3.1.3. Alfalfa

Alfalfa, fenugreek, and lentil all belong to the legume family. The contents of AGM (11 mg/kg), PUT (12 mg/kg), CAD (1 mg/kg), SPD (140 mg/kg), and SPM (34 mg/kg) in ungerminated alfalfa seeds were lower than those for lentil, but higher than for fenugreek. Similar to lentil sprouting, alfalfa sprouting did not result in a higher content of SPM (34 mg/kg), while the content of SPD (323 mg/kg) doubled compared to ungerminated seeds. Sprouting resulted in two orders of magnitude greater contents (Figure 2c) of AGM (2669 mg/kg) and PUT (1015 mg/kg), and the accumulation of CAD (1910 mg/kg) resulted in higher levels than that for lentil sprouts and lower levels than for fenugreek. Polyamines PUT and CAD present in sprout samples could be formed as a result of microbial spoilage [16] or endogenous synthesis by plants. Insufficient hygienic standards could impair microbial spoilage. However, even with 11 log colony-forming units/g and five orders of magnitude higher microbial load after the end of the 12-day storage period of four sprout varieties, only a minor increase in CAD and PUT content was observed [69]. In order to test whether some polyamines were outside the plant cells, due to microbial spoilage, the sprouts were rinsed with extraction buffer (30 s), and only about 10% polyamines were found in such extracts. The ratios of the polyamine contents reflected those in sprouts, strongly suggesting that polyamines in the "rinsing solution" originated from ruptured cells (0.4 M HCl).

The contents of PUT (1034 mg/kg), SPD (292 mg/kg), and SPM (30 mg/kg) in microgreens remained in similar ranges to those in sprouts. The content of CAD (257 mg/kg) was lower by an order of magnitude, as was also observed in lentil and fenugreek microgreens. Alfalfa sprouts, and especially, microgreens (Figure 2c), stood out as extremely rich sources of AGM (5392 mg/kg). Such high contents were found only in some samples of fermented soybean pastes [70]. As noted above, there are relatively few data on the content of AGM in foods [39]. This is rather surprising, considering the many health benefits of AGM supplements and the fact that long-term intake of relatively high doses of AGM should be safe [53]. Dietary AGM can be absorbed in the small intestine and pass the blood–brain barrier [71], where it can interfere with some important central nervous system disorders. The majority of the experiments were conducted in a rodent model with ingested daily doses of 10 mg/kg or higher [34]. Human trials are rare; daily doses of about 10 mg/kg [44] have been shown to be effective in relieving pain in lumbar disc-associated radiculopathy. An intake of more than 1 kg of alfalfa microgreens (FW) would be necessary to reach an equivalent level, which is unrealistic. However, some rodent experiments [42] have shown that oral administration of 0.1 mg/kg of AGM alone produces the antidepressant effects. If similar results are confirmed in human studies, this would put a different perspective on alfalfa sprouts as a source of dietary AGM.

#### 3.1.4. Daikon Radish

The absolute content of AGM (52 mg/kg), PUT (11 mg/kg), CAD (4 mg/kg), SPD (96 mg/kg), and SPM (56 mg/kg) in ungerminated seeds of daikon radish was similar to that in lentil seeds. The germination of this cruciferous vegetable (Figure 2d) resulted in some differences from the analyzed legumes. There was only a slight increase in the content of CAD (21 mg/kg), and the values in sprouts were one or two orders of magnitude lower than in legume sprouts. On the other hand, the contents of AGM (4270 mg/kg) and SPD (423 mg/kg) in daikon sprouts were the highest of all four plants analyzed. In combination with the relatively low content of CAD, this indicates that daikon radish sprouts are an excellent source of nutritionally beneficial polyamines. Previous reports on the change in polyamine content during sprouting [16,72], were consistent with the current results and showed a large accumulation of AGM. In the same studies, it was found that SPD content in sprouts decreased on an FW basis, but, assuming 10% DW, the recalculated values were consistent with the observations of our study. An increase in SPD content was also observed in [73], but the reported levels are an order of magnitude lower in both ungerminated seeds and sprouts.

In contrast to legumes, in which microgreens were nutritionally better than sprouts, due to a lower content of CAD and a higher content of AGM, SPD or SPM, daikon radish microgreens were nutritionally inferior to sprouts. Contents of the nutritionally beneficial polyamines AGM (3951 mg/kg), SPD (250 mg/kg), and SPM (17 mg/kg) were lower in microgreens than in sprouts. The content of SPM in microgreens was even lower than in ungerminated seeds.

#### *3.2. The E*ff*ect of Thawing on The Determined Polyamine Contents in Frozen Sprouts*

The general protocol for the extraction of biogenic amines, described in materials and methods (Section 2.4.1), explicitly requires immediate extraction of freshly harvested microgreens and sprouts. The reason for this rigor is that the freeze/thaw cycle initiates extensive transformations of polyamines. Consequently, the polyamine contents determined depend strongly on the time delay of sampling after the start of thawing (Figure 3, Figure A1). Freezing (in liquid nitrogen) and storage for a few hours at −20 ◦C was in itself not problematic, but thawing at room temperature was. The contents of the majority of the polyamines analyzed already decreased on a time scale of minutes. In the case of fenugreek (Figure 3), approximately 30% lower contents of PUT and CAD were found in frozen sprouts (−20 ◦C) that were left at room temperature for only 5 min before homogenization in 0.4 M HCl. During longer thawing times, considerable amounts of SPD and AGM were also degraded. Similar results were observed for lentil and alfalfa sprouts, in which CAD and PUT were most susceptible to degradation (Figure A1a,b). Daikon radish sprouts were much less a ffected by freeze/thawing (Figure A1c).

**Figure 3.** Time-dependent changes in the content of polyamines (agmatine (AGM), putrescine (PUT), cadaverine (CAD), spermidine (SPD), and spermine (SPM)) during thawing of the frozen fenugreek sprouts.

Experimental artifacts, resulting from sample preparation, are not unusual and are often related to the enzymatic conversion of metabolites. A typical example is the oxidation of ascorbic acid to dehydroascorbic acid, catalyzed by ascorbate oxidase, which can lead to a distorted ratio of both forms of vitamin C when samples are homogenized under conditions where the enzyme remains active [74]. As shown in Figure 3, the polyamines are also highly susceptible to degradation, which can lead to experimental artifacts if the samples are not handled properly. Indeed, there is little overall consistency in the sample preparation protocols for the analysis of polyamines in sprouts. Fresh sprouts could be ground or homogenized in a grinder before extraction in acid [13,15,16], stored in a frozen state before extraction [75], lyophilized before extraction [68], or directly homogenized in acid [14]. Any protocol that disrupts tissue integrity prior to extraction could result in a significant transformation of polyamines if the criteria for enzyme activity are met.

It has been observed that freezing and thawing of soybean sprouts [76] resulted in significant accumulation of γ-aminobutyric acid (GABA), which is formed in two enzymatic steps from PUT, including the action of amine oxidases as the first step. Recently it was shown that GABA accumulation in thawed sprouts is most probably the result of ruptured cellular structures rather than the higher enzyme activity itself [77]. In neither of these two studies, where such enzymatic activity could be observed with certainty, was the change in polyamine profiles assessed. Since the transformation of polyamines in legume sprouts was much more pronounced than in radish, the most likely candidates are the diamine oxidases with copper ion in the active site, termed copper amine oxidases (CuAOs). CuAOs are expressed in high levels in legumes [22] and are localized in apoplasts, intercellular spaces, or loosely bound to the cell walls [23,24]. The freezing and thawing of plant tissue is detrimental to its structural integrity and leads to rapid changes in the content of some secondary metabolites, often resulting in lower sensory quality and nutritional value [78]. However, this enzymatic potential can also be used to increase the content of desired bioactive constituents [77].

#### *3.3. Degradation of Exogeneous Biogenic Amines by Homogenized Sprouts*

Copper amine oxidases (CuAO) from the legume family have, in general, optimal activities at neutral or slightly alkaline pH in general and relatively broad substrate specificity, as they catalyze the oxidation of diamines and polyamines, with PUT and CAD as optimal substrates [79–82]. A similar substrate specificity could be derived from the degradation pattern of polyamines observed in thawed fenugreek (Figure 3), lentil, and alfalfa seeds (Figure A1a,b). According to some older studies [83], the substrate specificity of CuAO could be even broader, including monoamines. The monoamines TYR, PEA, and TRP are often present in fermented foods together with PUT, CAD, and HIS. The content of PEA and TRP in foods is generally low, while HIS and TYR may be present in relatively high concentrations and may have adverse health e ffects associated with a local immune response (HIS) or increased blood pressure and migraine (TYR). PUT and CAD are less toxic to intestinal cells than TYR and HIS, and only some foods with the highest content [31] could potentially have an adverse effect. Of the sprouts included in the study, even those with the highest CAD/PUT content do not pose a direct risk if consumed in the hydrated form. PUT and CAD are nutritionally problematic, mainly due to their interference with the activity of intestinal amine oxidases with broad substrate specificity, since, at high concentrations of these diamines, less HIS and TYR is oxidized [32]. Even low dietary doses of biogenic amines are problematic for persons taking monoamine oxidase inhibitors as part of their antidepressant therapy [84].

Optionally, biogenic amines can be degraded in the food matrix before consumption. Accordingly, it was investigated as to whether homogenized sprouts could be used as a source of the enzyme for the oxidation of biogenic amines in complex mixtures. Homogenized fenugreek sprouts (less than 0.5% DW in suspension) e fficiently oxidized PUT, CAD, and even TYR, when all analyzed biogenic amines were present in the mixture at concentrations of 50 mg/<sup>L</sup> and in the pH range of 6 to 8. TYR was even more e fficiently oxidized than PUT and CAD (Figure 4). After 5 min of incubation at pH 6, 53% of TYR, 32% of PUT, and 22% of CAD were oxidized (Figure 4a–c). At the end of the incubation period (2 h), only traces of these 3 polyamines were observed. At pH 5, the activity was much lower so that after 2 h, the values observed were closely similar to those at pH 6 after 5 min. At pH 4, the enzyme was not active against any of the substrates.

**Figure 4.** Time-dependent degradation of (**a**) putrescine, (**b**) cadaverine, and (**c**) tyramine by homogenized fenugreek sprouts at different pH values.

Three other substrates were much less susceptible to oxidation (Figure A2). Under optimal conditions, only 40% of TRP was oxidized after 2 h of incubation, while homogenized sprouts had practically no effect on the stability of HIS and PEA. The substrate specificity of the amine oxidases in fenugreek appears to differ from that of the white pea, for which HIS is a better substrate than TYR [29]. Fenugreek sprouts could, potentially, be used to reduce PUT, CAD, and TYR loads of fermented high protein food products that stabilize the pH of the matrix above 5. Some examples are sausages and cheeses or, when more acidic fermented vegetables and pulses are added to the matrix, with pH above 5. On the other hand, the content of biogenic amines PUT and CAD in legume sprouts could be reduced simply by freezing and thawing for several dozen minutes. Under these conditions, considerable amounts of PUT and CAD are degraded, while the content of nutritionally beneficial SPD is only slightly reduced (Figure 3).
