Estimation of Free and Immobilized Protease Efficiency in Inhibiting of Food Spoilage Bacteria

 
The effect of proteolytic enzymes as antimicrobial alone or synergy with plant-derived compounds or antibiotics was reported by several authors, such as effective against Listeria monocytogenes, Bacillus sp, Pseudomonas sp, Staphylococcus simulans and Escherichia coli ATCC 25922 (Salinas Ibanez​ et al., 2021), Bacillus subtilis and Staphylococcus aureus (Indarmawan et al., 2016); Acinetobacter sp. KC119137.1 and S. aureus NCIM 5021 (Manohar et al., 2015). Currently, immobilized enzymes are considered a promising technology in enzymology that aims to be less wasteful in using free enzymes due to the relatively high costs of production (Guler et al., 2020). The benefit of immobilization is represented by the reuse of enzymes many times without any loss in activity and the ability to complete the required reaction (Alsoufi, 2021). Therefore, protease immobilizations are studied using different ways and carriers such as covalent binding, encapsulation, adsorption and entrapment, as immobilization with polylactic acid an eco-friendly (Calzoni et al., 2021), Celite 545 (de Oliveira et al., 2021), Materium 540 (MAT 540) (de Oliveira et al., 2020), as-spun nanofibrils (Guler et al., 2020), Ca-alginate (Qamar et al., 2020), Eupergit CM (Aslan et al., 2018), cellulose monoacetate/chitosan (Demirkan et al., 2018), cysteine-functionalized MNPs (Masi et al., 2017), hollow core-mesoporous shell silica nanospheres (Ibrahim et al., 2016) and sodium alginate beads (Geethanjali and Subash 2013). Thus, this study aims to partial purification of protease from Prickly lettuce leaves using ammonium sulfate, immobilization bentonite, characterization and application as antimicrobial against the growth of some food spoilage bacteria as a lone or synergistic with Welsh onion Allium fistulosum L. 

Place of work
 
This study was conducted in the laboratories of the Department of Product Evaluation and Service Performance at the Market Research and Consumer Protection Center, University of Baghdad, Baghdad, Iraq and the Department of Science, College of Basic Education at Mustansiriyah University, Baghdad, Iraq.
 
Research period
 
The study was conducted from January 10, 2023, to December 10, 2023.
 
Crude protease preparation
 
Protease was extracted from leaves of Prickly lettuce (Lactuca serriola L.) using acetone powder. (NH4)2SO4 at 40-80% saturation was used for the concentration of crude extract. The precipitate was dialyzed against deionized distilled water through (Dialysis membrane Spectra/Por® 7 MWCO 10000 Da) for 24 h at 4°C, then concentrated by freeze dryer (Al-Soufi, 2013).
 
Protease proteolytic activity
 
The proteolytic activity of free protease (FP) and immobilized protease (IP) was estimated according to the method of Robinson (1975) by mixing 0.3 mL of FP and 0.3 g of IP with 0.7 mL of 0.1M sodium phosphate buffer activation solution pH (7.2) containing 0.1 M of cysteine and 0.001 M of EDTA, then heated for 5 min at 35°C. Then, 1 mL 0.1 M sodium phosphate buffer solution pH (7.2) containing 1% casein was heated for 5 min at 35°C. Then, the reaction was stopped by adding 3 mL of 5% Cl3CCO2H and filtered after 30 min through filter paper (Whatman No. 3), the absorbance of filtrates was measured at 280 nm. Control was prepared by adding Cl3CCO2H at zero time of reaction. One unit of the enzyme is defined as the protease activity that changed by 1.0 unit of absorbance min-1 under the experiment conditions.
 
Estimation of protein
 
Protein was estimated using Bradford’s method (1976).
 
Activation of clay
 
Clay (bentonite) was obtained from Baghdad’s local markets. The activation assay was done by adding 10% 3-APTES solution in acetone to clay (v w^-1) and stirring at 25°C for 1 h. After that, filtrating and washing using acetone consecutively three times and then drying by oven at 80°C; the dried bentonite was treated with 10% aqueous glutaraldehyde solution (w v^-1) for 60 min. This was followed by filtrating and washing using 0.1 mM of sodium phosphate buffer solution pH (7.2) consecutively three times, then stored in the same buffer solution at 4°C until using in the immobilization of enzyme according to the method of Alsoufi (2021). The pH modification for the buffer solution was (7.2) instead of (6) to suit the enzyme activity and experimental conditions.
 
Immobilization of protease
 
Protease was immobilized by mixing 10 mL of the enzyme (10 mg mL^-1) with 10 g of activated clay and slowly stirring (avoid foaming) for 2 h at 4°C, following that, adding 1% of glutaraldehyde in 0.1 mM sodium phosphate buffer solution pH (7.2) and stirrer for 1h at 4°C. The storage was in the refrigerator until use, according to Alsoufi (2019), with the modification of buffer solution to be 0.1 mM sodium phosphate buffer solution, pH (7.2) instead of 50 mM Tris-HCl buffer solution, pH (7).
 
Immobilization efficiency
 
The immobilization efficiency (IE) of protease was calculated using Eq. (1) by Alsoufi (2021).
   
 Where IM= Specific activity (U mg^-1) of immobilized protease; FA= Specific activity (U mg^-1) of free protease.
 
Optimum activity and stability of pH and temperature
 
The effect of optimum relative activity (%) for pH and temperature on the FP and IP was assayed using 1% casein in 50mM buffers solutions pH (5-10), sodium acetate (5-6.5), sodium phosphate (7-7.5), Tris-HCl (8-9.5) and Glycine-NaOH (10) for pH and (30-80°C) for temperature (Geethanjali and Subash, 2013). In contrast, the relative stability (%) for pH and temperature was estimated after incubating the enzyme for 30 min (Al-Soufi, 2013). 
 
Storage and reuse
 
The effect of storage and reuse of protease was estimated according to the method of Alsoufi (2019) by storing FP and IP in optimum pH of stability for 30 d at 4°C and studying the reuse of IP for 30 continued reuse.
 
Extraction and detection of phytochemicals from welsh onion
 
The phytochemicals (glycosides, alkaloids, terpenes, saponins, tannins, coumarins and flavonoids) of Welsh onion Allium fistulosum L. (WO) were extracted and detected according to the method of Alsoufi and Aziz (2022) using hot aqueous extraction through adding 50 g of chopped onion to 100 mL of deionized distilled water and put the mixture in the water bath at 100°C for 1 h. Then, it was left to cool at room temperature and filtered through filter paper (Whatman No.1). The filtrate was used to detect phytochemicals, then dried by lifolyzer and kept at 4°C until use.
 
Antimicrobial activity
 
Antimicrobial activity of FP and IP were studied against S. aureus and E. coli that were obtained from the Department of Science, College of Basic Education, Mustansiriyah University, Baghdad, Iraq, through activation of bacterial strains by incubation under sterilized conditions in 20 mL of nutrient broth at for 24 h at 37°C. Then, spread 100 µL on nutrient agar and incubate for 24 h at 37°C. An isolated colony from each strain was placed in physiology serum (deionized distilled water and 0.9% sodium chloride) until they reached 0.5 turbidity of McFarland (106 CFU mL^-1). The antimicrobial activity was estimated by adding 1mL of 25, 50, 75 and 100 mg of FP, IP, or WO to 40 mL of nutrient broth. Adding 1 mL of bacterial culture medium to each type of them and incubating the mixture at 37°C for 24 h. Absorbance was measured by UV-Vis spectrophotometer at 610 nm in 2 h time intervals for FP and IP according to the method by Bayazidi et al. (2018). After 24 h for WO and synergistic mix, the synergistic antimicrobial activity was distributed (Table 1) according to the method of Alsoufi and Aziz (2022).
The changes in absorbance (%) of all treatments were calculated using Eq. (2) (Alsoufi and Aziz, 2022). 
   
Where:
a= Absorbency of control after incubation at 37°C for 24 h. b= Absorbency of treatment at zero time of incubation at 37°C.
c= Absorbency of treatment after incubation at 37°C for 24 h.
 
Statistical analysis
 
SAS (Statistical Analysis System) program version 9.6th ed., was used to detect the difference factors in the experiments of this study using LSD (Least significant difference) test (Analysis of Variation-ANOVA) to compare between means in results (SAS, 2018).

Immobilization efficiency
 
The efficiency of protease immobilization was 78% of the original amount of enzyme used. In this context, the immobilization efficiency of protease with different sources was 50-55% with as-spun nanofibrils (Guler et al., 2020), 68% on polylactic acid an eco-friendly (Calzoni et al., 2021), 45% with sodium alginate beads (Geethanjali and Subash, 2013), 71% with Eupergit CM (Aslan et al., 2018), 83% on cellulose monoacetate/chitosan (Demirkan et al., 2018) and 75.6% onto hollow core-mesoporous shell silica nanospheres (Ibrahim et al., 2016).

The main aim of immobilization was to get an enzyme kit with high activity, stability, reusable and low cost (Alsoufi, 2021), which was achieved by binding the highest amount of enzyme with inert materials unescorted by any losses of activity and stability such as polymers and inorganic materials (Alsoufi, 2019) that provided non-effect of catalytic property in the active site due the binding with immobilization materials (Masi et al., 2017 ).
 
Effect of optimum pH and temperature
 
The optimum activity pH for FP and IP was (8.5) (Fig 1). The stability for 30 min of FP (the losses less than 10%) was at the pH range of (7.5-9) with loss of 21 and 14% of its original activity at pH (5 and 10), respectively. The stable of IP was at the pH range (5-10) with a loss of 15 and 7% of its original activity at pH (5 and 10), respectively (Fig 2). The optimum activity temperature for FP and IP was 60°C (Fig 3). The FP was stable for 30 min at 55°C, while IP was stable for 30 min at 60°C (without any losses of its activity) (Fig 4).



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Many research studies refer to variances in the optimum temperature of protease from different sources. On this basis, Calzoni et al. (2021) observed that the optimum conditions of IP on eco-friendly polylactic acid were at a temperature of 55°C and a pH of (8.6). At the same time, de Oliveira et al. (2021) found that the highest activity of temperatures and pH for IP from Rhodotorula oryzicola on Celite 545 were 60°C and (6.5), respectively. de Oliveira et al. (2020) noticed the same results for IP from Moorella speciosa on Materium 540 (MAT 540). Qamar et al. (2020) showed that the optimum pH of protease from Bacillus brevis was (8 and 10) for FP and IP on Ca-alginate, respectively and the optimum temperature was at 45 and 65°C, respectively. Aslan et al. (2018) noted that the optimum pH and temperature for IP from Bacillus licheniformis on Eupergit CM were (7-8) and 70°C, respectively. Similarly, Masi et al. (2017) observed that the optimum pH of FP and IP from (P. aeruginosa and Enterococcus hirae) with cysteine-functionalized MNPs was (9) and the optimum temperature was 60°C for both types of enzyme, with recorded more stability to IP than FP during incubation for 5 h at 60°C and pH (9). Geethanjali and Subash (2013) reported that the optimum pH and temperature of protease from Labeo rohita visceral were (9) and 60°C, respectively, for FP and IP with sodium alginate beads, noting that the IP was a high activity of 98% even at 50°C compared to FP.

The pH and temperature values of protease were varied relying on the source, type, pI, molecular weight and the content of carbohydrate and substrate (Al-Soufi, 2013), each enzyme has an optimum value of these parameters and the enzyme can incur ionizations and acquire charges due pH of reaction solutions. Temperature may affect the amino acid bonds, leading to a loss of enzyme activity (de Oliveira​ et al., 2021). Generally, immobilization increases the stability of the enzyme towards pH and temperature, which were used in the reaction due to strengthening the enzyme’s three-dimensional structure via covalent bonds (Aslan et al., 2018; Ibrahim et al., 2016).
 
Storage and reuse
 
The IP did not lose activity for 15 d while losing 57% over 30 d of storage at 4°C; the FP was stable for 4 d while losing all activity over 17 d at 4°C (Fig 5). The IP activity was stable at up to 15 reuses, losing 24% of its initial activity after 30 reuses (Fig 6).





The stability of immobilized enzymes through reuse and storage is an essential requirement for the success of its applications, which reduces the price and increases suitability for commercial applications (Alsoufi and Aziz, 2022; Geethanjali and Subash, 2013). The total enzyme activity of IP on polylactic acid was eco-friendly (4:1) to FP after 100 reuses (Calzoni et al., 2021). The IP from R. oryzicola on Celite 545 retained up to 73% of activity after 15 reuses (de Oliveira​ et al.,  2021). de Oliveira et al. (2020) found that IP from M. speciosa on MAT540 retained 60% of the initial catalytic activity after 15 reuses in this context. Qamar et al. (2020) showed that the IP from Bacillus brevis on Ca-alginate retained more than 80% of original activity up to 8 reuses. Aslan et al. (2018) observed that IP from Bacillus licheniformis on Eupergit CM has not lost activity through 20 reuses. The IP from Bacillus sp. on hollow core-mesoporous shell silica nanospheres retained up to 55.6% of activity after 12 reused (Ibrahim et al., 2016). Geethanjali and Subash (2013) noted that IP from Labeo rohita with sodium alginate beads was more stable at 4°C compared to 25°C during storage for 6 d with near retention of 90% and 20% from original activity, respectively. The losing activity of immobilized enzymes during reuse constitutes a central problem affecting their applications due to the leakage of an enzyme from carrier material used for immobilization. The effect of mechanical forces for centrifugation after all use and cover catalytic site in the enzyme’s active site by accumulating substrate oxidation products (Qamar et al., 2020; Alsoufi, 2019; Ibrahim et al., 2016). Therefore, this parameter was used to choose the appropriate application and ensure marketing success (Alsoufi, 2021). Immobilization generally improves enzyme stability, while losing protease activity is attributed to autolysis at a high temperature (Geethanjali and Subash, 2013).
 
Phytochemicals of welsh onion
 
The detection of phytochemical substances found in Welsh onion refers to the hot aqueous extract of contain (glycosides, alkaloids, terpenes, saponins, tannins and flavonoids).

Welsh onion has many pharmacological activities (antimicrobial, antifungal, anti-inflammatory, antioxidant, anticancer) and traditional uses as an herbal medicine (Sung et al., 2018) due to the phytochemical substances found in the plant, such as flavonoids, terpenes, glycosides, saponins, alkaloids and tannins (Alsoufi and Aziz, 2022).
 
Application
 
The result showed that the antimicrobial effect of IP was greater than that of FP on S. aureus and E. coli. The optimum concentration of enzyme used was 100mg over incubation of each type of bacteria for 24 h at 37°C (Fig 7; 8). The antimicrobial effect of hot aqueous extract (WO) on inhibition growth of E. coli was more than S. aureus; the optimum concentration of extract used was 100 mg over incubation of each type of bacteria for 24 h at 37°C (Fig 9). The synergistic effect from 50 mg of IP and 50 mg of (WO) showed a noticeable increase in inhibition growth of S. aureus and E. coli (Fig 10). The absorbance (%) at 610 nm for growth of bacteria over 24 h at 37°C of incubation showed significant change (P≤0.01) compared to the control to be 22.48±1.37, 45.74±2.06, 33.33±1.79, 63.75±3.64 and 82.17±4.05% for S. aureus, while it was 9.52±0.71, 68.71±3.52, 45.59±2.75, 77.55±3.81 and 92.52±3.98% for E. coli when using 100, 100, 75+25, 25+75, 50+50 mg of IP and (WO), respectively (Table 2).











The achievement of food safety represented the main aim of food production due to consumers’ health concerns related to food spoilage and the increasing demand to enhance the shelf life of food using natural ingredients and preservatives and reduce the chemical materials use (Alsoufi and Aziz, 2022). Therefore, many authors reported that the effectiveness of proteases an antimicrobial and forming of biofilm as alone or synergistic with herbal drugs, phytochemicals and synthetic antibiotics (Salinas Ibanez et al., 2021) due to the ability of lyses cell walls by cleavage the pentaglycine bridges between the peptidoglycan and reduce the amount of carbohydrate and protein contents in the biofilms formed (Manohar et al., 2015). Such as proteases from Dunal fruit against Helicobacter pylori (Salinas Ibanez et al., 2021), protease from Xylaria psidii KT30 against B. subtilis and S. aureus (Indarmawan et al., 2016), Papain cross-linked polymers against Acinetobacter  sp. KC119137.1 and S. aureus (Manohar et al., 2015), subtilisin against Pseudomonas fluorescens (Molobela et al., 2010) and L. monocytogenes (Longhi et al., 2010).

The study shows the possibility of immobilization of partial purification protease from Prickly lettuce Lactuca serriola L. leaves on bentonite with good efficiency of immobilization, storage, reuses and suitable pH and temperature of proteolytic activity of enzyme, for using it as an antimicrobial agent against food spoilage bacteria, particularly Staphylococcus aureus and Escherichia coli, either alone or in synergistic with extract of Welsh onion Allium fistulosum L.

The authors declare that there is no conflict of interest regarding this work.