Natural Inhibitors of the Polyphenol Oxidase Activity Isolated from Shredded Stored Iceberg Lettuce (Lactuca sativa L.)

Abstract

Polyphenol oxidase (PPO) is the key enzyme responsible for enzymatic browning. To extend the shelf life of shredded lettuce, knowledge about biochemical PPO properties is required. The characterization of the enzyme from shredded, cold-stored lettuce was performed using pyrocatechol and the endogenous substrate (ES) (lettuce phenolics). The optimum pH and temperature for PPO activity were 5 and 50 °C, respectively. Natural infusions used as the PPO inhibitors (IC₅₀) were ranked as follows: lovage (0.09%), marjoram (0.13%), orange peel (0.14%), oregano (0.15%), basil (0.22%), lemon peel (0.24%), parsley leaves (0.58%), and wheat bran (1.06%). Among well-recognized PPO inhibitors, kojic acid (0.00043%), ascorbic acid (0.00053%), and L-cysteine (0.00085%) were the most effective. Among the metal ions, MgCl₂, FeCl₂, and CaCl₂ at 0.5 mM inhibited the PPO activity most effectively (by 28%, 27%, and 21%, respectively). The substrate used (pyrocatechol/ES) significantly influenced the enzyme inhibition. Using pyrocatechol, the lovage extract acted in a mixed mode (K_mi = 27.8 mM, V_maxi = 2.03 mU), while the ES acted according to the non-competitive mode (K_mi= 0.57 mg GAE/mL, V_max = 0.0046 U). The study confirms that natural extracts are more effective than L-cysteine when the ES is used. A pre-storage treatment with an infusion may be potentially used to improve the quality of shredded lettuce.

1. Introduction

In recent years, low-processed foods, called “ready-to-eat” foods, enjoy great consumer interest. Among these types of products, the most popular are low-processed fruits and vegetables such as lettuce mixes, germinated seeds, and peeled and shredded vegetables. Their popularity is related to their high nutritional quality and the fact that they are fresh, taste good, and easy to use, and enable a quick preparation of the meal [1].

Unfortunately, the main problem with low-processed fruits and vegetables is their short shelf life, usually limited to a few days under cold storage. The unfavorable changes are mainly caused by the activity of endogenous enzyme systems causing so-called enzymatic browning. The appearance deterioration (enzymatic browning) of low-processed fruits and vegetables is caused by polyphenol oxidase (PPO) and peroxidase (POD) [2,3]. PPO catalyzes the hydroxylation of monophenols to diphenols, the oxidation of diphenols to quinones, and the polymerization of quinones to dark pigments—melanins. PODs catalyze the oxidation of various substrates, including phenolic compounds, in the presence of hydrogen peroxide to the final dark-pigmented products [4]. Our preliminary studies have shown that, in shredded iceberg lettuce, the main enzyme involved in enzymatic browning is PPO—in the cut lettuce during storage, the activity of this enzyme was significantly higher than peroxidases, and a significant increase was also recorded compared to the fresh samples [5].

Considering that the activity of those enzymes negatively affects the food quality, it is desirable to limit their activity. Currently, some physical and chemical treatments are used to extend the shelf life of low-processed food. Among the physical methods, the most noteworthy are blanching, pulsed electric field, high hydrostatic pressure, cold plasma technology, and radiation technologies. Unfortunately, despite the numerous advantages of these methods, they have certain limitations; namely, they may negatively affect the texture of the final product and cause significant losses of vitamins. Due to the above limitations, they can be applied to a limited group of products, such as liquid products (e.g., juices and smoothies); however, they are not recommended for preserving low-processed food. Chemical methods limiting enzymatic browning include treatments with reducing agents, chelating agents, and acidulants. Even though these compounds preserve the natural properties of low-processed food and are relatively inexpensive, in recent years, more and more producers have consciously stopped using them. Furthermore, a significant drawback of these agents is the lack of biodegradability (e.g., sulfites) [6,7]. Acidulants, such as ascorbic acid and citric acid, decrease the pH below the optimal value for enzyme activity. Moreover, our previous studies have shown that the acidification of low-processed vegetables could stimulate the growth of molds and yeasts [8]. Finally, enzyme inhibitors, such as kojic acid or EDTA, are successfully applied, but, recently, they have consequently been eliminated by consumers more aware of their effect on health and the natural environment. In light of that, new technologies or tools, especially those of natural origin, are wanted.

The quality of low-processed products can be preserved or enhanced by the application of natural plant extracts [9,10]. This approach also has additional advantages; instead of inhibiting the enzymatic browning process, it may improve the pro-health properties, maintain a high microbiological purity, or simultaneously fortify with desirable components (e.g., phenolics and microelements). To use this new technology consciously, it is important to approach the subject from several key perspectives. In the beginning, a detailed characterization of enzymes involved in the generation of undesirable changes is required. Of course, special attention should be placed here on the inhibitory profile and substrate specificity. It was previously proven that the inhibition pattern (the effectiveness of the action) is strongly tailored by the substrate used. Karakus, Yildirim, and Acemi [11] have reported that the type of substrate used to carry out the reaction determines the effectiveness of the tested fennel (Foeniculum vulgare) PPO inhibitors and also has a significant impact on the inhibition type. The 0.1 mM ascorbic acid was the most effective using pyrogallol (25% inhibition of PPO activity), and the weakest using 4-tertbutylcatechol (10% inhibition of enzyme activity) as a substrate. Moreover, the 0.1 mM L-cysteine solution inhibited approximately 36% PPO activity with catechol and almost twice more with pyrogallol. Karakus, Yildirim, and Acemi [11] also showed that non-competitive inhibition was observed for sodium metabisulfite and glutathione using catechol, and 4-tertbutylcatechol as a substrate, while uncompetitive and competitive inhibitions were detected using pyrogallol, respectively. Similarly, for truffle PPO, it has been reported that sodium metabisulfite acted as a non-competitive inhibitor in the presence of catechol, while it was uncompetitive when tyrosine was used [12]. Furthermore, uncompetitive inhibition was found for benzoic acid and thiourea using catechol and 4-tertbutylcatechol, but non-competitive inhibition was found when pyrogallol was used. The research results cited above allow for the hypothesis that, for studying the activity and kinetics of enzymatic reactions, as well as the enzyme activity model, it would be more appropriate to use an endogenous substrate (i.e., polyphenolic compounds isolated from the studied plant material). This would enable a better representation of the natural environment in which these enzymes exhibit activity, allowing for a more accurate characterization of the enzymes and a deeper understanding of various processes. It is also obvious that both shredding and storage cause stress, which may promote the expression of new isoenzymes inactive in fresh tissues [13,14]. Importantly, these isoenzymes usually differ in the case of the inhibitory pattern.

In sum, the aware development of new tools reducing enzymatic browning in cut vegetables and fruit needs extended knowledge about enzymes charged in that process. Therefore, our research is focused on the partial purification and the biochemical characterization of PPO isolated from stored shredded iceberg lettuce (enzymatic pattern responsible for enzymatic browning). It is important that different classes of potential inhibitors (commonly used PPO inhibitors, and natural plant extracts) were tested using lettuce phenolics as endogenous substrates.

2. Materials and Methods

2.1. Chemicals

L-cysteine, kojic acid, ascorbic acid, 4-methylocatechol, pyrocatechol, ammonium sulfate, dihydrogen phosphate (V), hydrogen phosphate (V), acetic acid, phosphoric acid, boric acid, sodium azide, ammonium sulfate, Bradford reagent, guaiacol, MgCl₂, KCl, ZnCl₂, FeCl₂, NaCl, CaCl₂, CuCl₂, and polyvinylpolypyrrolidone were purchased from Sigma–Aldrich Company (Poznan, Poland). All other chemicals were of analytical grade.

2.2. Materials

Dried basil (Ocimum basilicum L.), lovage (Levisticum officinale), oregano (Origanum vulgare), marjoram (Origanum majorana L.), chamomile (Matricaria chamomilla), thyme (Thymus vulgaris L), wheat bran from common wheat (Triticum aestivum L.), lemon peel and juice (Citrus limon), orange peel and juice (Citrus sinensis), and fresh and dried parsley leaves (Petroselinum crispum) were obtained from a local market in Lublin, Poland. Fresh iceberg lettuce (Lactuca sativa L. var. Maximo) was purchased from a conventional farm in the spring of 2021. After harvesting, lettuce was placed in polypropylene boxes and stored at 8 °C until tests. The material (shredded iceberg lettuce) was stored under refrigeration for 10 days, until clearly visible changes in color were observed (especially leaves browning). We have focused on the PPO isolated from stored vegetables because, as it is known, the activity of this enzyme increases during storage.

2.3. Partial Purification of PPOs

2.3.1. Preparation of Crude Extracts

The extraction of stored (10 days in polypropylene boxes in refrigerated conditions) iceberg lettuce PPO was carried out according to the method by Terefe, Delon, Buckow, and Versteeg [15], with slight modifications. The 100 g of iceberg lettuce was homogenized by using laboratory homogenizer H 500—POL-EKO (Wodzislaw Slaski, Poland) with a microprocessor speed and time controller (rotational speed: 5000× g for 10 min) with 400 mL phosphate buffer (0.1 M, pH 5.0), which contained 0.5 % polyvinylpolypyrrolidone. The homogenate was then stored at 4 °C for 12 h before being centrifuged at 12,000× g at 4 °C for 30 min.

2.3.2. Salting Out

The crude extracts from stored iceberg lettuce were precipitated with (NH₄)₂SO₄ 80% salt saturation at 4 °C. Then, the samples were centrifuged at 12,000× g, at 4 °C for 30 min. The salt was removed by dialysis in the cellulose bag (MW cut off > 12,000) at 4 °C in the 5 mM phosphate buffer, pH 6.8, for 24 h.

2.3.3. Gel Filtration Chromatography Using Sephadex G-75

The partially purified enzyme was placed into Sephadex G-75 columns (1.6 cm × 80 cm) equilibarted with 0.01 M phosphate buffer (pH 5), and the collection parameter was adjusted to 0.25 mL/min and 2 mL/tube. High-activity fractions were collected based on the detected PPO activity. The high-activity fractions were placed into ultrafiltration centrifuge tubes Amicon^® Ultra Centrifugal Filter (Miami, FL, USA) 10 kDa MWCO of sample volume 15 mL, regenerated cellulose membrane of Sigma–Aldrich) for desalination and concentration. The concentrated enzyme samples were frozen and freeze-dried.

2.4. PPO Assay

For the PPO assay, 10 μL of the enzyme solution (concentrated freeze-dried enzyme with 0.05 M phosphate buffer pH 5) were incubated with 240 µL 0.05 M phosphate buffer (pH 5) and 50 μL of 0.05 M pyrocatechol at 24 °C for 5 min, and absorbance at 420 nm was measured. The PPO activity was expressed as U, where 1U is defined as enzyme activity which catalyzes the conversion of 1 µmol of substrate in 1 min under optimal conditions [16].

2.5. Protein Assay

Protein content was determined using the Bradford [17] and expressed as bovine serum albumin equivalents.

2.6. Characterization of PPO

2.6.1. Effect of Temperature on Enzyme Activity

To measure the effect of changes in temperature on enzyme activity, the reaction buffer, and substrate solutions were kept at different temperatures (10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 °C) for 1 h. Next, the activity was measured according to the procedure outlined in point 2.4. PPO activity was expressed as residual PPO activity compared to the results at the optimum temperature.

2.6.2. Effect of pH on Enzyme Activity

To determine the effects of different pH on PPO activity, enzyme activity was measured at pH from 2 to 12 (Britton and Robinson’s buffer) using 50 mM pyrocatechol as a substrate. The reactions were performed according to the procedure outlined in point 2.4. The pH value corresponding to the highest enzyme activity was taken as the optimal pH. PPO activity was expressed as residual activities.

2.6.3. Inhibition of PPO Activity with Catechol as Substrate

Preparation of Inhibitory Solutions

The common chemical inhibitors (L-cysteine, kojic acid, and ascorbic acid), and metal ions (MgCl₂, KCl, ZnCl₂, FeCl₂, NaCl, CaCl₂, and CuCl₂) were dissolved in water and analyzed at concentrations ranging from 0.01 mM to 10 mM (in the reaction mixture 0.002 mM to 2 mM, respectively) and 5 mM to 20 mM (concentration in the reaction mixture: 0.5 mM to 2 mM), respectively. Plant materials—dried basil (Ocimum basilicum L.), lovage (Levisticum officinale), oregano (Origanum vulgare), marjoram (Origanum majorana L.), chamomile (Matricaria chamomilla), thyme (Thymus vulgaris L.), wheat bran from common wheat (Tricum aestivum L.), lemon peel (Citrus limon), orange peel (Citrus sinensis), and fresh and dried parsley leaves (Petroselinum crispum)—were ground in a laboratory mill and sieved (60 mesh), and 10 g of samples were mixed with 90 mL of boiling distilled water (95 °C) and then allowed to cool. Then, the samples were centrifuged 5000× g at 4 °C for 30 min and used for treatments. The infusions were tested at concentrations ranging from 0.0001% to 10%.

Inhibition of PPO Activity (Exogenous Substrate)

Then, 10 μL of the enzyme solution (concentrated freeze-dried enzyme dissolved in 0.05 M phosphate buffer pH 5) were incubated with 180 µL 0.05 M phosphate buffer (pH 5) and 60 µL inhibitor solution. The reaction was started by adding 50 μL of 0.05 M pyrocatechol. The absorbance at 420 nm was measured at 24 °C. Next, IC₅₀ value was calculated and expressed in % of dry mass.

Model of Inhibition and Kinetic Constants

Data were plotted as the 1/V and 1/S concentration according to the method proposed by Lineweaver and Burk [18]. The reaction was conducted using 50 mM pyrocatechol as a substrate. Based on Lineweaver–Burk graphs, a mode of inhibition and kinetic parameters of reaction (V_max, K_m, V_maxI, and K_mi) were defined.

2.6.4. Inhibition of PPO with Endogenous Substrate

Preparation of Endogenous Substrate

The white part of the iceberg lettuce was frozen and lyophilized. Subsequently, 0.5 g of the raw material was homogenized with 12.5 mL 70% acetone, shaken (1 h, 4 °C, 150 rpm), centrifuged (12,000× g at 4 °C for 30 min), and concentrated in a concentrator. Then, it was frozen, lyophilized, and dispersed in 100 mM phosphate buffer pH 5 immediately before testing. It was used as a substrate for determining the IC₅₀ values for the most promising natural extracts selected with pyrocatechol as substrate in the previous step of the study (basil, lovage, oregano marjoram, and orange peel).

Inhibition of PPO Activity (Endogenous Substrate)

Then, 10 μL of the enzyme solution (concentrated freeze-dried enzyme with 0.05 M phosphate buffer pH 5) were incubated with 70 µL 0.05 M phosphate buffer (pH 5), and 30 µL inhibitor solution (L-cysteine solution, kojic acid solution, ascorbic acid solution, lovage extract, oregano extract, and orange-peel extract). The reaction was started by adding 90 μL of the endogenous substrate and left at 24 °C for 30 min. The reaction was stopped with 96% ethanol. The sample was centrifuged (centrifuge with rotor for tubes and PCR strips by Benchmark (Sayreville, NJ, USA), 5500 rpm/2000× g, 1 h), and the absorbance was measured at 380 nm (the wavelength was selected based on the absorption spectrum). Similarly, samples T0 were prepared (enzyme stopped with ethanol before substrate adding). Next IC₅₀ value was calculated and expressed in % of dry mass.

Mode of Inhibition and Inhibition Kinetics Parameters (Endogenous Substrate)

For the three most effective natural inhibitors (oregano extract, lovage extract, and orange peel extract) and pure chemical inhibitor, kinetic parameters and inhibition mode were determined using the Lineweaver and Burk method as described previously in the Section “Model of Inhibition and Kinetic Constants” with some modifications (endogenous substrate was used). The PPO activity was expressed as U, where 1U = 0.001 ΔOD380/min under the assay conditions.

Serial dilutions of the endogenous substrate were made. The total content of polyphenolic compounds in substrate solution was determined [19]. The effect of inhibitors on PPO activity was measured using the procedure described previously in the Section “Inhibition of PPO Activity (Exogenous Substrate)”.

2.7. Statistical Analysis

All experimental results were means (±standard deviation) of at least four independent measurements. Analysis of variance (ANOVA) and Tukey’s post hoc test were used to compare the groups (STATISTICA 13, StatSoft, Inc., Tulsa, OK, USA). Differences were considered significant at α = 0.05.

3. Results and Discussion

Table 1. Partial purification procedure of PPO from stored (10 days) iceberg lettuce.

Step	Total Activity [U]	Total Protein [mg]	Specific Activity [mU/mg]	Yield [%]	Purification Fold
Crude extract	0.35	65.79	5.29	100	1
Salting out	0.17	25.8	6.43	47.65	1.21
Sephadex G-75	0.05	0.65	75.65	14.21	14.3

shows the sequential purification of PPO isolated from stored material. The procedure started with preparing a crude extract from stored iceberg lettuce, precipitation with (NH₄)₂SO₄ (80% salt saturation), followed by anion exchange chromatography and gel filtration chromatography. This procedure gave partially purified iceberg lettuce PPO. Ammonium sulfate fractionation as the first step of iceberg lettuce PPO purification proved convenient and effective in removing large amounts of non-targeted proteins and brown pigments, by reducing the total proteins by almost 54% and 61% and yielding 50% and 48% in fresh and stored samples, respectively. Further purification by loading the dialyzed enzyme on Sephadex G-75 columns (1.6 cm × 80 cm) with 0.01 M phosphate buffer (pH 5) containing 0.02% sodium azide eliminated much of the remaining non-targeted proteins. As a result, the PPO from material was purified over 12-fold, with a 13.35% activity recovery; in turn, PPO from stored was purified over 14-fold, with a 14.21% yield. Similar results were obtained by Yuzugullu et al. [11], who reported the purification fold for PPO from fennel seeds was 20-fold.

Based on the obtained results (Figure 1A), it can be concluded that PPO exhibits activity over a relatively wide pH range (from 4 to 9), with the optimum pH identified in the range of 5–7 (using pyrocatechol as a substrate). The highest activity was observed at pH 5. The enzyme very quickly lost its activity in an alkaline condition. Similar results were obtained by Gawlik-Dziki [20], who reported that the optimum pH for butter lettuce is 5.5 using catechol and 6.8 using 4-methylcatechol as substrate. Moreover, Xinglong and Xingfeng [21] showed that PPO activity is the highest in pH 4.5 using catechol as the substrate. In turn, Gonzales, De Ancos, and Cano [22] have reported that the optimal pH value for blackberry fruits was 6.5 using catechol as the substrate, and 5.5 and 8.0 for raspberry fruits using catechol as the substrate [23]. Generally, most vegetables and fruits show the highest activity at or near pH 7 in the literature [24,25], but these values may be different for PPO obtained from various sources of the enzyme and may be dependent on the substrate used in the reaction. In most cases, the pH optima have been reported to be between 4.0 and 8.5; it should be noted that they are dependent not only on the vegetable raw material but also on the purity of the enzyme being tested. Figure 1B shows the influence of temperature on PPO activities in the assay conditions (pH 5 and 0.05 M pyrocatechol as a substrate). The optimum temperature for PPO is 50 °C. Above this temperature, a rapid loss of enzyme activity was observed. What is surprising is that the enzyme was still active at 80 °C; however, the activity was relatively low (20% relative activity). The optimal value of the temperature for PPO is dependent on the source of the enzyme and also the kind of substrate used for the reaction. According to the literature data, the optimum temperature for PPO is in the range of 40–60 °C depending on the raw material. Similar results concerning the same enzyme, but isolated from a different substrate, were presented by Serradell, Rozenfeld, Civello, and Chaves [26]; Gao, Liu, and Xiao [27]; and also Navarro, Tárrega, Sentandreu, and Sentandreu [28]. They studied the optimum temperature for PPO isolated from strawberries (50 °C), Shona cabbage (60 °C), and persimmons (55 °C), respectively.

The inhibitory effect of the studied pure chemical compounds and plant infusions is presented in

Table 2. Effect of selected chemical compound and natural plant extracts on the iceberg lettuce PPO activity.

Inhibitor	IC50
	Pyrocatechol	Endogenous Substrate
Chemical compounds	[%]
L-cysteine	0.00085 ± 0.00002 k	0.34 ± 0.01 c
kojic acid	0.00043 ± 0.00001 k	0.0067 ± 0.0002 k
ascorbic acid	0.00053 ± 0.00001 k	0.026 ± 0.007 jk
Plant infusions	[%]
basil (Ocimum basilicum L.)	0.22 ± 0.01 de	0.21 ± 0.01 e
lovage (Levisticum officinale)	0.09 ± 0.003 hi	0.09 ± 0.004 i
oregano (Origanum vulgare)	0.15 ± 0.007 fg	0.04 ± 0.002 j
marjoram (Origanum majorana L.)	0.13 ± 0.005 fg	0.16 ± 0.07 f
orange peel (Citrus sinensis)	0.14 ± 0.002 fg	0.11 ± 0.03 gh
lemon peel (Citrus limon)	0.24 ± 0.01 d	na.
dried parsley leaves (Petroselinum crispum)	0.58 ± 0.02 b	na.
fresh parsley leaves (Petroselinum crispum)	dose-independent	na.
chamomile (Matricaria chamomilla)	dose-independent	na.
wheat bran from common wheat (Triticum aestivum L.)	1.06 ± 0.045 a	na.

Means (± standard deviation) with different letters are statistically significant (n = 9; α = 0.05, 2-way ANOVA). IC50—the concentration of an inhibitor that inhibits the enzyme activity by 50%; na.—not analyzed (the reactions with endogenous substrate were carried out only for the most active inhibitors).

. The lowest values of the IC50 (pyrocatechol as a substrate) were noted for chemical compounds. Even 0.03% solutions of ascorbic acid and L-cysteine inhibit the activity of an enzyme by 50% (with pyrocatechol as a substrate). Interestingly, the application of natural extracts in low concentrations also effectively limited the activity of the tested enzyme from the oxidoreductase class. Among the water plant extracts, the best results were obtained for lovage (IC50 = 0.09%), marjoram (IC50 = 0.13%), and orange peel (IC50 = 0.14%) with pyrocatechol as a substrate. Usually, synthetic substrates (catechol and its derivatives) are used to study PPO activity. In our research, to confirm the inhibitory properties of the selected extracts, additionally, an endogenous substrate isolated from iceberg lettuce was used. Similar results were obtained; the lowest IC50 values were in the case of water extract obtained from oregano (0.04%), lovage (0.09%), and orange peel (0.11%). Interestingly, in the case of L-cysteine, kojic acid, and ascorbic acid, the obtained IC50 values in reaction with the endogenous substrate were significantly higher than in the assays conducted using pyrocatechol.

To clarify the inhibitory effects of the studied extracts on PPO activities, kinetic studies were performed with the Lineweaver–Burk method (

Table 3. Mode of inhibition and kinetic parameters of process using pyrocatechol and endogenous substrate.

	Pyrocatechol as a Substrate
Inhibitor (IC50 Value)	Vmaxi [mU]	Kmi [mM]	Vmax [mU]	Km [mM]	Mode of Inhibition
L-cysteine	0.87	174	0.91	21.4	competitive
kojic acid	0.38	20	0.61	12.5	mixed
ascorbic acid	1.37	202	1.39	52.4	competitive
basil infusion	1.71	8.72	2.08	8.16	mixed
lovage infusion	2.03	27.8	2.05	11.11	mixed
oregano infusion	1.77	8.28	1.74	5.42	competitive
wheat bran infusion	2.39	3.35	2.45	3.17	competitive
lemon peel infusion	2.21	15.96	2.16	12.77	non-competitive
orange peel infusion	625	3.2	714	3.6	uncompetitive
marjoram infusion	0.37	5.38	0.66	2.13	mixed
parsley infusion	1.12	9.07	3.93	25.18	uncompetitive
	endogenous substrate
	U	mg GAE/mL	U	mg GAE/mL
L-cysteine	0.104	17.24	0.067	4.5	competitive
kojic acid	0.0067	1.85	0.067	4.5	non-competitive
ascorbic acid	0.034	2.94	0.067	4.5	competitive
lovage infusion	0.0046	0.57	0.0098	0.625	non-competitive
oregano infusion	0.0059	0.46	0.0098	0.61	uncompetitive
orange peel infusion	0.0083	0.59	0.0098	0.0625	competitive

IC50—the concentration of a substance at which 50% inhibition of enzyme activity is achieved, i—inhibitor, V_max—maximal velocity of reaction, K_m—Michealis constant, U—unit, GAE—gallic acid equivalents.

, Figure 2). The majority of the studied extracts suppressed PPO activities in a dose-dependent manner; only in the case of chamomile extract and fresh parsley was it impossible to determine the IC50 values because the inhibition was dose-independent.

The results from the Lineweaver–Burk plots indicated that the L-cysteine and ascorbic acid acted as competitive inhibitors of PPO with a K_mi of 174 mM and 202 mM, respectively. The competitive inhibitor resembles the substrate molecule in its structure. It binds to the free enzyme in the active centre, preventing the substrate from binding to the enzyme molecule. Increasing the substrate concentration partially eliminates the inhibitor’s effect. In the case of non-competitive inhibition, the inhibitor binds in a place other than the active centre. Increasing the substrate concentration does not eliminate the inhibitor’s effect. In turn, uncompetitive inhibitors bind to the resulting enzyme–substrate complex, leading to the formation of an inactive enzyme–substrate–inhibitor complex. Moreover, in this case, increasing the substrate concentration does not eliminate the inhibitor’s effect. Mixed inhibition is a case of partially competitive and non-competitive inhibition. The inhibitor can bind to both the free enzyme and the ES complex.

In this case, kojic acid was observed to be of the mixed inhibition type. These results are in agreement with Altunkaya and Gökmen [29], who reported that both cysteine and ascorbic acid are competitive inhibitors of PPO in fresh lettuce (Lactuca sativa). Ali, El-gizawy, El-bassiouny, and Saleh [30] also report that ascorbic acid in concentrations <1.5% acted as a competitive inhibitor, while, in a higher concentration, it could reduce the formed quinone instantly to the origin substrate (catechol).

The most popular PPO inhibitors are ascorbic acid and citric acid. Their mechanisms of action are mainly based on decreasing pH. Therefore, these compounds have a negative effect on the texture and taste of food. Moreover, our previous studies have shown that the acidification of low-processed vegetables could stimulate the growth of molds and yeasts [8]. Food preservation without the use of chemicals is becoming more and more popular. Consumers consciously eliminate products preserved with synthetic preservatives from their diets. This is due to the increased care for one’s health, but also the desire to care for the natural environment.

It is worth noting that the cost of producing and utilizing natural water extracts is relatively low compared to pure chemicals. Moreover, the application of natural plant extracts can have a positive effect on human health, because they are a rich source of low-molecular-weight antioxidants. Natural plant extracts, especially received from herbs such as basil, marjoram, lovage, or oregano, are accepted by consumers because they have a positive effect on the taste and aroma of iceberg lettuce and are commonly used in the seasoning of salads. Because of the above, other inhibitors, especially of natural origin, are wanted. Yu et al. [31] showed that the addition of Rosa roxburghii juice effectively improved the appearance of apple juice by inhibiting the enzymatic browning and PPO activity. In turn, Altunkaya and Gökmen [10] used waste material such as grape seeds. Their research showed that the treatment of fresh-cut lettuce (L. sativa) with extracts received from grape seeds could cause limited darkening (lower values in lightness values during storage). Our studies have shown that wheat bran from common wheat (Triticum aestivum L.) and orange peel (Citrus sinensis), also being a waste material, could effectively inhibit the activity of PPO isolated from iceberg lettuce. Yee, Chen, and Wong (2018) [32] confirm that natural anti-browning agents (honey, red onion extract, red chili pepper extract, and pineapple juice) have the potential to be used to control the browning of ginger as well as other vegetables and fruits.

Kinetic constants (K_m and V_max) are calculated for six substrates using the Lineweaver–Burk plot. According to the Km values, as a determinant of the enzyme’s affinity for its substrate, the PPO enzyme exhibited the highest affinity pyrocatechol (K_m = 2.39 mM) and 4-methylocatechol (K_m = 10.01 mM). In connection with the above for further studies, pyrocatechol was selected as a substrate for iceberg lettuce PPO. Our results are in accordance with Gawlik-Dziki, Szymanowska, and Baraniak [33], who reported that the broccoli PPO had the highest affinity to catechol and 4-methylcatechol. Generally, to detect the PPO activity among various plants, catechol and 4-methylcatechol are usually used as a substrate. Moreover, Aydemir [34] showed that the K_m value for PPO isolated from artichoke and for catechol was 10.2 mM min⁻¹, and that for 4-metylocatechol was 12.4 mM min⁻¹.

The results from the Lineweaver–Burk plots (Table 3) received in the results reaction carried out with pyrocatechol indicated that, among the examined functional solutions, the extracts of oregano and wheat bran act according to the competitive model, the extract of lemon peel represents a non-competitive inhibition type, and the parsley and orange peel represent the uncompetitive type, while the extracts of basil and lovage, and marjoram represent the mixed type. In turn, L-cysteine and ascorbic acid represent competitive types of inhibition, and kojic acid is mixed.

In turn, reactions carried out with an endogenous substrate showed that the orange peel extract represented a competitive mode of inhibition, the oregano extract uncompetitive, and the lovage extract non-competitive. In the case of chemical compounds (L-cysteine and ascorbic acid), the received results were similar (regarding the type of inhibition). Only in the case of kojic acid was a different type of inhibition observed, dependent on the substrate used.

In most scientific publications, synthetic substrates (pure chemical compounds such as catechol, pyrocatechol, or 4-methylcatechol) are used to conduct reactions with PPO (determining its activity, determining the IC₅₀ values, identifying the type of inhibition, and determining the kinetic constants of the process). In our study, we aimed to verify whether similar results would be obtained using an endogenous substrate obtained from plant material. IC₅₀ values were determined for selected inhibitors in the presence of the endogenous substrate. Interestingly, in the case of natural extracts, no statistically significant differences were observed in the reaction with exogenous (pyrocatechol) and endogenous substrates. However, it is worth noting that the IC₅₀ values determined for chemical compounds (L-cysteine, kojic acid, and ascorbic acid) in reaction with the endogenous substrate were significantly higher than in reaction with pyrocatechol as a substrate.

Additionally, for the three most effective inhibitors (with the lowest IC₅₀ values: lovage, oregano, and orange peel), the inhibition model was determined in reaction with the endogenous substrate, and, next, the values of the kinetic constants were calculated. The obtained results are not identical to those obtained in the reaction with pyrocatechol. Some examples of Lineweaver–Burk plots for PPO inhibition (reaction carried out with endogenous substrate and pyrocatechol) were presented in Figure 2. In turn, the inhibition model and kinetic parameters of the process using an endogenous substrate were presented in Table 3.

The results suggest that the use of endogenous substrates may be more appropriate for studying enzyme kinetics, especially in practical applications where reactions occur under natural conditions. The use of synthetic substrates may introduce certain distortions in the analysis of enzymatic kinetics because the reaction conditions may differ from those occurring in real biological systems. Therefore, studies demonstrating the effectiveness of endogenous substrates may suggest the need to consider alternative research methods that better reflect the natural environment of enzymes. Results regarding enzyme kinetics can also vary when different chemical compounds are used as substrates. Yee et al. [32] examined the effect of natural anti-browning agents on PPO from ginger (Zingiber officinale Roscoe) and they report that the mechanism of honey in inhibiting PPO varies depending on the variety of substrates used. The V_max value was unchanged and Km value was increased for PPO with honey using 4-methylcatechol. It can be concluded that honey showed competitive inhibition; however, honey exhibited non-competitive inhibition towards ginger PPO when pyrocatechol was used as the substrate, whereby the K_m value was unchanged. This is another argument supporting the use of endogenous substrates for conducting reactions with PPO. Without more detailed research, including an analysis of the plant’s phenolic profile, it is difficult to determine which phenolic compounds are utilized as substrates by enzyme.

The effect of the selected metal ions at different concentrations varying from 20 to 5 mM (concentration in the reaction mixture: 2 mM to 0.5 mM) on iceberg lettuce PPO activity using pyrocatechol as a substrate was presented in

Table 4. Effect of selected metal ions on the iceberg lettuce PPO activity.

Inhibitor	Relative Activity [%] *
Metal Ions	Concentration (in the Reaction Mixture) [mM]
	0.5	1	2
MgCl2	72 ± 4 g	100 ± 5 efg	123 ± 6 defg
KCl	82 ± 4 fg	100 ± 5 fg	130 ± 6 defg
ZnCl2	97 ± 5 fg	123 ± 6 defg	197 ± 10 bcd
FeCl2	73 ± 4 g	178 ± 24 cde	266 ± 13 ab
CuCl2	118 ± 6 defg	210 ± 10 bc	316 ± 16 a
NaCl	83 ± 4 fg	112 ± 6 fg	125 ± 6 defg
CaCl2	79 ± 4 fg	115 ± 6 efg	161 ± 8 cdef

* 100% activity of enzyme—activity of enzyme without inhibitors, value lower than 100% activity—enzyme with inhibitors, value higher than 100%—some metal ions act as activators of PPO. Means (±standard deviation) with different letters are statistically significant (n = 9; α = 0.05, 2-way ANOVA).

. Unfortunately, the IC50 value for metal ions could not be determined, because they act in a dose-independent manner, whereas it was observed that, in the smallest studies concentrations (0.5 mM), MgCl₂ and FeCl₂ inhibit the activity of PPO by almost 30%, and KCl and NaCl by 18% and 17%, accordingly. As seen in Table 3, almost all tested metal ions in the concentrations 1 mM and 2 mM were enzyme activators. On the other hand, the highest increase in enzyme activity (over a three-fold increase in activity) was observed with CuCl₂ at the highest studied concentration (2 mM). Ma et al. [35] and Karakus, Yildirim, and Acemi [11] have reported that the activating effect of metal ions (Mg²⁺ and Cu²⁺) can be explained by the stabilization of the enzyme structure by them causing an increase in affinity to the substrate. In contrast, some metal ions (Ca²⁺, Na⁺, and K⁺) inhibit the enzyme activity by changing the conformation of the enzyme through the changing chemical bonds in the active site. Moreover, Sun et al. have observed that adding Fe²⁺ and Na⁺ into the substrate–enzyme system increased the PPO activity. The plant PPO is suggested to have one binuclear Cu site per protein molecule for the reaction centre and various metal ions may affect the substrate by combining this reaction centre [36]. Similar dependencies were observed in our results (1 mM and 2 mM solutions of FeCl₂ and NaCl). Yuzugullu et al. [11] reported that CuSO₄ (in the same concentration) also activates the PPO activity isolated from fennel (by 1.5-fold). In general, the way metal ions interact with the enzyme activity is strictly dependent on the concentration used. The addition of metal ions while extending the shelf life of low-processed foods may contribute to the prevention of nutritional deficiencies. PPO is an enzyme containing copper atoms in the active site that are essential for its catalytic activity. This could explain why the copper ions activate the enzyme. Cu²⁺ could cause an increase in enzyme stability and activity by supporting more enzyme–substrate contact [37].

4. Conclusions

The results suggest that using endogenous substrates may be more appropriate for studying enzyme kinetics, especially when studying enzymes isolated from plant material. This study concluded that oregano extract was the best natural inhibitor to inhibit iceberg lettuce PPO. Moreover, other natural inhibitors such as lovage, marjoram, and orange peel can be used to replace the sulfite-containing agents that could harm human health. Among metal ions, 0.5 mM MgCl₂ and 0.5 mM FeCl₂ decreased enzyme activity by nearly 30%. The addition of metal ion solutions while extending the shelf life of low-processed foods may contribute to the prevention of the most common nutritional deficiencies. The obtained research results are very promising and it is possible that, in the future, they will allow us to limit the use of synthetic preservatives. Moreover, as a result of the obtained results, it is possible to more precisely determine the kinetic constants in the reaction of PPO with the endogenous substrate and to more precisely determine the type of inhibition.