Subjects baseline characteristics
Ten subjects (n = 3 men; n = 7 women) with a mean age of 42 ± 11.8 (years) and BMI of 26 ± 5.8 (kg m−2) (reflecting the average BMI of the Scottish adult population) were recruited for this study.
Composition of the study test meals
The protein, fat, soluble and insoluble NSP content of the meals are presented in Fig S1A–D (ESM_1).
Amino acid composition of the test meals is presented in Table 1 including ANOVA Fisher’s test values for each amino acid for each test meal. The concentrations of histidine (His), serine (Ser), arginine (Arg), glycine (Gly), aspartic acid (Asp), glutamic acid (Glu), threonine (Thr), alanine (Ala), proline (Pro), lysine (Lys), tyrosine (Tyr), valine (Val), isoleucine (ILeu), leucine (Leu), phenylalanine (Phe), methionine (Met) and cysteine (Cys) are presented in mmoles/ portion test meal. Overall, the quantity of individual amino acids was significantly different across the test meals: His, Ser, Arg, Asp, Thr, Ala, Pro, Lys, Tyr, Val, Ile, Leu, Phe, Met (p < 0.001), Gly, Cys (p = 0.002) and Glu (p = 0.005). The meat meal had significantly higher levels of Lys, Val, Ile and Met and was richest in Ala, Thr and His compared with the other plant-based meals The buckwheat meal was richest in Cys; the fava bean meal significantly higher in Pro and richest in Tyr; the green pea meal significantly higher in Gly, Ser, Asp, and richest in Phe and Glu and the hemp meal significantly higher in Arg.
The micronutrients content of the meals are presented in Fig S 2A–C.
Non-nutrient plant metabolites
The ten most abundant phytophenols found in each test meal are presented in Fig. 2. Buckwheat meal had the highest amount of total plant metabolites measured (164.6 mg/portion), followed by hemp and fava bean meals (82.6 and 81.1 mg/portion, respectively) and PCA (Fig. 2) of all metabolites showed clear discriminations of buckwheat and hemp from the rest of the meals. The clustering of meat with the green pea, lupin and fava bean meals in the top left quadrant is likely to be due to the similarity of the profiles with the wheat portion of the meals; specifically the presence of ferulic, benzoic and sinapic acid. However, ferulic acid was the most abundant phytophenol in the fava bean meal and was significantly higher (p < 0.001) in comparison with the meat meal. The clear segregation of buckwheat and hemp meal is likely to be due to the presence of anthocyanins (pelargonidin) being present only in the buckwheat meal and syringaresinol being significantly higher in the hemp meal (p < 0.001). The complete profile of plant metabolites is presented in Table S2 A-D from supplementary data ESM_1.
For the meat meal, a higher average intake (93.5% of the meal served) was recorded. This was followed by lupin (80.9%) and hemp (69.7%). The least quantity consumed of all the intervention meals was fava bean, with an average intake of 65.0% of the meal served; green pea and buckwheat meals had similar (p > 0.05) average intakes of 67.1 and 69.7%, respectively.
Overall, the quantity of individual amino acids consumed is significantly higher in the case of the meat meal in comparison with other meals, with very few exceptions where there is no significant difference (Fig. 3). Despite the significantly higher quantity of amino acids being consumed, the amount of individual amino acids measured in plasma over 300 min following meals consumption was not significantly higher for the meat meal compared to the plant-based meals, with few exceptions (see Fig. 3). These included significantly lower levels of His for the fava bean and buckwheat meals, Lys for the fava bean, hemp and buckwheat meals, Tyr, Ile, leu, Phe and Pro for the buckwheat and Met for all meals except hemp (see Fig. 3).
The average (n = 10) plasma concentration for each of the amino acids (in nmoles/g plasma) following consumption of test meals at 30, 60, 90, 120, 150, 180 and 300 min together with the AUC and iAUC as well as the plasma amino acid concentrations over 300 min are presented in Table S3 from Online Resource ESM_1. Overall ANOVA found a significant effect of the diet on iAUC for the plasma amino acids with few exceptions such as Ala, Gly, Asp, Glu, Gln, and Trp where no significant effect was found. When ANOVA compared iAUC for meat vs the other plant-based meals, no significant difference was found for the amino acid plasma concentrations over 300 min with the exception of Leu, Met, Asp, Lys, Arg, His and Tyr, where significant differences were found. When ANOVA compared iAUC between the plant-based meals, again significant differences were found in the amino acid plasma concentrations over 300 min with the exception of Ala, Gly, Thr, Asp, Glu, Gln and Trp where no significant differences were found. ANOVA analysis showed that iAUC for the plasma concentrations of all BCAAs (Val, Leu Ile) over 300 min only after consumption of buckwheat meal from the plant-based meals was significantly lower (p < 0.001) when compared with plasma distribution after meat meal consumption, exception was iAUC for Leu (p < 0.05) after hemp consumption (see Fig. 4). In case of AAAs, ANOVA analysis showed that iAUC for plasma concentrations of Phe was significantly lower after buckwheat (p = 0.05) and green pea (p < 0.05); Tyr was significantly lower after buckwheat (p < 0.05) and lupin (p < 0.001) consumption when compared with meat. ANOVA analysis showed no significant differences in Trp plasma concentrations following plant-based meal when compared with meat (Fig. 4).The concentrations of di- and polyamines (spermidine, cadaverine, putresine) as well as tyramine and histamine were found to be significantly increased (p < 0.001) in 24 h plasma samples after fava bean meal consumption (Table S5).
The major phenylpropanoid‐derived metabolites have been shown to arise from microbial fermentation of protein . Some of these metabolites have been shown to have beneficial effects for human health, including anti‐oxidant, anti‐inflammatory and anti‐cancer activities [38, 39], however, some of these compounds can be toxic, including acting as co‐carcinogens [40, 41]. On this study, the volunteers were fed high-protein meals, so it was also important to measure the main metabolites derived from the aromatic amino acids to assess potential differences in their plasma concentrations. Moreover, the 24-h plasma sample gives an indication of products produced by microbial metabolism. It was observed that only after consumption of the fava bean meal that there was a significant increase (p < 0.001) of phenylpropionic acid, phenylacetic acid and phenylpyruric acid in the 24-h plasma sample suggesting higher levels of microbial metabolism of phenylalanine (Table S4 A, ESM_1). After the consumption of the buckwheat meal, a significant increase (p < 0.05) of phenylpropionic acid and phenylacetic acid was observed in plasma at 3- and 5 h (Table S4 A, ESM_1). Again, only after consumption of the fava bean meals was a significant increase (p < 0.001) of plasma 4-hydroxyphenylacetic acid, 4-hydroxyphenylpyruric acid, 4-hydroxyphenyllactic acid and p-cresol in the 24-h plasma sample observed, indicating high microbial metabolism of tyrosine (Table S4 B, ESM_1). There was a significant effect of diet (p = 0.009) and time (0 vs 24 h, p = 0.025) on indole-3-propionic acid (IPA). Specifically, there were significant increases in indole-3-propionic acid concentrations in the 24-h plasma after lupin (p = 0.028), green pea (p = 0.026), and buckwheat (p = 0.034) meal consumption (Table S4 C, ESM_1). Following the consumption of the fava bean meal, there was a significant increase (p < 0.001) of indole-3-acetic acid, indole-3-carboxylic acid and indole-3-lactic acid in the 24-h plasma sample compared with baseline (Table S4 C, ESM_1). Following the consumption of the meat meal, there was significant increase (p < 0.01) of plasma indole-3-carboxylic acid in the 24-h plasma sample and indole-3-pyruvric acid throughout the 5-h postprandial period (Table S4 C, ESM_1).
The highest number of overall plasma metabolites with a significant increase in concentration (within 5 h and mainly at 3 h) was observed following consumption of the buckwheat meal (Table S5). Following the consumption of fava bean meals was observed the highest number of plasma metabolites with a significant change after 24 h (Table S5) suggesting that fava bean meal was metabolised late on GI tract at colon level. Ferulic acid was the metabolite with a significant increase at the 3 h plasma samples following the consumption of all the test meals apart from lupin meal, suggesting that it could be bioavailable from the wheat flour ingredient of the meals. In case of the lupin meals, the concentrations of ferulic acid in plasma at 180 min postprandially is comparable with the concentrations measured in the other test meals, but the increase is not significant (0 vs 180 min).
Gastrointestinal hormones and their correlation with plasma plant metabolites
Overall ANOVA analysis found no significant effects of the test meals on the ghrelin plasma concentration for AUC (p = 0.266), and mild significance for iAUC (p = 0.037). ANOVA found no significant changes in ghrelin concentration with time and between plant-based meals. However, following consumption of the hemp meal, the lowest ghrelin concentrations (lowest AUC) in the volunteers’ plasma was recorded and a significant effect on iAUC (p = 0.014, ANOVA) when plant-based meals vs meat meal were compared (Fig. 5).
Overall ANOVA showed no significant time effect on PYY (p = 0.753), and no diet effect (p = 0.793), AUC (p = 0.400) and iAUC (p = 0.325). ANOVA analysis also found no significant changes in PYY concentration with time. After consumption of the hemp meal, plasma PYY had the lowest AUC in average and after consumption of buckwheat meal had the highest AUC amongst all the test meals (Fig. 5).
For GLP-1, overall ANOVA analysis found significant effect of diet (p = 0.0045) and time (p < 0.001) and between diet effects (p = 0.0120), with buckwheat vs hemp (p = 0.049) and buckwheat vs fava bean (p = 0.011). Significant effect on overall AUC iAUC (p = 0.02). ANOVA analysis found significant plasma concentration differences between the fava bean and buckwheat meals (p = 0.017 at 90 min, p = 0.050 at 120 min and p = 0.037 at 150 min), hemp and buckwheat meals (p = 0.007 at 90 min, p = 0.011 at 120 min, and p = 0.051 at 150 min), and the meat and buckwheat meals (p = 0.005 at 300 min). There were significant effects on AUC and iAUC (p = 0.01, ANOVA), with the AUC (51,228.20) after the hemp meal being significantly higher (Fig. 5).
Overall ANOVA analysis found a time effect (p = 0.007) for insulin but no effect on AUC and iAUC (p = 0.157 and p = 0.173). ANOVA analysis found a mild diet effect at 30 min (p = 0.048) and significant insulin plasma concentration differences at 90 min. Insulin concentration after the hemp meal was significantly lower when compared with the buckwheat (p = 0.009), fava bean meal (p = 0.005) and meat (p = 0.012) meals and had the lowest, but not significant, AUC and iAUC.
PLS DA analysis highlighted several plasma metabolites potentially associated with the gastrointestinal hormones measured (Fig. 6 and Tables S6 A–D). Plasma 4-hydroxyphenylpyruvic acid, indole-3-pyruvic acid, 5-hydoxy tryptophan, genistein and biochanin A were present in significantly higher amounts (p < 0.001) after hemp consumption (at 1 and 3 h) compared with meat and were positively associated with increased GLP-1 and PYY and negatively associated with increased insulin. Plasma 3-hydroxymandelic acid and luteolidin were positively associated with the effect of hemp on GLP-1 and ghrelin.
Secoisoresinol, benzoic acid, 4-hydroxy mandelic acid were found to be present in significantly higher amounts (p < 0.001) after hemp consumption (at 1 and 3 h) when compared with meat and were positively associated with the decrease of insulin and ghrelin (Tables S6 A–D). Similarly, significantly higher plasma concentrations (p < 0.001) of coumestrol were observed following hemp consumption when compared with meat and was associated with increased PYY and decreased Insulin concentrations in plasma. Furthermore, p-coumaric acid, daidzein, matairesinol (p < 0.01), phenyl acetic acid, luteolin, apigenin, and pinoresinol (p < 0.001) were detected at significantly higher amounts in plasma at 1 and 3 h after consumption of the hemp vs meat meals and were associated with decreased ghrelin.
Plasma lipids, glucose, urea and homocysteine
There was no significant diet effect on HDL (p = 0.359) and LDL (p = 0.280) when analyzed with ANOVA, there was no significant difference between meals for the AUC and iAUC values for HDL, p = 0.865, and p = 0.291, respectively (ANOVA). There was no significant difference between meals for the AUC, and iAUC values for LDL, p = 0.064, and p = 0.298, respectively (ANOVA). Overall ANOVA analysis found significant diet and time effect on postprandial plasma NEFA (p < 0.001, ANOVA) over 300 min, plasma concentrations being significantly higher after consumption of hemp and lupin meals than other test meals. ANOVA found significant effect of diet on AUC for NEFA (p < 0.001, ANOVA), AUC for hemp and lupin meals being significantly higher than rest of the test meals and no significant effect on iAUC (p = 0.055). Significant effect on NEFA concentration at 90 min (p = 0.005, ANOVA), NEFA plasma concentration after the hemp meals was significantly higher than after meat and fava bean meal, and NEFA plasma concentration after the lupin meal were significantly higher than after the green pea, fava bean and meat meals; NEFA plasma concentration after the lupin and hemp meals was significantly higher than after the green pea, fava bean, buckwheat and meat meals at 120 min (p = 0.004, ANOVA), at 150 min (p < 0.001, ANOVA) and 180 min (p < 0.001, ANOVA); at 300 min, NEFA plasma concentration after the lupin meal were significantly higher than after the green pea, fava bean and buckwheat meals and NEFA plasma concentration after the hemp meal was significantly higher than the buckwheat and green pea meals (p = 0.018, ANOVA). Overall ANOVA analysis found no significant diet effect on triglycerides (p = 0.060), AUC (p = 0.409) but a significant time effect (p < 0.001) and a mild effect on iAUC (0.041), significantly higher after the lupin meal iAUC (174.0) in comparison with fava bean, meat and buckwheat meals. The hemp and lupin meals are the highest in fat, however, we consider that the supplementation of the diet with these meals which are also high in insoluble fibre (NSP) should be done for a prolonged period in order to see any effect of this nutrient or their metabolites on the metabolic health biomarkers such as NEFA.
Overall ANOVA analysis found significant effects on diet and time on plasma glucose concentrations (p < 0.001), these being significantly higher after consuming of the buckwheat meal ANOVA analysis found a significant effect of the test meals on AUC and iAUC for glucose (p = 0.010, p = 0.032, respectively), AUC after the buckwheat meal was significantly higher than all other meals, iAUC was significantly higher after the buckwheat meal in comparison with the green pea, meat and hemp meals. After consumption of the buckwheat meal, plasma concentrations for glucose were significantly higher at 60 min (p = 0.015, ANOVA), when compared with the lupin, hemp, green pea, and meat meals; at 120 min (p = 0.028, ANOVA), when compared with all the other meals; and at 150 min (p = 0.001, ANOVA), when compared with the hemp, fava bean, green pea and meat meals.
Overall ANOVA analysis found a significant diet (p < 0.010) and time (p < 0.001) effect on postprandial plasma urea over 300 min. ANOVA analysis found no significant effect of test meals on AUC and significant effect for iAUC for urea (p = 0.091, p = 0.035, respectively), iAUC was significantly higher after the meat and lupin meals in comparison with the green pea, fava bean and buckwheat meals. After consumption of the lupin meal. plasma urea concentrations were significantly higher at 150 and 180 min (p = 0.047, p = 0.015, respectively ANOVA), when compared with the buckwheat, fava bean and green pea meals; and at 300 min (p < 0.001, ANOVA), plasma concentration of urea were significantly higher after consumption of the meat meals in comparison with the buckwheat, fava bean, green pea, hemp meals.
Overall ANOVA analysis found significant diet and time effect on postprandial plasma HCys (p < 0.001) over 300 min, plasma HCys concentrations being significantly higher after the fava bean meal in comparison with all the other test meals. ANOVA found no significant effect of test meals on AUC and significant effect for iAUC for urea (p = 0.536, p < 0.001, respectively), iAUC was significantly higher after the fava bean meals in comparison with all the other meals. After consumption of the fava bean meals, plasma HCys concentration was significantly at 300 min (p = 0.031, ANOVA) when compared with all the other test meals.
Satiety and hunger
Significant differences were found for all the VAS measures (hunger, fullness, desire, quantity, thirst, preoccupation) with time, from baseline to post-lunch (p < 0.001), data not shown. There were no significant differences between the intervention meals for most of the variables measured. The largest and only significant difference between the intervention meals occurs with hunger at 300 min (p = 0.018), see Fig. 7. Hunger score was significantly lower at 300 min after the buckwheat meal (35.60) vs meat meal (48.50), lupin meal (51.10) and fava bean meal (51.70). There was no significant difference in consumption of ad libitum lunch (p = 0.977). The results showed mild significance (p = 0.04) for the effect of protein (with a coefficient of − 2.08) for the hunger VAS values for all intervention meals when adjusted for protein intake (using consumed protein amount as a covariate with ANCOVA). The source of the protein did not show significant difference for inducing increased satiety (p = 0.434).