Comparative assessment of amino acids composition in two types of marine fish silage

Degree of hydrolysis

Organic silages prepared from fat fish (FFS) and lean fish (LFS) had a characteristic tawny brown colour which was accompanied with a strong characteristic salty-fishy odour. At the end of 5 DoF, both FFS and LFS exhibited sluggish liquefaction which increased progressively concomitant with the DoF (Table S1). Liquefaction is an indicator of tissue hydrolysis due to the action of acid. During 35 DoF, the degree of hydrolysis (measured in terms of liquefaction volume) increased progressively with the DoF in both types of ensilages and was relatively higher in LFS compared to FFS on all sampled DoF (Table S1). In general, lipolysis supersedes the proteolysis in all major biochemical processes²³. A relatively higher degree of hydrolysis recorded in LFS may be attributed to the presence of a greater proportion of light muscles compared to dark muscles. Relatively greater susceptibility of light muscles to hydrolysis compared to dark muscles might be due to lower lipid content in the former²³.

Irrespective of fish type, the measured pH values in both types of ensilages (FFS and LFS) were similar (data not shown) and the values showed a progressive increase from 1.0 ± 0.03 (0 DoF) to 6.0 ± 0.03 (35 DoF). Such an increasing trend in pH with the advancement in DoF could be attributable to gradual solubilisation of boney material with the advancement fermentation time^24,25,26.

Changes in principal biochemical constituents

During the 35 DoF, the concentrations of total protein (TP) in both FFS and LFS progressively increased with the DoF and showed significant differences with the advancement of DoF (p < 0.05) (Table 1). Irrespective of ensilage type, the measured concentrations of TP (FFS, 97.82 ± 0.01 mg/mL; LFS, 62.96 ± 0.06 mg/mL) on 30 DoF were significantly higher (p < 0.05) than those concentrations recorded on other DoF (Table 1). In contrast, Ramasubburayan et al. (2013)²⁷ reported a slight decrement in TP content (~ 4%) on 30 DoF fish silage. Similarly, TP concentrations in FFS on 30 DoF were significantly higher than in LFS on corresponding DoF (p < 0.05).

Although the total carbohydrate (TC) content was observed to be significantly higher in FFS compared to LFS on all DoF (Table 1), the differences, however were insignificant (p > 0.05). The TC content was significantly higher (p < 0.05) in FFS on 30 DoF than estimated from LFS on the corresponding DoF. Furthermore, the significant differences in TC concentrations with DoF in both types of fish silages were not discernible. In addition to contributing as an energy source for the growth and metabolism of plants, the carbohydrates are known to shield plants against abiotic stressors such as osmotic imbalance and salinity²⁸. In the absence of previous reports, the concentrations of TC recorded in the present study would serve as baseline information.

The total lipid (TL) content in FFS was relatively higher compared to LFS on all DoF (Table 1). It has been reported that the TL content of fish silage vary in accordance with the fat content of the fish species²⁹. Compared to other DoF, the concentrations of TL in FFS and LFS were significantly higher on 25 DoF and 30 DoF, respectively (p < 0.05). TL content in LFS did not follow any particular trend during 35 DoF (Table 1). On the other hand, concentrations of the TL in FFS progressively increased from 110.10 ± 0.04 mg/mL (10 DoF) to 200.98 ± 0.03 mg/mL (30 DoF) and dropped to 159.25 ± 0.03 mg/mL (35 DoF). This is in agreement with previous studies by Palkar et al.³⁰. Such an increase in the TL concentrations with the progress of the DoF until 35 DoF, particularly in FFS may be due to considerable release of stored lipid during the liquefaction of the fish tissue³¹. Furthermore, a substantial decrease in TL content in FFS on 35 DoF could be attributable to oxidation of lipid due to presence of higher proportion of long chain polyunsaturated fatty acids compared to LFS³². By virtue of crucial role played by lipid components in plants in providing structural integrity to the cell membrane, cell signalling related to biotic as well as protection from abiotic stressors and in mediating a variety of cellular metabolic reactions³³, a detailed study on fatty acids profiles in fish silage thus becomes of paramount importance.

Comparative assessment of total amino acid composition

Although the content of total amino acids (TAA) was relatively higher in FFS than in LFS during 35 DoF however, their profiles were almost similar (Fig. 1). The TAA content in FFS peaked on 30 DoF (41.2 ± 0.033 mg/g) from a lowest value of 28.9 ± 0.033 mg/g (10 DoF) and then dropped to 33.6 ± 0.033 mg/g (35 DoF) (Fig. 1). The profiles of TAA in FFS on 30 DoF in terms of concentration (mg/g) followed the order: glutamic acid and leucine (6.0 ± 0.033) > Arginine (5.3 ± 0.033) > lysine (3.2 ± 0.033) > glycine (2.9 ± 0.033) > phenylalanine (2.6 ± 0.033) > serine (2.4 ± 0.033) > aspartic acid (2.3 ± 0.033) > alanine (2.1 ± 0.033) > histidine (1.8 ± 0.033) > valine (1.6 ± 0.033) > methionine (1.5 ± 0.033) > isoleucine (1.5 ± 0.033) > threonine (1.4 ± 0.033) > cysteine (0.946 ± 0.033).

On the other hand, content of TAA in LFS also followed a similar trend with the concentrations peaking on 30 DoF (35.8 ± 0.066 mg/g) from an initial concentration of 31.1 ± 0.066 mg/g (10 DoF) (Fig. 1). The concentrations of profiles (mg/g) of TAA in LFS on 30 DoF followed the order, leucine (5.90 ± 0.033) > glutamic acid (4.97 ± 0.033) > arginine (4.5 ± 0.033) > phenylalanine (3.38 ± 0.033) > aspartic acid (2.92 ± 0.033) > alanine (2.23 ± 0.033) > methionine (2.19 ± 0.033) > lysine (1.882 ± 0.033) > serine (1.881 ± 0.033) > tyrosine (1.410 ± 0.033) > glycine (1.219 ± 0.033) > threonine (0.953 ± 0.033) > valine (0.945 ± 0.033) > isoleucine (0.864 ± 0.033) > histidine (0.417 ± 0.033).

A comparative assessment of profiles of TAA in both FSS and LFS during all DoF revealed a similar pattern, albeit with obvious differences in the concentration of few amino acids (Fig. 1). It has been hypothesised that the occurrence of decarboxylation that follows transamination of amino acids as a consequence of increase in pH during fermentation is known to cause a decrement in the concentration of few amino acids, especially valine and isoleucine³⁴. During the present study, the concentrations of histidine, valine, isoleucine, glycine and lysine were significantly higher (p < 0.05) in FFS compared to LFS (Fig. 1). The plausible explanation for lower levels of isoleucine and valine in LFS may be due to transamination of these amino acids^34,35,36. The reduction in the concentration of few amino acids during the fermentation attributable to the differential chemical reactions between alpha and aldehyde groups of amino acids has been highlighted^31,34,35,36. In contrast, a striking similarity in the composition of TAAs in fish silages prepared from a variety of raw materials with or without addition of molasses has been reported^34,35,36. Therefore, it does appear that concentrations of few TAA during the fermentation are directly dependent on the type of raw material and fermentation period.

In the present study, amino acids such as cysteine, tryptophan asparagine and glutamine, were conspicuously absent in both types of ensilages on all DoF. The usage of mild organic acid (formic acid) for the preparation of fish silage resulting in the partial and or complete destruction of cysteine has been reported^37.  Absence of tryptophan in both FFS and LFS during the 35 DoF could directly be linked to its highly unstable nature in acidic medium, thus rendering it to become the first limiting amino acid in formic acid-based fish silages³⁸. The increase in pH concomitant with the DoF enhancing the rate of transamination and protease-based degradation of TAAs such as asparagine and glutamine might explain the absence of these amino acids³⁹.

Content and composition of free amino acids

During the 35 DoF, the content of FAA in both types of fish silages followed the trend of TAA (Fig. 2). In FFS, the levels of FAA increased from 4.37 ± 0.003 mg/g (10 DoF) and attained a highest concentration of 31.35 ± 0.003 mg/g on 30 DoF (Fig. 2). The concentrations (mg/g) of 18 FAAs in FFS on 30 DoF followed the order: arginine (5.90 ± 0.003) > leucine (3.09 ± 0.003) > glutamic acid (2.61 ± 0.003) > alanine (1.83 ± 0.003) > phenylalanine (1.79 ± 0.003) > cysteine (1.67 ± 0.003) > histidine (1.56 ± 0.003) > aspartic acid (1.54 ± 0.003) > serine (1.32 ± 0.003) > lysine (1.16 ± 0.003) > threonine (1.09 ± 0.003) > valine (1.07 ± 0.003) > isoleucine (1.06 ± 0.003) followed by methionine (0.93 ± 0.003) > tyrosine (0.92 ± 0.003) > tryptophan (0.72 ± 0.003) > asparagine (0.57 ± 0.003) > glutamine (0.15 ± 0.003).

Profiles of FAA in LFS also followed a similar trend that of FFS. Concentrations (mg/g) of a sum of 19 FAAs peaked on 30 DoF (18.26 ± 0.003) which was almost 6 times higher than the content recorded on 10 DoF (2.87 ± 0.003). In contrast, the highest recorded amino acid in LFS was leucine (2.65 ± 0.003) followed by arginine (2.45 ± 0.003), alanine (1.94 ± 0.003), glutamic acid (1.47 ± 0.003), phenylalanine (1.37 ± 0.003 m), tyrosine (1.11 ± 0.003), methionine (1.11 ± 0.003), aspartic acid (0.85 ± 0.003), tryptophan (0.74 ± 0.003), glycine (0.71 ± 0.003), cysteine (0.71 ± 0.003), lysine (0.58 ± 0.003), serine (0.57 ± 0.003), isoleucine (0.50 ± 0.003), valine (0.47 ± 0.003), threonine (0.46 ± 0.003), histidine (0.26 ± 0.003), asparagine (0.19 ± 0.003) and glutamine (0.10 ± 0.003).

Changes in amino acid composition during fermentation

The relative composition of FAAs in FFS and LFS varied in accordance with DoF, and the relationship was found to be highly significant (ANOVA, F value = 12.72; p < 0.00001). The amino acid profiles in LFS and FFS during 35 DoF were dominated by arginine, glycine, methionine, glutamic acid, phenylalanine, lysine and leucine (Fig. 2). At the end of 10 DoF, the concentrations of dominant amino acids such as arginine, glutamic acid, glycine, valine and leucine were significantly higher in FFS than in LFS (Mann–Whitney test, p < 0.05). Concentrations of tyrosine, lysine, glycine, glutamic acid were markedly higher in FFS than in LFS at the end of 15 DoF (Mann–Whitney test, p < 0.05). Furthermore, the concentrations of asparagine, histidine, threonine, valine, isoleucine, serine, glycine, cysteine, lysine, glutamic acid, arginine were significantly higher in FFS as compared to LFS at the end of 25 DoF and 30 DoF, respectively (Mann–Whitney test; p < 0.05) (Fig. 2). At the end of 35 DoF, the concentrations of amino acids such as asparagine, histidine, isoleucine, valine, cysteine, serine, lysine and arginine were significantly higher in FFS compared to LFS.

Results of the one-way analysis of similarities (ANOSIM) test and the non-metric multidimensional scaling (nMDS) plot (Fig. 3) indicated that the differences in the amino acid composition in two types of ensilages (FFS and LFS) during the 35 DoF were highly significant (Fig. 3; nMDS, stress 0.032, one-way ANOSIM R = 0.9881; p < 0.0001). The dissimilarity in amino acid composition between two types of ensilages (FFS and LFS) at the end of 10 DoF was mainly contributed by leucine, arginine, glycine, glutamic acid and histidine (Table 2). At the end of 15 DoF, tyrosine, tryptophan, leucine, cysteine, glycine and glutamic acid were majorly responsible for the dissimilarity observed between the two types of ensilages (FFS and LFS). A significant dissimilarity in amino acid composition between two types of ensilages (FFS and LFS) at the end of 25 DoF and 30 DoF is mainly attributable to arginine, glycine, histidine, glutamic acid. At end of 35 DoF, histidine, arginine, cysteine, lysine, glutamic acid were the main amino acids that contributed to the dissimilarity in amino acid composition between two types of fish ensilage (FFS and LFS) (Table 2). This spatial segregation suggested that fermentation period was majorly responsible for the dissimilarity in altering the amino acid profiles between two types of fish ensilages (FFS and LFS). Overall, the analyses suggest that fermentation period of 25‒30 days exerted a profound effect on the composition of amino acids in both types of ensilage compared to other fermentation periods (p < 0.05) (Table 2).

The observed decline in TAA and FAA contents beyond 30 DoF could possibly be due to solubilisation of the bony materials which results in the production of amines as a consequence of an action by formic acid. Amines being alkaline, might have resulted in a drastic increase in pH of the silage with the progress of the DoF, eventually leading to lower yields of amino acids^40,41. A drastic decrease in the concentration of amino acids with the progress of fermentation due to the formation of major biogenic amines (cadaverine, tyramine and ornithine) has been reported⁴². Furthermore, other amines such as histamine (from histidine), 2-phenylethylamine and tyramine (from phenylalnine and tyrosine decarboxylation, respectively), tryptamine from tryptophan, putrescine obtained from decarboxylation of arginine and cadaverine from lysine have also been reported to be responsible in decreasing the concentration of amino acids in fish silages^{43,44,45,46,47}.

The transformation of FAAs into biogenic amines via decarboxylation, oxidative deamination or by other undefined pathways during the extended fermentation has been reported to be the major factor responsible for the reduction in the content and altering the composition of amino acids⁴⁸. Örlygsson (1995)⁴⁹ attributed the reduction in the levels of alanine as a consequence of the Stickland reaction, wherein alanine gets converted to acetic acid upon reaction with glycine and water. By-products of this reaction (ammonia and carbon dioxide) may render further increase in the pH and thus advancing the degradation of FAA to biogenic amines and eventually resulting in decreased FAA levels.

Amino acids as plant growth promoters

Amongst all FAAs, the levels arginine, leucine and glutamic acid were highest during 25‒30 DoF, particularly in FFS (Fig. 2). In addition to its role as nitrogen storage and transport in many plants, arginine also promotes the synthesis of phytohormones that are responsible for fruiting and flowering of the plants⁵⁰. Amino acids such as glycine and glutamic acid help in the development of plant tissue and synthesis of chlorophyll. Acting as an osmotic agent in the cytoplasm, L-glutamic acid aids in the opening of stomata. Similarly, leucine is actively involved in plant defence against environmental stressor and also in protecting the reproductive tissues of plants from pathogens⁵¹. The enzymatic locus in plants involved in protein synthesis can only recognise the L-form of amino acids⁵². L-form of amino acids (methionine, tryptophan and proline) acting as precursors of phytohormones (ethylene, espermine and espermidine)—involved in inhibition of senescence^53,54,55, energy generation and plants’ defence against various environmental stresses^18,19. Another phytohormone, auxin involved in promoting cell elongation, phototropism, root and shoot elongation⁵⁶ and pollen fertility protection of plant from water stresses is produced from tryptophan⁵⁷ The enzymatic locus involved in protein synthesis in plants is capable of recognising the L-form of amino acids⁵². The fish silages produced during this study were rich in L-amino acids, especially the free amino acids could be an inexpensive and excellent alternative as a plant growth enhancer or as an organic fertiliser for boosting crop yields.

Untreated fishery waste has potential to trigger eutrophication consequent to its release into the coastal marine waters. Transforming this biowaste into a useful product could minimise both environmental and disposal problems. An application of organic foliar formulations derived from fish silage on okra (Abelmoschus esculentus) and red amaranth (Amaranthus tricolor) resulting in enhanced plant growth, leaf pigmentation and greater yields in comparison to that of the chemical fertilizer has been demonstrated⁸. Furthermore, effectiveness of liquid fish silage (concentration, of 5–10%) as an organic fertiliser for the growth of pakchoy (Brassica rapa L. subsp. chinensis) has been documented⁹. Results of the present study points out that fish biowaste can be transformed into an eco-friendly supplementary plant growth enhancer due to the high yield of free amino acids. A comparative assessment of amino acids (both total and free) in silages produced from two different fish types based on the fat content with different days of fermentation forms the novelty of the present study.

Source: Ecology - nature.com