3.2. Nutritional Analysis
In terms of physicochemical properties, unlike other pulses, such as chickpea, fava bean or pea, lentil germplasms are relatively stable and their pasting properties or hydration capacity vary little across different accessions/varieties [23
]. This is relevant since these characteristics are significantly correlated to the nutritional quality of the seeds, herein analysed.
In the case of protein concentration, in the present study, a significant variation was found between the lentil varieties (p
≤ 0.05), and this intraspecific variation was also observed in a different group of 12 lentil varieties analysed in a different study [25
]. The mean protein concentration amongst seeds was 24.4% and the highest measured varieties (with values above the mean) were Du Puy, Kleine Schwarze, Rosana, Flora, Große Rote, Kleine Späths II.
Furthermore, after seed germination, the protein concentration was highly increased in all lentil varieties (Figure 2
), and mean protein concentration in seed sprouts was 29%. For varieties with significant variation between protein levels in seeds and seed sprouts, the ones with the highest percentage increases were Dunkelgrün Marmorierte (23%), Du Puy (20%), Große Rote (22%) and Kleine Späths II (20%). Comparative studies using different pulses also showed that lentil has the highest protein content and that total protein values increase after germination [22
]. In the present study, protein was measured as total N content, which has been reported to remain unaltered by germination [26
]. Thus, the higher values of protein here reported are promising but should be confirmed in future studies.
Regarding seed and seed sprout mineral concentrations, six nutrients were selected for the present analysis. The selected micronutrients were Fe, Zn and Mn and the macronutrients Mg, Ca and K (Table 1
As observed in other studies [27
], Fe concentration values were on average ~50 µg/g (Table 1
). The lentil varieties with highest Fe concentration (both in seeds and seed sprouts) were Thessalia, Dimitra, Rosana, Flora and Kleine Rote. Germination process leads to a significant decrease in Fe concentration of Samos (36%), Große Rote (11%) and Kleine Späths II (14%) varieties. This effect of germination in Fe concentration has been reported in previous studies using different legumes seeds, namely, soybean and kidney bean [28
]. These studies showed that the seed Fe concentration decrease was counterbalanced by a major improvement in the availability of Fe. The concentration of the micronutrients Zn and Mn, on the contrary, were shown to be positively impacted by germination, as obtained here (Table 1
More specifically, of the 12 varieties under analysis, only five did not register a significant increase in Zn concentration after sprouting. In general, the varieties with higher Zn concentration were Thessalia, Dimitra, Kleine Schwarze and Kleine Rote. Higher Zn concentration increases (p
≤ 0.0001) were found in Dunkelgrün marmorierte (29%), Du Puy (21%), Samos (22%) and Kleine Späths II (24%) varieties. In regards to Mn concentrations (Table 1
), Thessalia, Große Rote and Kleine Rote demonstrated the highest both in seeds and seed sprouts; and the varieties that presented a significant increase after germination were Du Puy (108%), Rosana (64%), Große Rote (52%) and Kleine Rote (42%). Legume germination is associated with a drastic reduction in phytate content, which in seeds bind with minerals, forming insoluble complexes, making them unavailable [29
Amongst the analysed macronutrients, Mg concentration was not affected by germination (Table 1
), similarly to what was found in a study using soybean seeds [29
]. Calcium concentration, on the other hand, was reported to increase approximately 55% in legume seeds after germination [30
], as well as its bioavailability [28
]. In the present study, the varieties with the highest Ca concentration (Samos and Kleine Rote) did not show variations after germination. The varieties in which Ca concentration was significantly increased were Dunkelgrün Marmorierte (58%), Du Puy (56%), Kleine Schwarze (41%) and Rosana (49%).
In terms of K concentrations, it was reported that soybean seed sprouts present a concentration five-times higher when compared to dry seeds [29
]. In the present study (Table 1
), the varieties with the highest K concentration were Thessalia, Kleine Schwarze, Santa and Große Rote. Seed K concentration has been identified as a possible marker for germination capacity due to its role in initiating the imbibition of water and facilitating the associated physiological processes [32
]. Here, the varieties with the highest K concentration (Table 1
) are not amongst the ones with significantly higher germination rates (Figure 1
); however, significant differences amongst varieties were few. Only Rosana variety presented significant increases (30%) in K concentration after germination (Table 1
As all 12 varieties were grown in the same field conditions, the differences detected in this study are mainly genotypic. Interestingly, Dunkelgrün Marmorierte and Du Puy varieties, that share the same genetic background, showed no significant differences for the analysed factors, both in the seeds and seed sprouts. Knowledge on the correlation between genotypic variation and nutritional traits can contribute to future breeding programs as well as for a targeted selection of the most appropriate varieties for human consumption.
Between the four varieties with the highest germination rate—Rosana, Kleine Späths II, Kleine Rote and Du Puy—and based on their percentage of protein and potential for mineral increase after germination, the following studies proceeded with the Rosana variety.
3.3. Microbial Counting and Disinfecting Methods
Firstly, the impact of the seed disinfection treatments on lentil germination percentage was tested (Table 2
). It was found that in general, the germination efficiencies for all treatments and controls were high, with most seeds germinating at over 90%. In general, there was no negative impact of disinfection on germination efficiency, albeit the hot water treatment for Salmonella
inoculated seeds seems to have lowered the germination values by about 32.6% when compared to the non-disinfected non-inoculated seeds and non-disinfected and inoculated seeds.
In order to test the different disinfection treatments, the seeds were artificially inoculated with Salmonella
spp. and E. coli.
As shown in Table 3
, disinfection with only hot water was efficient for reducing E. coli
(from 2.7 × 108
to 2.7 × 107
UFC/mL), as was SDS treatment for Salmonella
spp. (from 1.1× 108
UFC/mL to ≤1.0 × 108
UFC/mL). Regarding sprouts, the reduction was more accentuated when the combination of Amukine and SDS was applied, reducing 2 logs (corresponding to a 99% reduction of bacterial load) for E. coli
when compared to sprouts inoculated with no disinfection and 1 log logs (corresponding to a 90% reduction of bacterial load) for Salmonella
In 1999, the U.S. Food and Drug Administration recommended the utilization of 20,000 ppm of calcium hypochlorite for seed disinfection [34
] and this is the method commonly used by sprout manufacturers. However, this treatment was considered potentially hazardous to the environment and industrial workers, and new efficient strategies are needed to improve sprout safety [16
The products selected for this experiment are of easy access and are usually utilized for disinfection since they have chemical constituents, such as hypochlorite (found in common household bleach and Amukine) and ammonia, that act by denaturing bacterial proteins, similar to what happens when exposed to high temperatures resulting in bacterial death [35
], or when exposed to ethanol and sodium hypochlorite. Here, some combinations of these treatments were tested to mimic and improve the disinfections done in a domestic environment.
As shown in Table 1
, in general, the germination rate was not affected by the treatments or the inoculation with the pathogens, as they presented similar germination rates. However, seeds inoculated with Salmonella
spp. treated with only hot water showed lower rates of germination, of about 65%. This could denote higher sensitivity of the seeds to this method. Also, the length of the sprouts was not affected overall (data not shown). Hence, these treatments could be applied on the seeds with relatively little impact on germination and growth processes.
The effect of the disinfection treatments on the bacterial load was also evaluated. In this study, the reduction of microorganisms was expected, not only on the seeds but also after germination. Overall, the treatment more suitable for microorganism reduction was disinfection of seeds with SDS followed by Amukine treatment after germination.
In the case of seed decontamination, the protocol that showed better results—less microbiological growth—in E. coli
contamination was the one used in [4
]. Treatment with hot water reduced the growth of E. coli
, showing that heat inactivation is effective for this pathogen, as also observed elsewhere [36
]. On the other hand, Salmonella
was more susceptible to SDS treatments, which reduced colony count to below the detection levels.
Concerning seed sprouts, the same was verified. For E. coli
hot water was more effective than SDS as in Barampuram et al. [37
] and for Salmonella
spp. the best results were obtained with a combination of ethanol, sodium hypochlorite and SDS. This could be due to the fact that Salmonella
spp takes longer to be inactivated by the heat, making treatment with SDS more suitable for this pathogen.
Considering the seeds that suffered treatment before and after germination, Amukine combined with SDS presented a reduction of 1 log for Salmonella spp. and 2 logs for E. coli, being the better treatment for disinfection. Amukine works by denaturation of the cell’s proteins making them inactive while washing the sprouts with water only dilutes the microorganism existent in the plant, making this treatment not enough for proper disinfection.