Mushrooms (tails and nonmarketable mushrooms) were dried by hot air (50°C) and ground to for a powder (42 mesh) that we called MTM. As shown in Table 1, the basic composition of MTM is characterized by an important carbohydrate and protein content: 53.62 ± 3.9% and 28.76 ± 1.63%, d.w., respectively, and a low fat content (3.21 ± 0.22%), which makes it a good substrate for the preparation of protein hydrolysates that are potentially usable in the food and/or agronomic industry.
Enzymatic Hydrolysis Of Mtm
The proteins present in MTM were hydrolyzed by two procedures: single hydrolysis with one protease (Alcalase®, Flavourzyme®, L-450 or papain) and sequential hydrolysis with two proteases (“Alcalase® + Flavourzyme®” or “L-450 + Flavourzyme®”). Figure 1A and 1B show the hydrolysis curves as a function of base consumption (ml of 1 M NaOH) vs. hydrolysis time (min) and DH vs. hydrolysis time (min) obtained by single and sequential hydrolysis, respectively.
As Fig. 1A shows, the highest DH was obtained with Alcalase® and L-450, and both were similar at 23.7 ± 0.5 and 23.0 ± 0.7, respectively, which was not surprising because both enzymes are highly active endopeptidase from Bacillus licheniformis, one from Novozyme® and the other from Biocon®, with serine-type activity. Lower DHs were obtained with Flavourzyme® and papain, 13.9 ± 0.5 and 9.6 ± 0.4, respectively, where the first was a mixture of exo- and endoproteases with a predominance of exoprotease: activity, and the second was an endopeptidase with mainly cysteine-type activity.
Figure 1B shows that a significantly higher HD was observed in the sequential process with two enzymes than in the single hydrolysis, increasing DH from 23.7 ± 0.5 to 28.7 ± 0.9 (p < 0.01) and from 23.0 ± 1.2 to 27.3 ± 1.6% (p < 0.01) for “Alcalase® + Flavourzyme®” and “L-450 + Flavourzyme®”, respectively. This result can be attributed to the action of the exoprotease, which showed that a greater number of attack points led to greater activity and, therefore, broke a greater number of peptide bonds. Consequently, both combinations of enzymes were applicable. Although the sequential process is more expensive than single hydrolysis, due to the use of two proteases, the content of FAAs and oligopeptides (> 5 kDa) is much higher than that in single hydrolysis, as we will discuss below. This scenario can be explained by a synergistic effect of endopeptidase and exopeptidase activities.
Characterization Of Mb-pphs
Table 1 also shows the basic composition of the dry Mb-PPHs obtained by single (Alcalase®) and sequential (“Alcalase®+Flavourzyme®”) hydrolytic processes. These results showed that no significant differences were observed in the basic compositions, although significant differences were observed for FAAs, oligopeptides (> 0.2 kDa and < 5 kDa) and peptides + proteins (> 5 kDa), as well as for β-glucans, between the hydrolysates obtained by single and sequential processes and with respect to MTM. This can be explained by the enzymatic activity of the endo- and exoenzymes used in the study, as we will discuss below.
Table-1 Basic compositions of MTM and dried Mb-PPHs obtained by single (Alcalase®) and sequential (“Alcalase®+ Flavourzyme®”) enzymatic hydrolytic processes
|
MTM
|
Mb-PPH(Alcalase®)
|
Mb-PPH(Alcalase®+Flavourzyme®)
|
Moisture (%)
|
7.32±1.27
|
9.30±0.63
|
9.44±0.55
|
Dry matter (%)
|
92.75±1.23
|
90.70±0,63
|
90.56±0.55
|
Ash (% d.w.)
|
8.81±2.32
|
15.31±0.97
|
16.03±0.84
|
Organic matter (% d.w.)
|
91.19±2.32
|
84.69±0.97
|
83.97±0.84
|
Nt (g/100 g d.w.)
|
5.23±0.39
|
10.11±0,41
|
10.24±0.28
|
Protein (% d.w.)
|
28.76±1.63*
|
63.19±1.36 **
|
64.01±1.48 **
|
FAAs# (% d.w.)
|
1.04±0.08
(3.62%)
|
3.85±0.22
(6.09%)
|
7.28±0.63
(11.37%)
|
Oligopeptides# (% d.w.)
|
9.49±0.12
(33.00%)
|
18,12±0.51
(66.64%)
|
18.93±1.76
(67.15%)
|
Peptides+Proteins# (% d.w.)
|
18.23±0.98
(63.38%)
|
5,20±0.38
(19,12%)
|
4.13±0,18
(14.65%)
|
Total Carbohydrates (% d.w.)
|
53.62±3.91
|
11.63±3.22
|
11.98±4.12
|
β-Glucans (% d.w.)
|
14.14±1.62
(26.37%)
|
7.74±0.62
(66.55%)
|
8.02±0.54
(66.94%)
|
Crude Fat (% d.w.)
|
3.21±0.22
|
n.d.
|
n.d.
|
Others (% d.w.)***
|
5.8
|
9.89
|
8.78
|
*Nt x 5.5; **Nt x 6.25; ***Determined by difference in the means; FAAs = free amino acids; n.d. = not detectable. Data are expressed as the mean±standard deviation.
#FAAs: <0.2 kDa; Oligopeptides: >0.2 kDa and <5 kDa; Peptides+Proteins: >5 kDa
These results also showed that almost half of the dry matter contained carbohydrates (mainly single sugars, oligosaccharides and soluble polysaccharides). It should be noted that soluble β-glucans represented 7.74 ± 0.62% and 8.02 ± 0.54% of the product obtained by single and sequential hydrolysis, respectively, representing 16.25 and 17.07% of the total soluble carbohydrate content. Although the content of β-glucans in the MTM was significantly higher (14.14 ± 1.62%) than that found in Mb-PPH, the β-glucans that occurred in the MTM included both soluble and insoluble β-glucans, while in the Mb-PPH, there were only soluble β-glucans. The presence of β-glucans and β-oligoglucans in Mb-PPHs is of great importance since they can act as elicitors against certain pests [28, 29].
Figure 2 shows the molecular distribution obtained by size-exclusion chromatography for MTM, Mb-PPHAlcalase® and Mb-PPHAlcalase®+Flavourzyme®, grouped into three groups: FAA (< 0.2 kDa), oligopeptides (> 0.2 and < 5 kDa) and peptides + proteins (> 5 kDa). As these results show, the main protein components of MTM are molecules with Mw > 5 kDa (peptides + proteins; 63.38 ± 1,21%), while in Mb-PPHAlcalase® and Mb-PPHAlcalase®+Flavourzyme®, the main components are molecules with Mw > 0.2 and < 5 kDa (oligopeptides, 66.64% and 67.15%, respectively), obtaining the highest FAA concentrations in Mb-PPHsAlcalase®+Flavourzyme®, as expected.
This type of protein hydrolysate with high oligopeptide and/or FAA concentrations has different beneficial effects in modern agriculture. Protein hydrolysates can improve crop tolerance to abiotic stresses; therefore, root applications of plant-derived protein hydrolysate have been observed to improve salinity tolerance by improving nitrogen metabolism and a higher K/Na ratio and proline accumulation in leaves [30]. Biostimulants in the presence of oligopeptides could also act as plant regulators; in this respect, several bioactive oligopeptides produced from a variety of plants have been found to have phytohormone-like activities [31, 32].
Compositional analysis shows (see Table-1) that the FAA content of MTM (1.04 ± 0.08%, d.w.) was significantly lower (p < 0.01) than that of the Mb-PPHs at 3.85 ± 0.22%, d.w. for the hydrolysate obtained by simple hydrolysis and 7.28 ± 0.63%, d.w. for that obtained by sequential hydrolysis. As expected, the highest content of FAAs was found in the hydrolysate obtained by sequential hydrolysis due to the exoproteasic activity of Flavourzyme®.
Table 2 shows the amino acid composition, after acid hydrolysis, of the protein material present in MTM, Mb-PPHAlcalase® and Mb-PPHAlcalase®+Flavourzyme®. As these results show, the content of essential amino acids for plants (Trp, Thr, Val, Lys, Leu, Met, His, Phe and Ile) increased significantly in the Mb-PPHs compared to content in MTM, showing that the use of these hydrolytic enzymes resulted in an increase in the content of plant essential amino acids.
Table 2
Amino acid composition of proteins found in MTM and Mb-PPHs obtained by single (Alcalase®) and sequential hydrolysis (Alcalase®+Flavourzyme®)
| MTM | MbPPHL−450 | MbPPHLA450+Flavourzyme® |
Ala | 28.96 ± 0.83 | 54.97 ± 2.12 | 59.42 ± 1.17 |
Arg | 14.66 ± 0.25 | 34.39 ± 1.48 | 38.48 ± 2.06 |
Asx | 27.94 ± 1.02 | 52.50 ± 1.98 | 58.61 ± 2,01 |
Cys | 0.95 ± 0.62 | 1.43 ± 0.71 | 1.02 ± 0.32 |
Glx | 53.75 ± 1.04 | 125.19 ± 4.34 | 130.32 ± 5.21 |
Gly | 15.12 ± 0.42 | 32.12 ± 1.89 | 30.18 ± 1.45 |
His | 8.08 ± 0.30 | 15.91 ± 0.87 | 14.58 ± 0.56 |
Ile | 11.90 ± 0.47 | 26.33 ± 1.43 | 23.78 ± 1.64 |
Leu | 19.66 ± 0.54 | 74.87 ± 2.21 | 73.85 ± 1.98 |
Lys | 20.81 ± 0.78 | 21.15 ± 1.54 | 24.85 ± 1.09 |
Met | 5.89 ± 0.23 | 3.08 ± 0.26 | 2.96 ± 0.31 |
Phe | 10.63 ± 0.58 | 25.61 ± 1.93 | 22.82 ± 2.01 |
Pro | 12.10 ± 0.43 | 30.56 ± 2.11 | 24.74 ± 1.84 |
Ser | 15.83 ± 0.52 | 29.41 ± 1.75 | 31.12 ± 1.16 |
Thr | 16.58 ± 0.72 | 31.53 ± 2.23 | 28.82 ± 1.43 |
Trp* | 5.58 ± 0.32 | 5.33 ± 0.44 | 5.39 ± 0.61 |
Tyr | 4.26 ± 0.29 | 16.10 ± 1.21 | 15.76 ± 2.02 |
Val | 17.42 ± 0.51 | 51.37 ± 4.32 | 53.28 ± 3,85 |
PEAAs | 116.55 | 255.18 | 250.33 |
The results are expressed as the mean ± standard deviation of mg/g of product. *Determined by basic hydrolysis. PEAAs: Plant essential amino acids (Trp, Thr, Val, Lys, Leu, Met, His, Phe, and Ile). |
Regarding FAAs, practically all (18 AAs) were detected in both hydrolysates, while only 10 AAs were detected in the MTM (Ala, Asx, Glx, Gly, Leu, Lys, Phe, Ser, Tyr and Val).
Due to the amino acid, oligopeptide, peptide and carbohydrate contents, these hydrolysates could be rooting and defense protein enhancers for plants [16, 17]. Therefore, these potential activities will be studied in detail in future works, anticipating preliminary results in relation to the effect of seed priming on maize seed germination and root growth.
Effect Of Seed Priming With Mb-pphs On Germination And Growth
Seed priming was assessed at 0.00% (control), 0.01%, 0.05%, 0.10, 0.50%, 1, 2, 5 and 10% Mb-PPHs (single and sequential) overnight (approximately 12 h), which was found to be the best period for this study (data not shown). As shown in Table 3, all concentrations assayed, except 0.01%, showed a higher germination percentage than that of the control. Of the treatments, the 1% treatment had the highest percentage of germination (98.3 ± 2.3%). Seed priming with concentrations higher than 1% (2%, 5% and 10%) did not show any improvement in maize germination and even had a negative effect. Seed priming with all concentrations of Mb-PPH other than 0.01% significantly increased the length of the maize seed radicle, obtaining the highest value (4.3 ± 0.2 cm) for the 1% treatment; no increase was observed at higher concentrations.
Table 3
Effect of seed priming with Mb-PPHs on germination and radicle length.
[Mb-PPH] | % of seed germination | Radicle length (cm) |
0.01% | 81.3 ± 4.6 | 3.1 ± 0.2 |
0.05% | 89.0 ± 2.3 | 3.4 ± 0.2 |
0.10% | 93.3 ± 4.6 | 3.5 ± 0.1 |
0.50% | 94.7 ± 2.3 | 4.1 ± 0.2 |
1.00% | 98.3 ± 2.3 | 4.3 ± 0.2 |
2.00% | 97.3 ± 2.3 | 4.1 ± 0.4 |
5.00% | 94.7 ± 6.1 | 4.0 ± 0.3 |
10.00% | 92.0 ± 4.0 | 4.0 ± 0.3 |
Control | 81.3 ± 1.2 | 2.9 ± 0.2 |
Data are expressed as the mean ± standard deviation. |
For the hydroponic study, the concentration of Mb-PPH was reduced to the following concentrations: 0.05%, 0.10%, 0.50%, 1 and 2%. The results obtained for root/shoot length, fresh root/shoot weight and dry root/shoot weight are shown in Tables 4 and 5. These data show that in comparison to the control, seed priming with Mb-PPH had a positive effect on root/shoot length and this effect was statistically significant for concentrations > 0.05% for the three parameters measured (root/shoot length, fresh root/shoot weight and dry root/shoot weight).
Table 4
Effects of seed priming treatment with Mb-PPHs on the root length and fresh and dry weight of maize seed growth by the hydroponic method over 21 days
[Mb-PPH] | Root length (cm) | Root fresh weight (g/plant) | Root dry weight (mg/plant) |
0.05% | 17.22 ± 4.12 | 0.29 ± 0.12 | 17.11 ± 2.55 |
0.10% | 19.56 ± 4.15 | 0.33 ± 0.11 | 19.31 ± 3.58 |
0.50% | 20.98 ± 3.13* | 0.55 ± 0.16* | 28.21 ± 2.23** |
1.00% | 22.48 ± 3.31** | 0.67 ± 0.24** | 33.22 ± 3.71** |
2.00% | 20.93 ± 3.08* | 0.61 ± 0.21** | 28.53 ± 3.11** |
Control | 14.65 ± 3.12 | 0.23 ± 0.08 | 13.74 ± 2.16 |
Data are expressed as the mean ± standard deviation. *p < 0.05 **p < 0.01 |
Table 5
Effects of seed priming treatment with Mb-PPHs on the shoot length and fresh and dry weight of maize seed growth by the hydroponic method over 21 days
[Mb-PPH] | Shoot length (cm) | Shoot fresh weight (g/plant) | Shoot dry weight (mg/plant) |
0.05% | 29.07 ± 4.92 | 1.33 ± 0.33 | 78.89 ± 5.77 |
0.10% | 31.21 ± 3.98 | 1.34 ± 0.34 | 82.31 ± 4.34 |
0.50% | 33.22 ± 4.01 | 1.45 ± 0.37 | 88.39 ± 7.86 |
1.00% | 34.28 ± 4.23 | 1.65 ± 0,22 | 99.26 ± 6,86 |
2.00% | 33.87 ± 5.12 | 1.59 ± 0.42 | 92.35 ± 8.34 |
Control | 23.83 ± 2.44 | 0.92 ± 0.17 | 74.82 ± 3.86 |
Data are expressed as the mean ± standard deviation. |
Taking into account that the vigor index (VI) was a parameter that gave information about the effect of seed priming on the development of the plant, as it combined germination data with shoot and root length, its value was determined from the results shown in Tables 3, 4 and 5. The results are shown in Fig. 3, showing that seed priming with 1.00% Mb-PPH presented the highest increase in vigor index (VI); concentrations of 0.10%, 0.50% and 2.00% also yielded a significantly different VI value from that in the control, while priming with 0.05% had no effect.