Assessment of (‐) epicatechin as natural additive for improving safety and functionality in fresh “Piel de Sapo” melon juice

Abstract Epicatechin (EC) is a very abundant flavonoid in vegetable tissues that presents high antioxidant activity in living systems. The minimum inhibitory concentration (MIC) of (‐)EC was determined in three species of bacteria commonly associated with foodborne illness of plant origin: Listeria (L.) monocytogenes, Escherichia (E.) coli ‐serogroups O157: H7 and O111‐ and Bacillus (B.) cereus; two strains of probiotic‐type lactic acid bacteria (PT‐LAB) and two control strains. All 10 strains were assayed under three temperature conditions (30º, 10º, and 4ºC) and at each temperature under two pH conditions (6.7 and 5.5). Mean EC MIC values were generally lower at refrigeration (4º and 10ºC) temperatures and at standard pH (6.7). By inoculating with each of the strains separately, both melon juice (MJ) and MJ supplemented with EC (ECSMJ), at the accepted maximum sensorial limit, and storing them at 4ºC for 10 days; the final counts (CFU/mL) were lower for ECSMJ than for plain MJ both for pathogenic bacteria and for PT‐LAB. The presence of EC during refrigerated storage counteracted the ability of MJ as a growth medium for all the pathogenic bacteria. ECSMJ increased the antioxidant activity of MJ significantly to levels similar to those of EC alone. (‐) Epicathechin would be a promising ingredient for increasing the functional properties of “Piel de Sapo” MJ (phenolic compounds and antioxidant ability) while contributing to improving the safety of this type of juice during prolonged refrigerated storage at 4ºC.

involved are Salmonella enterica (particularly associated with the netted Cantaloupensis varietal group), although other bacterial agents have also been reported, among which are verotoxin-producing E.
In order to increase the safety of fresh juices, the addition of natural antimicrobials found in edible plants and herbal extracts has recently been proposed among other alternatives. (Rudra et al., 2020) Epicatechin (EC) is a very abundant flavonoid in vegetable tissues that presents high antioxidant activity in living systems. Specifically, it is included in the flavanols group, one of the five major polyphenolic groups found in tea leaves and various apple varieties. (Mendoza-Wilson & Glossman-Mitnik, 2006;Tsao et al., 2003) Flavanols exhibit the highest radical scavenging activity (4.21 mM), significantly more than other flavonoids. However, little is known about the anti-microbial effect of EC on foodborne pathogens, although it has been reported that EC displays anti-bacterial activity against Helicobacter pylori. ( Escandón et al., 2016) Also, to our knowledge, no information exists on the effect of EC in PT-LAB, although polyphenols have recently been recognized as a candidate category of prebiotic compounds. (Gibson et al., 2017).
Previous studies in our lab have indicated that "Piel de Sapo" melon juice (MJ) enables the viability of verotoxin-producing E. coli (VTEC) and growth potential (δ) of PT-LAB at 4ºC for 4 days. (Rúa et al., 2018(Rúa et al., , 2019 The aim of the present research was to assess the effect of a natural phytochemical EC on viability and growth in "Piel de Sapo" MJ of pathogenic (VTEC -O157:H7 and O111-, L. monocytogenes and B. cereus) and of PT-LAB (Lactobacillus [Lb.] rhamnosus GG and Lactococcus [Lc.] lactis subsp. cremoris 660) bacteria at 4ºC for 10 days and also to analyze the potential effect of EC in reinforcing the antioxidant capacity and phenolic content of this MJ.

| Bacterial strains and growth conditions
The strains used to carry out the study belong to three groups:  (Table 1; Caro, 2004;García-Armesto et al., 1993;García-Armesto & Sutherland, 1997;Monteagudo-Mera et al., 2011  The EC stock solution (10 mg/ml in 40% ethanol) was stored frozen. Before working with this solution, the sensitivity of the strains to ethanol in microtiter plate was checked by serial dilutions, starting from 40% ethanol up to 0.65% (v v -1 ); thus was found that the antimicrobial effect was found to be due to EC only.

| MIC and MBC assays
The antimicrobial microdilution assay to estimate the minimum in-

| Preparation and characterization of MJ
"Piel de Sapo" MJ was prepared and characterized as previously reported. (Rúa et al., 2018) Samples of MJ, both plain (MJ) and supplemented with 5,000, 2,500 or 1,250 µg/ml EC (ECSMJ), were subjected to sensory evaluation at the beginning (time 0) of the experiment and after 10-day storage at 4ºC by six trained panelists from the Institute of Food Science and Technology of the University of León. Also physical-chemical analyses (pH, titratable acidity and ºBrix) was performed on MJ and ECSMJ at time 0 and after 10-day storage at 4ºC. (Rúa et al., 2018).

| Antioxidant activity and total phenolic contents
Antioxidant activity was determined by the ABTS, DPPH, and FRAP methods and total phenolic contents (TPC) was determined using the Folin-Ciocalteau method (FC) and the Fast Blue BB (FBBB) method. Results for antioxidant activitiy were expressed as µmol Trolox per 100 ml of sample, and the results for TPC were expressed as mg of gallic acid equivalents per 100 ml. These methods were described previously (Rúa et al., 2018) Stock samples of MJ, EC, and ECSMJ (2,500 µg/ml of EC in the juice) were stored frozen. Duplicate aliquots of these samples were thawed, kept for 0 (2 hr), 1, 3, 5, 7, and 10 days at 4ºC and analyzed in duplicate for their antioxidant activity and TPC.

| Survival and growth of inoculated bacteria in melon juice
Freshly squeezed MJ and ECSMJ at a final EC concentration of 2,500 µg/ml were inoculated separately with 10 5 CFU/mL of one of PT-LAB and 10 3 -10 4 CFU/mL for the rest of the bacteria strains used in this study. Survival and growth of the strains were assessed at 0, 1, 3, 5, 7, and 10 days at 4ºC by counting on TBX for E. coli strains, Plate Count Agar (PCA) for B. cereus and En. faecalis strains, and TSA for L. monocytogenes and PT-LAB strains. The experiments were done in two batches of MJ, each under two conditions (MJ and ECSMJ). The growth potentials (δ) for each condition and each of the 10 strains were calculated as the difference between growth after 10-day storage in comparison with that at the beginning of the experiment (t = 0). The highest growth potential value obtained for the two lots in duplicate was considered. The results were interpreted according to criteria used by Beaufort et al. Beaufort et al., 2014), considering that a δ > 0.5 log 10 indicates that MJ and ECSMJ are able to support the growth of the bacteria tested.

| Statistical analysis
Statistical analysis was carried out using a one-way analysis of variance (ANOVA) for comparison of more than two different groups, using the posthoc Tuckey and Duncan method. The ANOVA analysis was performed using the statistical analysis program SPSS Statistics version 24 for Windows, available on the IBM website.

| Antimicrobial activity of EC in growth media under different temperature and pH conditions
Two PT-LAB, six strains with potential pathogenic ability, and two control bacteria were used in this study (Table 1). Under the tested conditions of pH and temperature, in the culture media used (CA-MHB), significant differences were found in the antimicrobial activity (MIC values) of EC (  (Table 3). MBCs were also generally higher at pH 5.5 than at pH 6.7, regardless of the temperature used. At pH 6.7, there was more diversity in the behaviour of the strains (two groups at 10 and 4ºC; and three at 30ºC) compared to that obtained at pH 5.5 (one group at 30 and 4ºC and two groups at 10ºC).
Taking into account that an antimicrobial compound is considered bactericidal when the MBC has a value less than double that of its MIC, and bacteriostatic when it is higher than double, (Moody et al., 2007)  MIC values for EC were lower at 4ºC than at 30ºC and at pH 6.7 with respect to pH 5.5; this effect was observed in a previous study (Rúa et al., 2018), but using PLX ® as antimicrobial against these PT-LAB.
The strains of L. monocytogenes used in this study are of different origin (cheese and sausage), and this could explain the differences in MICs obtained in some of the studied conditions. In general, L. monocytogenes CECT 4,032 is more resistant to EC than the L74 strain.  et al., 1993) The use of green tea extract (≤ 4,000 µg/ml) inhibits the growth of E. coli fron urinary tract. (Reygaert & Jusufi, 2013).
It has been described that the mode of antibacterial action of green tea extract, EGCG, and EC caused damage to the membranes. (Ikigai et al., 1993) Also, the stability of tea catechins is pH-and temperature-dependent. Tea catechins in aqueous solutions are very stable when pH is below 4; whereas, they are unstable in solutions with pH > 6.0 (Ananingsih et al., 2013), which could partly explain the lower antimicrobial effect of EC (MICs) under the conditions used in our study.

| Sensorial and physicochemical analysis of MJ and ECSMJ
Two batches of fresh "Piel de Sapo" MJ were sensory evaluated, without supplementation and supplemented with different EC concentrations (5,000, 2,500, and 1,250 µg/ml) (ECSMJ) in order to determine the concentration that the consumer allow. According to the tasting panel, 2,500 μg/mL of EC did not generally modify many of the sensorial parameters tested at the onset of addition and at the end of storage (10 days at 4°C) for fresh MJ or ECSMJ (Table 4)

| Antioxidant activity and phenolic content of ECSMJ
The antioxidant activity was determined in MJ, a solution of EC (2,500 µg/mL) and in ECSMJ for 10 days stored at 4°C, using three methods: ABTS, DPPH, and FRAP (Table 5). The values for antioxidant activity in MJ were similar using the three methods. The addition of EC to MJ produced a significant increase in the antioxidant activity of ECSMJ, with values similar to those for EC, which was ten times more than that produced by PLX ® in MJ, according to a previous study. (Rúa et al., 2018).

Bacteria tested Minimum inhibitory concentration (MIC) (µg/mL
In addition, values for EC and ECSMJ antioxidant activity were different for the three methods, but similar for the two samples in each method. Also, these values were approximately 120 (ABTS), 100 (DPPH), and 30 times higher (FRAP) than those of MJ. EC is a flavanol that exhibits the highest radical scavenging activity, significantly more than other flavonoids, as previously reported by Cai et al. (Cai et al., 2006).
TPC was determined in MJ and ECSMJ during the storage refrigeration period by the FBBB and FC methods (Table 5). In MJ, the average values were 6 or 14 mg of GAE 100 ml -1 , with FBBB or FC, resulting in a quotient value of 0.4. Previously, we determined a quotient value of 2.4, (Rúa et al., 2018) which could be due to the fact that a different batch of melons were used. In this sense, it has been described that although fruits and vegetables are recognized as the best source of the antioxidant diet, the amount and type of each is influenced by a number of factors, including genotype, ontogeny, environment, and postharvest hand (for review refer Salandanan et al.). (Salandanan et al., 2009).
The high TPC in ECSMJ, with mean values of 2,184 or 123 mg of GAE 100 ml -1 , according to the method used, FBBB or FC, respectively, is noteworthy. The value obtained by the FBBB method of 2,184 mg GA 100 ml -1 is much higher than the added amount of EC (250 mg 100 ml -1 ), which seems to indicate that the TPC is being overvalued by this method. To check this interference, a test was performed simultaneously with GA and EC, obtaining a linear relationship in both cases with a value of the equation of y = 3.62 × 10 -3 × + 0.29 with GA and y = 2.85 × 10 -2 × + 8.17 × 10 -2 with EC, the value of the slope ratio being approximately 8.
Therefore, this possible interference was avoided by correcting the value of TPC in ECSMJ giving an average value of 273 mg EC 100 ml -1 , which is similar to the one added (refer values in brackets in Table 5). We calculated the highest growth potential (

| CON CLUS IONS
The addition of EC to "Piel de Sapo" MJ provides it with antimicrobial properties that make it a safe food during storage at 4ºC for 10 days.
In general, this phenolic compound produces a decrease in counts (log CFU/mL) of the seven pathogenic bacteria during storage, reaching undetectable values for E. coli O157: H7 after seven days of storage at 4ºC, while for both PT-LABs the addition of EC does not prevent their survival during storage. The addition of EC to MJ resulted in a final log concentration of between 4.3 and 2.0 CFU/ mL, hence growth was prevented but its effect was largely bacteriostatic, except for E. coli O157:H7, which is bactericidal. Also the addition of EC greatly increases antioxidant activity and the low phenolic functional content of this juice, which could have a potential application in its presser vation, also increases the nutritional value of the product. TA B L E 5 Antioxidant activity and total phenolics of epicatechin, "Piel de Sapo" plain melon juice and supplemented with (-) epicatechin (2,500 µg/ml), stored for 10 days at 4 ºC (mean ± standard deviation) Note: Values in brackets are calculated using the relation between the standard curves with gallic acid or EC. Mean values in the same column and for each method following by different letters are significantly different (p <.05).