GC-MS analysis
Table 1 summarizes the major chemical components obtained by GC-MS of the Persicaria hydropiper leaf paste: Phytol (55.327%), 9,12-Octadecadienoic acid (8.669%), Phthalate (diethyl) (7.623%), Di-n-octyl phthalate (5.182%), Limonene (4.789%), n-Hexadecanoic acid (4.094%) and 9-Octadecenamide (3.877%). The presence of phytol is supreme with a maximum concentration of 55.327% which is found to have both antimicrobial and antioxidant properties (Costa et al. 2012). Agoramoorthy et al. (2007) observed that both 9,12-Octadecadienoic acid and n-Hexadecanoic acid creates unfavorable conditions for the growth of Gram-negative bacteria. Although Phthalate (diethyl) and Di-n-octyl phthalate are considered carcinogenic pollutants, these compounds did demonstrate antifungal and antibacterial activity against Aspergillus flavus, Bacillus subtilis, Escherichia coli, and Staphylococcus aureus (Ortiz and Sansinenea, 2018). Limonene is found to have antimicrobial properties and is used as a preservative (Hąc-Wydro et al. 2017). Other chemical compounds like Iso E Super (1.108%), Octanal, 2-phenylmethylene (0.972%), 9,9-Dimethoxybicyclo nona-2,4-dione (0.628%), Phthalic acid, butyl undecyl ester (1.1916%), and Diisooctyl phthalate (1.027%) are also present in Persicaria hydropiper leaf paste. Due to having these phytochemicals in the Persicaria hydropiper leaf, it could be used to preserve raw animal skin.
Table 1
Chemical composition of methanol extracted Persicaria hydropiper leaves paste
No.
|
Name of the compound
|
Concentration (%)
|
Molecular formula
|
1
|
Limonene
|
4.789
|
C10H16
|
2
|
Phthalate (diethyl)
|
7.623
|
C12H14O4
|
3
|
Iso E Super
|
1.108
|
C16H26O
|
4
|
Octanal, 2-phenylmethylene
|
0.972
|
C15H20O
|
5
|
n-Hexadecanoic acid
|
4.094
|
C16H32O2
|
6
|
Phytol
|
55.327
|
C20H40O
|
7
|
9,12-Octadecadienoic acid
|
8.669
|
C18H32O2
|
8
|
9,9-Dimethoxybicyclo nona-2,4-dione
|
0.628
|
C11H16O4
|
9
|
Phthalic acid, butyl undecyl ester
|
1.916
|
C23H36O4
|
10
|
9-Octadecenamide
|
3.877
|
C18H35NO
|
11
|
Diisooctyl phthalate
|
1.027
|
C24H38O4
|
12
|
Di-n-octyl phthalate
|
5.182
|
C24H38O4
|
FTIR analysis
The methanol-extracted Persicaria hydropiper leaf showed several peaks at a different wavelength (cm− 1) as illustrated in Fig. 3. The peaks indicate the presence of functional groups from different compounds. Stretching and bending of O-H functional group were observed at 2918, 3356, and 1382 cm− 1 wavelengths, respectively, strongly suggesting there are weak and strong intermolecular bonds of an alcohol group. A peak at 844, 1045 cm− 1 interacts strongly with the C-Cl group from halo compound and CO-O-CO stretching of anhydride. The presence of C-O stretching was also observed at 1134, 1292 cm− 1 and these numbers reveal strong aliphatic ether, aromatic ester groups. Stretching of S = O group at 1045, 1382 cm− 1 introduces the sulfoxide and sulfone group. C-N, C = C stretching of aromatic amine and conjugated alkene at 1292 and 1620 cm− 1 represent strong and medium interaction, respectively. The functional group N-H bending and N-H stretching was found at 1620, and 2918, 3356 cm− 1 which indicating the amine, an amine salt, aliphatic primary amine groups. N-H stretching was also reported by Balasubramanian (2019) during the preservation of skin using sodium polyacrylate. C-H bending and C-H stretching at 1826, 2918 cm− 1 demonstrate weak aromatic compound and medium alkane compound.
Optimization of preserving agent
Different percentages of Persicaria hydropiper leaf paste with or without common salt was applied (Table 2) to find the ideal amount of leaf paste that could preserve goat skins. The efficacy of leaf paste was assessed by both visual observation and with physically hand feel. Visual observation reveals that Persicaria hydropiper leaf paste could preserve goatskin in any condition without odor creation and no hair slip (Fig. 2). However, physical feeling was different according to the leaf paste percentage. Preservation of goatskin with only 10% plant paste makes the skin hard which could create difficulties in further processing. It should be noted that 10% plant paste with 12% salt produces very soft and flexible leather after 14 days which could enhance the damaging possibilities if it is processed in a drum. It seems that preservation with 10% plant paste along with 8% salt followed a similar trend to conventional salt curing and after 14 days of preservation the skins became flexible. It means they could be easily processed in a drum and there will be no deterioration (Fig. 2). Hence, 10% plant paste + 8% salt was chosen as the optimized combination for experimental purposes.
Table 2
Leaf paste optimized for the preservation method (14 days)
No.
|
% of curing agents
|
Hair slip
|
Odor
|
Physical feel
|
01
|
10% plant paste
|
No
|
No
|
Hard
|
02
|
10% plant paste + 4% salt
|
No
|
No
|
Medium hard
|
03
|
10% plant paste + 8% salt
|
No
|
No
|
Flexible
|
04
|
10% plant paste + 12% salt
|
No
|
No
|
Soft and flexible
|
Bacterial load
Bacterial count reveals the number of bacteria present in the preserved skins which helps in determining the efficacy of the new preservation method. The identified bacterial colonies that exist in the control and experimental skins during different time intervals of the short preservation period are presented in Fig. 4. In the early days of preservation, the bacterial load rose for both experimental and control samples. A possible explanation for this is that there might be certain types of bacteria present which can tolerate both the leaf paste and common salts for a certain period of time. By this time salt and leaf paste tolerant bacteria increased the number of bacteria in the colony through binary fission (Masoodi et al. 2021). Although the bacterial count rose for both methods up to the 7th day, results were comparable and in some cases experimental samples confirmed better results. From the 14th day, the bacterial count gradually declined which indicates that salt and leaf paste tolerant bacteria had gone astray. At the end of the 30th day of the preservation period the bacteria load was higher in the control samples compared to the experimental samples. This means that Persicaria hydropiper leaf paste showed better results when compared to conventional preservation methods. This phenomenon was observed due to the presence of an antimicrobial agent in the Persicaria hydropiper leaf paste (Prota et al. 2014).
TKN content
The value of the TKN content is serving as an indicator of the breakdown of the polypeptide chain of the collagen matrix. Due to bacterial degradation, the polypeptide chain of the goatskin is the breakdown by which components with ammonia and the amino group increase, and this generates a bad odor and promotes hair slip defects in the preserved skin (Vankar and Dwivedi, 2009b). Figure 5 depicts the extractable nitrogen content during preservation of the experimental and control skins. The figure shows that the value of the TKN for both the experimental and control samples increased during the initial 4 days of preservation. From the 5 day to 14 day the extractable nitrogen content value fell drastically which indicates that preservation efficiency was significantly increased for both samples. On the 21st day for the experimental and control samples, extractable nitrogen content was 3.12 g/kg and 2.87 g/kg, respectively. From the 21st day to the last day of preservation (30th day) the values remained almost unchanged for both samples. Throughout the preservation period the experimental sample showed slightly higher nitrogen value compared to the control sample, but there was no sign of hair slip and odor. Consequently, it can be stated that using 10% Persicaria hydropiper leaf paste with 8% salt goat skin makes preservation for 30 days possible.
Moisture content
The percentage of moisture content is considered one of the major parameters when assessing the preservation efficiency. Moisture content in the raw skin is a major parameter for assessment of the curing competency. Raw skin contains 65–70% moisture which is a favorable state for bacterial survival. One of the main aims is to reduce the moisture content to a significant level. The moisture content (%) of the experimental and control goatskins during time intervals of preservation is illustrated in Fig. 6. It is observed from Fig. 6 that for both samples in the first 7 days the moisture content fell remarkably. On 14th, 21st, and 30th days the moisture content remained static for both samples. For the experimental samples, moisture content decreased from 62.13–44.7%, which indicates that Persicaria hydropiper leaf paste has dehydrating properties. Sivabalan and Jayanthi (2009) have used Cassia fistula for the preservation of skins and observed a similar outcome. At the end of the 30 days’ preservation, moisture content in both samples was 44.7% (experimental) and 42.4% (control), which are far below the critical moisture content (50%) (Kanagaraj et al. 2014). Despite larger moisture content in the experimental samples compared to the control sample, there was no symptom of putrefaction. This reveals the effectiveness of the preservation methods.
Hydrothermal stability
The shrinkage temperature value acts as an index to determine the hydrothermal stability of the skins/hides. Shrinkage temperature indicates the structural stability of the collagen matrix which fluctuates due to the disintegration of stable linkages (Babu et al. 2012, Kannan et al. 2009). Natural plant extracts can alter the structural properties of collagen matrix negatively or positively which depends on the kinds of reaction involved (Vijayalakshmi et al. 2009). Shrinkage temperature value decreases if the hides/skins undergo detritions and increases if the plant extracts can give some tanning properties. The shrinkage temperature value during 30 days’ preservation of the skin using experimental curing agent (10% plant paste + 8% salt) and conventional curing agent (50% NaCl) is depicted in Fig. 7. It was observed in this figure that throughout the preservation period, no crucial change in the shrinkage temperature of both samples was evident. For experimental and control samples, the shrinkage temperature values rose slightly from 65.41°C to 65.43°C and 65.25°C to 65.37°C, respectively, after 30 days’ preservation. This slight increase in the shrinkage temperature value suggests that Persicaria hydropiper leaf paste positively alters the hydrothermal stability of the collagen matrix and can be used to preserve the skin protein.
Estimation of pollution load
Both the experimental and control preserved goatskin samples were subjected to a soaking operation. The main purpose here was to restore the moisture content in the goatskins which was lost during the preservation period. Apart from water different detergents, enzymes, and fat remover are used as chemicals in this operation (Sawalha et al. 2019). The pollution loads emitted during the soaking operation for the experimental and control samples are listed in Table 3. The soaking wastewater of the control sample contains a high level of chloride, TDS, BOD and COD compared to the experimental sample. The main pollution loads of the tannery wastewater are chloride content and TDS which diminished to 53.56% and 53.13%, respectively, because the common salt was replaced by 10% Persicaria hydropiper leaf paste with 8% salt. It is observed that due to high Cl− and TDS wastewater, treatment cost increases and it is estimated that for every 4 kg of wastewater the approximate extra expense in the treatment process is US$1 (Balasubramanian et al. 2019). Other pollution parameters like BOD and COD revealed a significant reduction to 50.92% and 47.10%, respectively, in the experimental soaking wastewater when compared to the control. The results demonstrate that the developed Persicaria hydropiper leaf paste-based preservation method not only retains the goatskin but is also environmentally friendly.
Table 3
Reduction of pollution load during soaking operation
Parameters
|
Experimental
|
Control
|
Unit
|
Reduction (%)
|
Cl−
|
8965 ± 23
|
19303 ± 156
|
mg/L
|
53.56
|
TDS
|
21293 ± 61
|
45431 ± 72
|
mg/L
|
53.13
|
BOD
|
586 ± 31
|
1194 ± 23
|
mg/L
|
50.92
|
COD
|
2987 ± 32
|
5647 ± 59
|
mg/L
|
47.10
|
Physical properties of processed leather
The type of preservation methods applied in the preservation process has a profound effect on the physical properties of the final leather product. The physical quality of the leather is assessed by deciding how it is used in the final product’s preparation. The value of the leather mainly depends on the physical characteristics of the end product (Kim et al. 2018). The physical properties of the processed upper crust leather of the experimental and control samples are reported in Table 4. Judging by the results, it was observed that physical properties viz. tensile strength, elongation at break, and grain crack load of experimental skins preserved with 10% Persicaria hydropiper leaf paste along with 8% salt, showed better physical properties compared to the control samples. Tensile strength of the finished leather is considered a prime feature and it plays a significant role in how well manufactured products function (Sudha et al. 2009). The tensile strength of the experimental and control finished leather samples were 218.7 kg/cm2 and 210.6 kg/cm2, respectively. Though experimentally processed leather indicates lower grain crack distension value compared to the control sample, it fulfils the requirements of the standard. The physical properties of the produced leather demonstrate that Persicaria hydropiper leaf paste has a positive impact on the mechanical and structural properties of skin protein.
Table 4
Physical properties of the processed experimental and control leather
Parameters
|
Experimental
|
Control
|
Required (Kanagaraj et al. 2001)
|
Tensile strength (kg/cm2)
|
218.7
|
210.6
|
200
|
Elongation at break (%)
|
44.03
|
43.5
|
40–65
|
Bursting strength:
Distension, grain crack (mm) Load, grain crack (kg)
|
7.8
25.3
|
8.1
24.8
|
7
20
|
Physical properties of processed leather
Fiber arrangement plays an important role when assessing the quality of the final leather since the physical strength of final leather depends on fiber orientation (Covington 2011). The fiber composition of the final leather produced from the experimental and control samples are assessed through SEM images which are depicted in Fig. 8. These images showed that the fiber structure of the experimental sample is very similar to the control sample, suggesting that Persicaria hydropiper leaf paste played no part in disordering the fiber orientation. However, any destruction of the fiber structure was not observed in the experimental sample, meaning that the recommended curing agent could be used in the preservation process.
Pilot trial
Moisture content of pilot-scale trial samples
Figure 9 illustrates the moisture content (%) during the 30-day preservation period of the pilot trial samples (curing agent: 10% Persicaria hydropiper leaf paste + 8% salt). The percentage of moisture content for all five pilot experiments showed a similar kind of situation. Up to the 7th day, moisture content was marginally reduced, while from the 14th day to 30th day the moisture content virtually remained in a steady-state condition in all five experiments. During this 30-day preservation period no damage to the skins like bad odor, or hair slip was observed.
Hydrothermal stability of pilot trial samples
Figure 10 presents the hydrothermal stability during the 30 days preservation period of the pilot-scale trial samples (curing agent: 10% Persicaria hydropiper leaf paste + 8% salt). Throughout the preservation period, no substantial change in the shrinkage temperature among the pilot-scale trial samples occurred. For instance, shrinkage temperatures of the fresh samples were 65.75°C, 66.32°C, 66.35°C, 65.98°C, and 65.65°C. These fell slightly to 64.68°C, 64.94°C, 64.98°C, 64.88°C, and 64.79°C, respectively, after 30 days of preservation for the pilot trial samples P1, P2, P3, P4, and P5.
Physical properties of pilot trial samples
Table 5 summarizes the physical properties of the final leather processed from the pilot trial samples. All the physical characteristics of the five trial samples fulfil the standard requirements. The tensile strength value ranged from 207 kg/cm2 to 219 kg/cm2 which is above the standard value. Tensile strength, elongation at break, and grain crack load were also in the standard range for all five pilot trial samples.
Table 5
Physical properties of the processed pilot-scale experimental leather
Parameters
|
P1
|
P2
|
P3
|
P4
|
P5
|
Kanagaraj et al. 2001
|
Tensile strength (kg/cm2)
|
209
|
213
|
219
|
207
|
215
|
200
|
Elongation at break (%)
|
45
|
43
|
46
|
44
|
42
|
40–65
|
Bursting strength:
|
|
|
|
|
|
|
Distension at grain crack (mm)
|
7.7
|
8.1
|
7.8
|
7.5
|
7.2
|
7
|