In the present study, we showed that RB consumption is followed by significantly different levels of various metabolites compared to those followed by WB consumption. Namely, levels of some amino acids, AA-derived compounds, TCA metabolites, and organic acids differed after intake of RB and WB. In addition we confirmed the earlier finding of lower postprandial insulin response to refined endosperm sourdough rye bread as compared to wheat bread .
The in vitro protein hydrolysis rate for the RB was slower than WB, and the relative content of soluble proteins before the hydrolysis was higher in RB than WB despite lower total protein content in RB. This finding, together with the different peptide profiles in the qualitative SDS-PAGE analyses of the test breads confirms that rye and wheat breads have different characteristics with regard to protein and peptide content. Consequently, we expected a different postprandial AA response. The analyses by GC×GC-TOF-MS confirmed significant postprandial changes in four essential AAs (phenylalanine, methionine, tyrosine and glutamic acid) during the insulin decrease. Neither one of them was branched-chain AA, which have previously shown to affect insulin signalling in adipose tissue .
However, significant changes in two metabolites related with the amino acid tryptophan were found in our study. On one side, ribitol, a metabolic end product formed by the reduction of ribose, significantly increased after RB intake while it decreased after WB intake. Interestingly, the level of this compound in fasting serum samples has been recently found to be significantly increased in post menopausal women after an 8-week consumption of high-fibre rye bread diet . Since tryptophan is a precursor for the biosynthesis of serotonin which inhibits neuropeptide Y expression, thus depressing hunger , we hypothesize that increases in the tryptophan precursors ribitol and ribonic acid after the RB diet may mediate positive effects of higher tryptophan concentration, resulting in decreased appetite and food intake. This hypothesis would agree with recent postprandial studies that have shown that intake of rye is linked to a high subjective satiety [5, 6, 30]. On the other side, picolinic acid is a catabolite of the tryptophan metabolism reported as an activator of macrophage proinflammatory functions. This acid has shown to selectively induce the chemokines macrophage inflammatory protein-1alpha and -1beta in macrophages . In our study the levels of picolinic acid significantly decreased after RB and increased after WB intake even one hour after consuming the test breads. This finding supports our earlier results linking rye bread consumption to inflammatory status. Higher inflammation markers were detected in fasting serum samples of subjects with metabolic syndrome after a 12-week diet with oat and wheat bread (high postprandial insulin response diet) than with rye bread and pasta (low postprandial insulin response diet) , in addition to higher lysophosphatidylcholine levels . However, human studies are needed in order to confirm the inflammatory effects of picolinic acid reported in animal studies.
It is important to keep in mind that the physiological and biochemical response to a dietary perturbation is complex. The postprandial response per se depends on and involves multiple factors. Multiple processes related to metabolism, inflammation and oxidation are affected [32, 33]. Recently, Pellis et al.  introduced a metabolomics and proteomics based postprandial challenge test to quantify the postprandial response of multiple metabolic processes in humans in a standardized manner. The application of the test in a dietary intervention study aimed at studying the effects of an anti-inflammatory supplement, revealed differences in several metabolites, some of them associated with amino acid and carbohydrate metabolism such as citric acid cycle metabolites. Differences in TCA cycle metabolites were also observed in our study after the intake of the two types of breads. However, these findings are probably not connected to the lower insulin response observed after RB intake. The larger decreases in pyruvic and succinic acids after RB meal might reflect a significant reduction in the aerobic glycolysis after RB intake. However, this should have contributed to a higher glucose response after RB than WB. Nevertheless, it is clear that the postprandial response might be a useful tool to reveal multiple aspects of metabolic health that would not be apparent from studying the homeostatic parameters at fasting state .
The rye bread tested in our study was baked by the traditional sourdough procedure which includes a fermentation process known to produce many changes in the bread matrix [35, 36], and has a potential to reduce glycemic response [19, 37]. In fact, lactic acid, either produced during sourdough fermentation or added directly to the bread, has been reported to reduce the rate of in vitro starch hydrolysis, suggesting that lactic acid interferes with the digestive process . However, in our study the in vitro assays simulating the mouth phase digestion of starch of the test breads showed a higher initial starch hydrolysis rate for the RB than for WB. Nevertheless, since the in vitro starch hydrolysis was not monitored after the pepsin treatment in conditions mimicking the small intestine, precise conclusions about the effects of starch digestion cannot be made.
Bioprocessing has been shown to modulate the levels and bioaccessibility of bioactive compounds . Recent studies demonstrated that sourdough fermentation increases the amount of free phenolic compounds , which have also been reported to have an impact on lowering the glycemic and insulin responses [41, 42]. However, it is not likely that the higher phenolic acids content of the RB than in WB can explain the observed decreased insulin response after RB. Most phenolic acids in cereals are ester linked to polymers and require several biotransformations until they can exert their beneficial effects in the body. It is therefore very unlikely that they can have such acute postprandial effect on insulin metabolism as observed after the RB intake. On the other hand, lowered pH of sourdough bread compared with white bread is indicative of the increased level of organic acids in sourdough bread. It has been suggested that the presence of organic acids could reduce the GER . However, the GER remained unchanged after both test breads although several organic acids identified by the metabolomics platform showed significant increases in their contents after RB versus WB intake.
There are, naturally, many other food factors already known or speculated to influence the postprandial glycaemia and insulinemia after bread consumption. These are for example differences in fibre content, botanical origin and structural properties of the raw material in cereal products that may have an effect on the postprandial responses. In addition, the type and extent of the bread processing and structure of the final product have been shown or speculated to have an effect on the postprandial responses [37, 44]. In most of these studies, when insulin response is modified, usually glycemic responses are influenced in the same way, or vice versa. In the case of rye bread, however, the main difference from white wheat bread is the insulin response .
Our aim was to identify metabolites that are linked with the lowered insulin response after RB consumption in search for possible mechanism for this interesting response. We were unable to find molecules that might have an effect on lower insulin response, but we found other metabolites that could play a role in other 'rye-specific' effects shown by others such as higher satiety after rye bread consumption [6, 28].
The finding that no significant changes in the metabolites were observed before 30 minutes, at which time point the insulin responses between RB and WB started to differ, does not support a causal explanation. Nevertheless, the changes observed in some metabolites associated with tryptophan were consistent with the changes in insulin levels after these breads and may suggest a beneficial inflammatory response after a single meal containing rye bread.