40 ,} Reference Govers, Termont and Lapre´ ^{41 )}. A high-Ca diet also reduced FW cytotoxicity by precipitating cytotoxic surfactants, resulting in lower colonic epithelium damage and higher resistance to infections⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 ,} Reference Lapré, De Vries and Koeman ^{36 –} Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Govers and Van der Meer ^{40 ,} Reference Govers, Termont and Lapre´ ^{41 ,} Reference Schepens, ten Bruggencate and Schonewille ^{47 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 –} Reference Van Ampting, Schonewille and Vink ^{52 )}. High-Ca diets contained 30 mmol/l⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 )}, 225 μmol/g diet⁽ Reference Lapré, De Vries and Koeman ^{36 ,} Reference Govers and Van der Meer ^{40 )}, 120⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 ,} Reference Schepens, Schonewille and Vink ^{51 )}, 150⁽ Reference Govers, Termont and Van der Meer ^{37 )} or 180⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 ,} Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 )} mmol/kg diet and 4·8 g/kg diet⁽ Reference Van Ampting, Schonewille and Vink ^{52 )} in the rats studies, and 1200 mg/d in the human study⁽ Reference Govers, Termont and Lapre´ ^{41 )}. Some effects of cytotoxic surfactants are cell membrane disruption, inflammatory reaction activation and epithelium hyperproliferation enhancement⁽ Reference Imamura, Micha and Khatibzadeh ^{24 ,} Reference Berg ^{32 ,} Reference Ajouz, Mukherji and Shamseddine ^{53 )}. Guarner⁽ Reference Guarner ^{54 )} criticised the use of erythrocytes instead of intestinal epithelial cells to analyse FW cytotoxicity, as done in some studies⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 ,} Reference Lapré, De Vries and Koeman ^{36 –} Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Govers and Van der Meer ^{40 ,} Reference Govers, Termont and Lapre´ ^{41 ,} Reference Schepens, ten Bruggencate and Schonewille ^{47 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 –} Reference Van Ampting, Schonewille and Vink ^{52 )}. Erythrocytes are susceptible to changes in pH resulting from the production of SCFA after the fermentation of non-digestible carbohydrates. Therefore, haemolysis caused by changes in pH by SCFA may not reflect epitheliolysis⁽ Reference Guarner ^{54 )}. On the other hand, intestinal cells normally use organic acids as an energy source⁽ Reference Guarner ^{54 )}. However, the erythrocyte assay has previously been validated⁽ Reference Lapré, Termont and Groen ^{49 )}. In a mouse study, the type of dietary fat influenced faecal FA excretion. Ca-PUFA soaps were more soluble and, therefore, better absorbed than Ca-saturated FA soaps⁽ Reference Lapré, De Vries and Koeman ^{36 )}. The decreased absorption of intestinal fat (mainly saturated fat) caused by dietary Ca is of interest for the improvement of obesity control. By contrast, FA precipitation was independent of the source of Ca (milk, calcium carbonate or calcium phosphate)⁽ Reference Govers, Termont and Van der Meer ^{37 )} and dietary phosphate content (75, 125 or 275 mmol/g diet)⁽ Reference Govers and Van der Meer ^{40 )}.

Increased faecal fat excretion after Ca supplementation seems to favour weight control. An average daily intake of 1200 mg of Ca results in the excretion of 5·2 g fat/d and a weight loss of 2·2 kg/year⁽ Reference Christensen, Lorenzen and Svith ^{55 )}. Therefore, it is possible that this mechanism contributes to obesity control but does not fully explain the effect of Ca on weight loss⁽ Reference Jacobsen, Lorenzen and Toubro ^{5 )}. We believe that the impact of dietary Ca on weight loss is also related to dysbiosis attenuation. In this context, a high-Ca diet (>1100 mg/d) seems to modulate gut microbiota by reducing the number of BA and FA available for bacterial metabolism. It is possible that this effect increases Lactobacillus and reduces bile-tolerant bacteria, as discussed below.

Resistance to pathogens and changes in gut microbiota composition

In rodents, high-calcium phosphate diets increased faecal lactobacilli excretion⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Van Ampting, Schonewille and Vink ^{52 )}, reduced faecal Enterobacteriaceae excretion⁽ Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Ten Bruggencate, Snel and Schoterman ^{56 )} and increased resistance to S. enteritidis after 6–7 d of infection⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Van Ampting, Schonewille and Vink ^{52 ,} Reference Ten Bruggencate, Snel and Schoterman ^{56 )}. Ca concentrations of these diets were previously mentioned⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Van Ampting, Schonewille and Vink ^{52 )}, except the concentration adopted in the study conducted by Ten Bruggencate et al.⁽ Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Ten Bruggencate, Snel and Schoterman ^{56 )}, which was 100 mmol Ca/kg diet. Rats were fed diets with 60 g/kg cellulose (control), fructo-oligosaccharides (FOS) or inulin with either a low (30 mmol/kg) or a high (100 mmol/kg) Ca concentration. After 2 weeks of adaptation, the animals were infected with S. enteritidis. During the following 6 d, FW cytotoxicity increased in the rats on inulin and FOS diets, but the high-Ca diet minimised this adverse effect⁽ Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 )}. In another study, rats infected with S. enteritidis were fed different sources of Ca supplements (calcium phosphate, milk Ca, calcium chloride or calcium carbonate (a total of 100 mmol Ca supplement/kg)) or a low-Ca diet (20 mmol calcium phosphate/kg). After an adaptation period of 2 weeks, all the Ca supplements reduced infection and increased resistance to Salmonella ⁽ Reference Ten Bruggencate, Snel and Schoterman ^{56 )}. Effects of Ca salts were similar to milk Ca⁽ Reference Ten Bruggencate, Snel and Schoterman ^{56 )}, suggesting a strategy to increase Ca intake in cases of restricted consumption of dairy foods – for example, lactose intolerance.

The authors suggest that the resistance to pathogens was because of BA and FA precipitation and, consequently, because of reduced cytotoxic surfactants in FW after calcium phosphate supplementation⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Van Ampting, Schonewille and Vink ^{52 ,} Reference Ten Bruggencate, Snel and Schoterman ^{56 )}. However, only Bovee-Oudenhaven et al.⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 )} evaluated BA and FA faecal excretion. gram-negative bacteria (such as Escherichia coli and Salmonella spp.) are more bile-tolerant than gram-positive bacteria (such as some species of bifidobacteria, Bacillus, Lactobacillus and Enterococcus)⁽ Reference Begley, Gahan and Hill ^{45 )}. Therefore, the reduction in cytotoxicity in the intestinal lumen (reduction in the lytic activity of luminal surfactants), induced by dietary calcium phosphate, could improve the growth of Lactobacillus and other gram-positive bacteria compared with that of gram-negative bacteria.

Another mechanism by which dietary calcium phosphate interferes with resistance to pathogens is by binding directly to Salmonella and, hence, increasing the excretion of faecal bacteria, which is also known as bacterial shedding. This reduces pathogen competition and thus enhances the growth of lactobacilli. In vitro,⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Ten Bruggencate, Snel and Schoterman ^{56 )} but not vivo,⁽ Reference Van Ampting, Schonewille and Vink ^{52 )} studies confirmed this mechanism. In an in vivo study⁽ Reference Van Ampting, Schonewille and Vink ^{52 )}, rats (n 8 per group) infected with Salmonella were treated with an antibiotic and were either fed the control diet (1·2 g/kg diet) or a high-Ca diet (4·8 g/kg diet). Both diets did not decrease Salmonella colonisation (measured by the excretion of faecal bacteria). The authors rejected the hypothesis that the binding of the calcium phosphate complex to Salmonella has a significant effect in vivo and, therefore, suggested that surfactant precipitation may increase endogenous microbiota.

Research on pigs showed different effects of calcium phosphate in intestinal lactobacilli colonisation. In general, high-calcium phosphate intake did not affect Lactobacillus spp. growth⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 –} Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )} in the pigs’ stomachs, ilea or colons. Only in the study by Mann et al.⁽ Reference Mann, Schmitz-Esser and Zebeli ^{60 )} did high- v. adequate-calcium phosphate diets increase Lactobacillus spp. growth in the stomach. These studies were performed with growing⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 )} or weaned⁽ Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 –} Reference Mann, Schmitz-Esser and Zebeli ^{60 )} pigs (n 8 per group) fed high-calcium phosphate diets: with an ileal pectin infusion⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 )}, with a high or low β-glucan content⁽ Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 )}, associated with a corn diet, or associated with a wheat–barley diet⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 ,} Reference Mann, Schmitz-Esser and Zebeli ^{60 )}. High-calcium phosphate diets contained 15 g Ca/kg diet⁽ Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 )}, 10 g Ca/kg diet⁽ Reference Schepens, Schonewille and Vink ^{51 )} and 14·8 g Ca/kg diet⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 ,} Reference Mann, Schmitz-Esser and Zebeli ^{60 )}. The main difference between these pig studies that explain the changes in gastrointestinal microbiota was the methodology used to quantify bacterial communities. Studies in which no effects on lactobacilli count were observed used quantitative PCR⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 –} Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )}, whereas Mann et al.⁽ Reference Mann, Schmitz-Esser and Zebeli ^{60 )} used the pyrosequencing of 16S rRNA genes. Because of the diversity and complexity of bacterial communities between species, the latter technique has been recommended. It has the capacity to sequence multiple fragments simultaneously and, thus, achieves more rapid and accurate bacterial genome sequences⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 ,} Reference Kim, Suda and Kim ^{61 )}. Although some authors criticise pyrosequencing for the phylogenetic classification of sequences obtained at the species level⁽ Reference Kim, Suda and Kim ^{61 )}, Mann et al.⁽ Reference Mann, Schmitz-Esser and Zebeli ^{60 )} did not use it for pyrosequencing the total genome; rather, they only used it for the 16S rRNA gene. Thus, they demonstrated increases on operational taxonomic units for Lactobacillus, indicating an increase in the number of microorganisms of this genus. This is extremely beneficial because of the effects of these bacteria on intestinal health.

Calcium phosphate diet modulated gastrointestinal microbiota in all pig studies, but the results were very different. Because of that, these studies will not be described in detail (see online Supplementary Table S1). In general, calcium phosphate intake over 14 d increased Clostridium cluster XI and XVIa in the ilea, caeca and colons of weaned pigs⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 –} Reference Mann, Schmitz-Esser and Zebeli ^{60 )}. Several species of Clostridium cluster IV and XIVa produce butyrate, which is an important energy source for colonocytes⁽ Reference Louis and Flint ^{62 )}.

A double-blind, placebo-controlled, crossover study evaluated the composition of the gut microbiota after calcium phosphate and probiotic supplementation in humans⁽ Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )}. Participants (thirty-two healthy men and women aged 25 (sd 5) years and BMI of 22 (sd 3) kg/m²) consumed a probiotic drink containing 10¹⁰ colony-forming unit (CFU)/d Lactobacillus paracasei alone or in combination with bread containing calcium phosphate (1 g/d) for 4 weeks. Calcium phosphate supplementation decreased total cholesterol, LDL-cholesterol and the LDL:HDL ratio, and increased faecal pH and the faecal excretion of secondary BA compared with supplementation with probiotics or placebo alone. Probiotic supplementation, alone or with calcium phosphate, increased faecal Lactobacillus excretion. The authors explained these effects as resulting from BA precipitation by amorphous calcium phosphate, particularly when BA are deconjugated by probiotics, and from the increased faecal excretion of these components. A less cytotoxic intestinal lumen, which contains a low concentration of cytotoxic surfactants, might favour the growth of lactobacilli and reduce blood cholesterol concentration because of the increased conversion of cholesterol into BA⁽ Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )}.

The results of the studies with mice, pigs and humans are difficult to compare because of the diversity of micro-organism species in different hosts. Within the same species (mouse, pig or man), the intestinal maturation stage can also influence gut microbiota composition. For instance, Enterococcus spp. in the ileum were observed to decrease and increase in growing and weaned pigs, respectively, after calcium phosphate supplementation⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 ,} Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )}. In humans, gut microbiota is dominated by bifidobacteria in the first 2–3 years of life (especially among breastfed children), remaining relatively stable in adults with 90 % of the bacteria from the Bacteroidetes and Firmicutes phyla. In the elderly, gut microbiota becomes less diverse again (higher Bacteroides:Firmicutes ratio, an increase in Proteobacteria and a decrease in Bifidobacterium)⁽ Reference Ottman, Smidt and de Vos ^{63 )}. Human gut microbiota also varies according to genetic background, diet, antibiotic use and the health status of the host⁽ Reference Nicholson, Holmes and Kinross ^{64 )}. Therefore, the interaction between Ca and other dietary nutrients (such as lactose, dietary fibre and probiotics) probably influences its effect on microbiota composition and activity.

Furthermore, the different results observed may be because of the variety of procedures used on microbiological analyses. These range from simple techniques such as counting CFU in mouse studies⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Van Ampting, Schonewille and Vink ^{52 )} to sophisticated molecular biology techniques such as quantitative PCR and pyrosequencing 16S rRNA genes in pig and human studies⁽ Reference Trautvetter, Ditscheid and Kiehntopf ^{43 ,} Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 –} Reference Mann, Schmitz-Esser and Zebeli ^{60 )}. The variety in materials or parts of the gastrointestinal tract used to evaluate microbiota composition may also make comparisons difficult. Mouse and human studies used faeces⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Van Ampting, Schonewille and Vink ^{52 )}, whereas pig studies used different viscera (stomachs, small intestines, caeca and colons)⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 –} Reference Mann, Schmitz-Esser and Zebeli ^{60 )}.

Differences between BA in mice (predominantly taurine-conjugated) and those in pigs (glycine-conjugated) also partly explain the diversity in results. In general, unconjugated BA and glycine-conjugated BA are more strongly precipitated by calcium phosphate than taurine-conjugated BA⁽ Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 )}. The taurine-conjugated:glycine-conjugated BA ratio in human bile is usually 3:1, which is more similar to that in pigs than to that in rodents⁽ Reference Begley, Gahan and Hill ^{45 )}.

Obesity is associated with changes in gut microbiota composition in animal and human research⁽ Reference Cani, Possemiers and Van de Wiele ^{10 ,} Reference Ridaura, Faith and Rey ^{65 )}. Some studies show an increase in the Firmicutes:Bacteroidetes ratio⁽ Reference Ley ^{66 )}, and others only show an overall decrease in Bacteroidetes and no change in Firmicutes⁽ Reference Turnbaugh, Hamady and Yatsunenko ^{22 )}. There are also differences between the gut microbiota of obese and lean subjects⁽ Reference Ridaura, Faith and Rey ^{65 )}. However, it is not yet clear whether obesity leads to dysbiosis or vice versa. If the effects of dietary Ca on lactobacilli growth are confirmed in human clinical trials, Ca supplementation will be a useful strategy in obesity treatment.

Change in faecal pH and in fermentation products

Most mouse⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Schepens, Rijnierse and Schonewille ^{67 )} and human⁽ Reference Govers, Termont and Lapre´ ^{41 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )} studies have found an increase in faecal pH after high-Ca diets (30 mmol Ca/l⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 )}, 100⁽ Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 )} and 120⁽ Reference Schepens, Rijnierse and Schonewille ^{67 )} mmol Ca/kg diet in mouse studies, and 1000⁽ Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )} or 1200 Ca mg/d⁽ Reference Govers, Termont and Lapre´ ^{41 )} in human studies). Other mouse studies⁽ Reference Lapré, De Vries and Koeman ^{36 )}, as well as pig studies⁽ Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 ,} Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )}, did not reveal such differences, and only one study showed a decrease in faecal pH as a result of Ca supplementation⁽ Reference Govers, Termont and Van der Meer ^{37 )} (225 μmol Ca/g diet⁽ Reference Lapré, De Vries and Koeman ^{36 )}, 10⁽ Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 )} or 14·8⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )} g Ca/kg diet, and 1 g/d⁽ Reference Govers, Termont and Van der Meer ^{37 )}). The decrease in faecal pH, caused by products of the colonic fermentation of non-digestible carbohydrates, supports the growth of beneficial bacteria (particularly bifidobacteria and lactobacilli)⁽ Reference Fooks and Gibson ^{68 )}. As previously mentioned, dietary Ca is soluble in acids, and it precipitates at alkaline pH. Gastric acidity (pH 1–3) is sufficient to release Ca complexed to salts or foods. Thus, the ionised Ca can be absorbed via transcellular active transport in the duodenum and proximal jejunum and via a passive paracellular process throughout the ileum. Less than 10 % of Ca absorption occurs in the colon, and it involves the paracellular and transcellular pathways⁽ Reference Bronner ^{69 )}. About 25 to 35 % of ingested Ca is generally absorbed. As pH increases from the ileum to the colon, the intestinal phosphate concentration also increases, causing Ca precipitation and Ca absorption reduction⁽ Reference Kopic and Geibel ^{28 )}. Therefore, an increase in Ca intake may increase the buffering capacity of faeces because of the formation of an amorphous calcium phosphate complex. In the studies discussed in our review, the acidification caused by bacterial fermentation may have been buffered by the high amounts of calcium phosphate in the colonic lumen (quantities described in the first paragraph of this session), causing no change or increase in faecal pH⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Lapré, De Vries and Koeman ^{36 ,} Reference Govers, Termont and Lapre´ ^{41 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 ,} Reference Schepens, Rijnierse and Schonewille ^{67 )}. The buffering effect is suggested by the increase in the faecal excretion of Ca and phosphate⁽ Reference Lapré, De Vries and Koeman ^{36 ,} Reference Govers, Termont and Van der Meer ^{37 ,} Reference Govers, Termont and Lapre´ ^{41 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )} and/or the decrease in BA and FA concentration in FW⁽ Reference Bovee-Oudenhoven, Termont and Dekker ^{26 ,} Reference Lapré, De Vries and Koeman ^{36 ,} Reference Govers, Termont and Van der Meer ^{37 ,} Reference Govers, Termont and Lapre´ ^{41 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )}.

In the animal research that evaluated the effect of Ca supplementation on the products of bacterial fermentation, there was an increase in acetate in the ilea⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 )} and caproate in the colons⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )} of pigs. There was also an increase in lactate in the caeca⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 )}, and in propionate and butyrate in the faeces of mice⁽ Reference Overduin, Schoterman and Calame ^{70 )}. Metzler-Zebeli et al.⁽ Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 )} observed a decrease in propionate in the caeca of pigs. The sources and quantities of fibre used to stimulate colonic fermentation varied among the studies (60 g pectin/d⁽ Reference Metzler-Zebeli, Vahjen and Baumgärtel ^{57 )}; 60 g FOS/kg diet⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 )}; 36 g crude fibre/kg diet⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )}; 6 % (w/w) galacto-oligosaccharides⁽ Reference Overduin, Schoterman and Calame ^{70 )}). The use of probiotics in humans⁽ Reference Whisner, Martin and Schoterman ^{71 )} and animals⁽ Reference Weaver, Martin and Nakatsu ^{72 )} increases Ca absorption, especially in the colon. However, even with this increase, it is possible that much of the ingested Ca transits through the colon without being absorbed⁽ Reference Whisner, Martin and Schoterman ^{71 )}.

In a crossover study, isoenergetic diets with similar macronutrient compositions, rich in semi-skimmed milk (1·7 g Ca/d) or cows’ semihard cheese (1·7 g Ca/d), and ingested by fifteen healthy adult men over 14 d increased faecal SCFA (acetate, propionate and butyrate) in comparison with the control diet (which was rich in butter with approximately 360 mg Ca/d)⁽ Reference Zheng, Yde and Clausen ^{44 )}. In comparison with the control diet, both experimental diets increased faecal fat excretion, mainly in the milk group. Cheese intake resulted in higher faecal concentrations of propionate and butyrate, whereas milk intake promoted greater faecal excretion of acetate. These results indicate that milk and cheese stimulate bacterial activity differently. According to the authors, further studies are needed to explore the reasons for this difference. A possible interference factor is the protein profile of cheese (mainly casein, absorbed slowly) and of milk (20 % casein and 80 % whey protein, with the latter absorbed faster)⁽ Reference Zheng, Yde and Clausen ^{44 )}.

Colonic fermentation of fibre mainly generates lactate and SCFA. The amount and type of these metabolites formed depends on gut microbiota composition, the substrate and the intestinal transit time⁽ Reference Van Langen and Dieleman ^{73 )}. Acetate is produced by several groups of bacteria and comprises about 60 to 75 % of total SCFA. In addition, acetate is metabolised in peripheral tissues (through the formation of acetyl-CoA) and/or used for butyrate synthesis⁽ Reference Van Langen and Dieleman ^{73 )}. The number of microorganisms that can produce propionate and butyrate is low. Propionate is mainly produced by Bacteroides spp., Clostridium cluster IX, Mitsuokella and Veillonella spp.⁽ Reference Metzler-Zebeli, Zijlstra and Mosenthin ^{58 )}. Proprionate is metabolised in the liver via glycogenesis and is a lipolysis inhibitor and an inhibitor of the formation of acetyl-CoA from acetate⁽ Reference Zheng, Yde and Clausen ^{44 )}. Butyrate, an inhibitor of acetate synthesis, is the main energy source for colonocytes, followed by acetate and then propionate⁽ Reference Zheng, Yde and Clausen ^{44 ,} Reference Pryde, Duncan and Hold ^{74 )}. Some SCFA production, such as butyrate, is important not only as an energy source for colonocytes but it also prevents the accumulation of potentially toxic metabolites such as d-lactate. In addition, butyrate acts in visceral sensitivity and intestinal motility, regulates transcellular fluid transport, reinforces the gut barrier and reduces mucosal inflammation and oxidative stress⁽ Reference Canani, Costanzo and Leone ^{75 )}. Eubacterium rectale, Clostridium coccoides and Roseburia, which belong to the genus Clostridium cluster XIVa, are the largest butyrate producers⁽ Reference Bronner ^{69 )}. Some species of Clostridium cluster XIVa can convert lactate to butyrate, whereas some cluster IX members can convert lactate to propionate⁽ Reference Louis, Scott and Duncan ^{76 )}.

Lactic acid is a primary metabolite of fermentation in the caecum⁽ Reference Van Langen and Dieleman ^{73 )}. The production of lactic acid and SCFA lowers the pH, inhibiting the activity of microorganisms that metabolise lactate, for example, propionate-producing bacteria⁽ Reference Duncan, Louis and Flint ^{77 )}. Excessive lactate production culminates in its accumulation in the colon as it has low intestinal absorption⁽ Reference Louis, Scott and Duncan ^{76 ,} Reference Duncan, Louis and Flint ^{77 )}. Overduin et al.⁽ Reference Overduin, Schoterman and Calame ^{70 )} suggest that dietary calcium phosphate supplementation influences this fermentation as the amorphous complex formed acts as a buffer against caecal acidification by lactate, thereby accelerating lactate conversion to SCFA in the caecum. According to the authors, colonocytes’ rapid uptake of butyrate may have masked SCFA production, which explains why they observed lower concentrations of butyrate in the colons of rats that had been fed prebiotics. Moreover, as lactate is less absorbable, it can accumulate in the colon in larger quantities⁽ Reference Overduin, Schoterman and Calame ^{70 )}.

Using the quantification of fermentation products to evaluate bacterial metabolic activity may be biased. Many of these products can act as intermediate substrates (i.e. lactate and acetate) and, therefore, may be associated with the metabolic activity of bacterial producers and/or bacterial users of these substrates. For example, an increase in lactate concentrations may indicate an increase in lactate producers or a decrease in lactate users. Therefore, it does not allow definitive conclusions. Moreover, about 95 % of the SCFA produced by bacterial fermentation are absorbed by colonocytes during the intestinal transit. Therefore, a lack in the alteration of these components may not represent real changes in the gut microbiota. Perhaps it represents the more effective use of the components, mediated by diet⁽ Reference Overduin, Schoterman and Calame ^{70 ,} Reference Louis, Scott and Duncan ^{76 ,} Reference Duncan, Louis and Flint ^{77 )}.

Obese subjects and animals have more SCFA in their caeca than lean ones. This seems to favour higher energy storage after the intake of non-digestible carbohydrate⁽ Reference Ley ^{66 ,} Reference Schwiertz, Taras and Schafer ^{78 )} and lower intestinal transit time induced by the hormone peptide YY⁽ Reference Musso, Gambino and Cassader ^{79 )}. Overall, this favours weight gain. Although the effects of Ca have not been confirmed, considering the results of the studies analysed, it is possible that the increased faecal fat excretion and the modulation of gut microbiota that resulted from high-Ca diets (approximately1100 mg Ca/d in the human studies⁽ Reference Govers, Termont and Lapre´ ^{41 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )}) counteracted these effects.

Effects on intestinal permeability and integrity

Paracellular permeability allows substance movement between adjacent cells, by a passive process. In contrast, through transcellular permeability, transport can occur across the cells, and it involves both active and passive processes⁽ Reference Teixeira, Moreira and Silva ^{17 )}. Several high-Ca salts (calcium phosphate, milk, calcium carbonate and calcium chloride) decreased intestinal permeability in rats (100⁽ Reference Ten Bruggencate, Snel and Schoterman ^{56 )} or 120 mmol Ca/kg⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 ,} Reference Schepens, Schonewille and Vink ^{51 ,} Reference Schepens, Rijnierse and Schonewille ^{67 ,} Reference Schepens, Vink and Schonewille ^{80 )}, and 1·5 % Ca⁽ Reference Rao ^{81 )}). Most of these studies used oral administration of EDTA chromium (Cr-EDTA) as a marker of intestinal paracellular permeability⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 ,} Reference Schepens, Schonewille and Vink ^{51 ,} Reference Schepens, Rijnierse and Schonewille ^{67 ,} Reference Schepens, Vink and Schonewille ^{80 ,} Reference Rao ^{81 )}. As Cr-EDTA is stable throughout the gastrointestinal tract, its excretion in the urine reflects total intestinal permeability⁽ Reference Schepens, Rijnierse and Schonewille ^{67 )}. Sugars, such as lactulose (reflecting paracellular permeability) and mannitol (transcellular permeability), are usually ingested to measure region-specific permeability. These sugars are readily degraded by colonic microbiota. Thus, the urinary excretion rate of these sugars (lactulose:mannitol ratio) is used to express the small intestine permeability⁽ Reference Arrieta, Bistritz and Meddings ^{82 )}.

Compared with the control diet, high-Ca diets (quantities described in the previous paragraph) decreased Cr-EDTA urine excretion, and increased the lactulose:mannitol ratio in rats⁽ Reference Schepens, Rijnierse and Schonewille ^{67 )}. However, there was no statistical difference when the urinary excretion of the lactulose and that of mannitol were analysed individually. On the basis of individual lactulose results, the authors concluded that dietary Ca did not affect the permeability of the small intestine. Consequently, they questioned the relevance of the lactulose:mannitol ratio and recommended measuring the excretion of each sugar individually⁽ Reference Schepens, Rijnierse and Schonewille ^{67 )}. This is relevant because, in some situations in which both sugars are excreted in large or small quantities, the ratio remains unchanged⁽ Reference Teixeira, Moreira and Silva ^{17 )}. Obese women appear to have higher urinary excretion of lactulose and mannitol, whereas their lactulose:mannitol ratio does not vary from that of lean women⁽ Reference Teixeira, Souza and Chiarello ^{83 )}. The use of large probes such as lactulose is the best way to analyse macromolecule passage through the intestinal barrier, such as dietary antigens and other components derived from bacteria⁽ Reference Teixeira, Moreira and Silva ^{17 )}. Another relevant example involves coeliac patients, who tend to have low mannitol excretion because of villous atrophy, whereas their lactulose excretion is high. When calculating the lactulose:mannitol ratio, this information does not lead to accurate results⁽ Reference Vilela, Torres and Ferrari ^{84 )}. Therefore, it is suggested that Ca supplementation mainly affects colonic permeability⁽ Reference Schepens, Rijnierse and Schonewille ^{67 )}, which is expected, considering the previous discussion about the effects of Ca on BA and FA precipitation in the colon. This effect reduces cell damage and, consequently, increases the integrity of the epithelial mucosa.

In a transgenic animal model of colitis induction, Ca supplementation prevented colitis-induced increase in the expression of extracellular matrix remodelling genes such as matrix metalloproteinases, procollagens and fibronectin. This suggests that Ca strengthened the integrity of the colonic mucosa⁽ Reference Schepens, Schonewille and Vink ^{51 )}. Even in animal models, high dietary Ca (90 mmol/kg) prevented the FOS-induced increase in intestinal permeability (measured by Cr-EDTA) only when phosphate content was medium (70 mmol/kg diet) or high (160 mmol/kg diet). This was not the case with low-phosphate diets (5 mmol/kg diet). The effect was attributed to the buffering capacity of the colonic lumen because of the formation of a calcium phosphate complex, which could reduce luminal cytotoxicity. In this respect, the phosphate content of the diet is not decisive, but it is necessary for the effect of Ca on intestinal permeability⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 )}. Therefore, based on Schepens et al.⁽ Reference Schepens, ten Bruggencate and Schonewille ^{47 )}, a ratio of about 1:1·3 of Ca:P can affect colon permeability.

Extracellular Ca (luminal Ca) was essential for intestinal tight-junction maintenance⁽ Reference Gonzalez-Mariscal, Contreras and Bolivar ^{85 )}. Tight junctions are apical intercellular joints, which contain transmembrane proteins, cytoskeleton components and cytoplasmic plaques⁽ Reference Gumbiner ^{86 )}. These junctions act on cellular adhesion, intracellular signalling, protection against extracellular entrance and paracellular transportation of substances to the intestinal lumen⁽ Reference Farquhar and Palade ^{87 )}. Among the various tight-junction proteins, the transmembrane proteins (such as occludin and claudin) and cytoplasmic plaques (como a zonula occludens (ZO)) are important for paracellular transport⁽ Reference Hwang, Yang and Kang ^{88 )}. Low-Ca and/or low-vitamin D diets reduce tight-junction gene expression in calbindin-null mice⁽ Reference Hwang, Yang and Kang ^{88 )}, suggesting the importance of this mineral for the synthesis of tight-junction proteins and, therefore, for paracellular permeability. The intact microbiome appears to be essential for normal gut–brain axis signalling and the expression of calbindin, restoring the intrinsic and extrinsic enteric nerve function in germ-free mice, and causing changes to intracellular calbindin concentrations⁽ Reference McVey Neufeld, Perez-Burgos and Mao ^{89 )}. Thus, it is believed that the microbiome may contribute to improve dietary Ca absorption.

Calcium phosphate supplementation (1 g/d) during 3 weeks increased GLP-1 and GLP-2 secretion in healthy adult men (n 10) in a double-blind placebo-controlled crossover study⁽ Reference Trautvetter and Jahreis ^{8 )}. Trautvetter and Jahreis⁽ Reference Trautvetter and Jahreis ^{8 )} suggest that Ca supplementation stimulates the secretion of gastrointestinal hormones (GLP-1 and GLP-2) through the modulation of the intestinal environment. GLP-2 has trophic effects in the intestinal mucosa and influences the tight-junction gene expression (occludin and ZO-1)⁽ Reference Dong, Zhao and Solomon ^{90 )}. On the other hand, Metzler-Zebeli et al.⁽ Reference Metzler-Zebeli, Mann and Ertl ^{91 )} observed a substantial down-regulation of occludin and ZO-1 protein expression in the jejunum of weaned pigs (n 8 per group) fed high-Ca diets (14·8 g Ca/kg) as compared with adequate-Ca diets (8·2 g/kg), whereas gene expression in the colon was unaffected by dietary Ca concentration. The authors suggest that alterations in gene expression were not translated into functional protein, as they did not observe higher intestinal permeability, measured by an enhanced serum acute-phase response or intestinal translocation of LPS⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 ,} Reference Metzler-Zebeli, Mann and Ertl ^{91 )}.

LPS or serum anti-endotoxin antibodies, gut barrier disintegration and endotoxaemia markers were lower after high-Ca diets than after the control diet in mice⁽ Reference Schepens, Schonewille and Vink ^{51 ,} Reference Van Ampting, Schonewille and Vink ^{52 )} (but not in pigs)⁽ Reference Metzler-Zebeli, Mann and Schmitz-Esser ^{59 )} (120 mmol Ca/kg⁽ Reference Schepens, Schonewille and Vink ^{51 )}, 4·8⁽ Reference Van Ampting, Schonewille and Vink ^{52 )} or 14·8 g/kg diet⁽ Reference Van Ampting, Schonewille and Vink ^{52 )}). Moreover, Ca supplementation reduced bacterial translocation after Salmonella infection, indicating increased mucosal integrity⁽ Reference Bovee-Oudenhoven, Termont and Weerkamp ^{27 ,} Reference Bovee-Oudenhoven, Wissink and Wouters ^{38 ,} Reference Ten Bruggencate, Bovee-Oudenhoven and Lettink-Wissink ^{50 ,} Reference Van Ampting, Schonewille and Vink ^{52 )}. However, we emphasise that differences in circulating endotoxin or bacteria may also reflect differences in detoxification or post-absorption clearance. Therefore, it is not related to intestinal translocation only⁽ Reference Creely, McTernan and Kusminski ^{92 ,} Reference Moreira, Texeira and Ferreira ^{93 )}.

Metabolic endotoxaemia, characterised by moderately elevated serum levels of LPS, is associated with obesity, T2DM and IR⁽ Reference Cani, Possemiers and Van de Wiele ^{10 ,} Reference Cani, Amar and Iglesias ^{20 )}. High-fat diets, which are generally associated with obesity, also seem to induce a reduction in II and low-grade endotoxaemia⁽ Reference Mani, Hollis and Gabler ^{94 )}. The factors involved in II breakdown in obese patients mainly consist of dysbiosis, adoption of dietary pattern characterised by foods rich in fat and fructose and deficiencies in the intake of nutrients⁽ Reference Teixeira, Collado and Ferreira ^{95 )}. In congruence with the mechanisms discussed in this review, we consider that the effects of Ca on II may involve gut microbiota and bacterial fermentation product modulation, in addition to direct action on tight junctions and a decrease in luminal cytotoxicity. For example, butyrate enhances the intestinal barrier because it facilitates the assembly of tight junctions⁽ Reference Ulluwishewa, Anderson and McNabb ^{96 )}. It is possible that a high-Ca (>1100 mg/d in the human studies selected for this review⁽ Reference Govers, Termont and Lapre´ ^{41 ,} Reference Trautvetter, Ditscheid and Kiehntopf ^{43 )}) intake contributes to the maintenance of II, especially in the obese. However, because no human clinical trials have been conducted to date, it is not possible to confirm this association yet.