ANTIMICROBIAL ACTIVITY OF ETHANOLIC EXTRACTS FROM WHEAT, SUNFLOWER AND MAIZE CROP RESIDUES

Large quantities of agricultural residues are generated every year. Most of the crop-based residues are underutilized, mainly left to decay on the land or to be burnt, which can lead to an increase in a load of environmental pollution. Considering this, diff erent strategies have been developed to use these renewable resources as raw materials for the production of bioactive compounds, their isolation and characterization, and potential application in a wide range of fi elds, particularly in the food industry as natural pre-servatives. In this study, the antibacterial effi cacy of wheat, sunfl ower, and maize crop residue ethanolic extracts against six bacterial strains ( Salmonella Typhimurium, Salmonella Enteritidis, Staphylococcus aureus , Escherichia coli , Listeria monocytogenes and Yersinia enterocolitica ) was evaluated by the broth microdilution method. Used extracts inhibited the growth of selected microorganisms with a minimal inhibitory concentration (MIC) o f 320 μg/mL for most of the tested bacteria . L. monocytogenes showed a MIC value o f 640 μg/mL for wheat ethanolic


INTRODUCTION
With the growth of the world's population, which in November 2022 reached 8 billion inhabitants, there is a great necessity for the production of a large amount of food (UN, 2022). Besides China and India, which are the two largest agricultural-producing countries, 157 million hectares of land were used for agricultural production in the European Union (EU) in 2020 (Eurostat, 2022). Th erefore, modern agriculture produces a considerable amount of residues every year, and its vast majority is currently dumped and accumulated in landfi lls or burned (Sadh et al., 2018). In Serbia, the total production of three of the most represented crops in 2021 was as follows: maize 6027131 tons, wheat 3442308 tons, and sunfl ower 607574 tons (Stat.YearB.Serb, 2022). Even though Serbia has a relatively developed agricultural sector (Zekić et al., 2010), agricultural waste is still an underutilized resource (Maksimović, 2022).
Th ere are two types of agricultural residues: fi eld (crop) and process residues. Field residues remain in the fi eld aft er crop harvesting and consist of leaves, stalks, straws, seed pods, stems, hulls, cobs, and weeds. Process residues are residues present even aft er the crop is processed into a valuable alternate resource, and these include husks, seeds, roots, bagasse, and molasses (Sadh et al., 2018). Th e limited and inadequate management of this agricultural waste causing environmental pollution is a global issue that emerged an urgent need to develop strategies based on new sustainable and circular models for waste timely utilization and valorization (Carpena et al., 2022).
Numerous studies have demonstrated that agro-industrial residues are essential sources of various complex and structurally diverse bioactive compounds, including fl avonoids, hydroxycinnamic acid derivatives, phenolic acids, tannins ascorbates, lignans, carotenoids, tocopherols, phytosterols and arabinoxylans (Babbar and Oberoi, 2014;Sadh et al., 2018). Hence, mainly process residues are shown to be raw materials with good prospects for extracting and identifying new compounds with antimicrobial and antioxidant potential (Sihem et al., 2015;Sheng et al., 2022). On the other hand, fi eld residues are abundant lignocellulosic biomass that varies slightly in composition with cellulose, hemicellulose and lignin as the major constituents, and the knowledge of their potential as a raw material for the extraction of diff erent bioactive phenolics is limited (Singh nee' Nigam et al., 2009). Only few data are found in the literature regarding phenolic compounds content in crop fi eld residues, such as wheat, maize and sunfl ower (Kumar and Goh, 2003;Vijayalaxmi et al., 2015;Alexandrino et al., 2021).
During the last two decades, extensive research has been devoted to discovering new antimicrobial agents, mainly from plants and other natural sources that could be applied in pharmaceutical and cosmetic products, as well as in the food industry (Nazzaro et al., 2013). With the development of the all-natural and green-label trend and consumer awareness of food safety and quality, meat and meat products that are highly susceptible to the growth of spoilage microorganisms and foodborne pathogens are of particular interest in regard to fi nding natural preservatives that could be used as safe antimicrobials and antioxidants in meat matrix and packaging (Ji et al., 2021).
Various basic standard methods and complex bioassays have been developed for in vitro antimicrobial susceptibility testing; the well-known and most commonly used methods include disk diff usion, well diff usion and broth or agar dilution (Balouiri et al., 2016). Th e microdilution method is considered a valuable tool for detecting the resistance to antimicrobials and comparing diff erent susceptibility since it is a fast method that does not require many resources and provides information on the lowest concentration that inhibits bacterial or fungal growth (Kolarević et al., 2016).
Considering all mentioned above, the present work is designed to investigate the antimicrobial eff ect of ethanolic extracts from three diff erent agricultural residues -wheat, sunfl ower, and maize against several microbial strains (Salmonella Typhimurium, Salmonella Enteritidis, Staphylococcus aureus, Escherichia coli, Listeria monocytogenes and Yersinia enterocolitica) which are the pathogens identifi ed as the most common causes of foodborne diseases.

Plant materials
Wheat, maize and sunfl ower harvest residues (maize and sunfl ower stalks, as well as wheat straw) originating from the territory of the Autonomous Province of Vojvodina (Serbia) were collected aft er the harvest time, between July and October 2021 and dried naturally in a shaded and well-ventilated place. A 3 kg quantity of each material was fi rst reduced to smaller particles using a grinder; then extracted with a six-fold weight of hexane for 1 h at 40 °C in industrial stainless steel 60 L extractor. Each of obtained hexane extracts was vacuum fi ltered through 87 g/m 2 fi lter paper to remove the hard residues and concentrated using a DLAB RE 200 Pro industrial rotary evaporator (60 °C, 60 rpm, 216-200 mbar, 150 min). Aft er extraction with hexane, plant material was left aside for 24 h in the open air, protected from direct sunlight, in order to remove the traces of residual solvent, and extracted again, for 1 h at 45 °C using a six-fold weight of 96% ethanol, followed by fi ltration and evaporation under the same working conditions. Ethanol extracts were used for further investigations. An aliquot of each extract was taken and diluted with DMSO before the analyses, as described further in the text.

Minimum inhibitory concentration (MIC)
Th e susceptibility of the selected isolates to active compounds was investigated by the broth microdilution method (CLSI, 1999; 2020; ISO, 2019). Each crop residue extract was tested in triplicates. Th e inoculum was prepared by the colony suspension method. Th e stock control culture of each of the six reference strains was sub-cultured on non-selective nutrient agar (NA) (Oxoid ® , UK) at 37 o C for 18 h to 20 h. Th ree to fi ve pure colonies of each microorganism were touched with a loop and suspended in 5 mL sterile saline. Th e suspension was adjusted to give a turbidity equivalent to a 0.5 McFarland standard using a spectrophotometer Cecil 2021 UV/VIS (Select Science, Bath, UK) where at 625 nm wavelength and a 1 cm path cuvette, the absorbance was in the range of 0.08 -0.13. Th e prepared adjusted inoculum (approximately 1×10 8 CFU/ mL) was diluted by transferring 0.1 mL of standardized isolate suspension to a tube containing 9.9 mL of Cation Adjusted Mueller-Hinton broth (CAMHB) (BBL™ Mueller Hinton II Broth, Becton, Dickinson and Company, Sparks, USA) (1:100 dilution) to obtain suspension of 1×10 6 CFU/mL, so when 50 μl is added to an equal volume (50 μl) of the examined solution, resulted in a fi nal inoculum of 5×10 5 CFU/mL. For L. monocytogenes modifi ed susceptibility medium, CAMHB without adding 5% lysed horse blood and 20 mg/mL β-NAD was used (Takahashi et al., 2013). Wheat, sunfl ower and maize crop residues extracts were diluted in DMSO (Fisher Scientifi c™, UK) and added to CAMHB at levels from 2560 μg/mL to 1.25 μg/mL by two-fold dilution in U-bottom 96-well microtiter plates (Kartell S.p.A., Italy). Aft er inoculation, plates were incubated at 37 °C for 18-20 h. MIC was determined as the lowest concentration of an active compound that prevented the visible growth of bacteria in the broth dilution susceptibility test (CLSI, 2012). Tetracycline (Fisher Scientifi c™, UK) was used as a control in the range of 64 to 0.03 μg/mL. Th e plates also included a negative control (media only) and a bacteria growth control (media and bacteria).

Minimum bactericidal concentration (MBC)
Following MIC determination of the crop residue extracts and antibiotic yielding a negative microbial growth aft er incubation, a well's content (10 μL) was sub-cultured on the surface of NA plates to determine the number of surviving cells (CFU/mL). Th e plates were then incubated overnight at 37 °C. Th e minimum bactericidal concentration (MBC) endpoint was defi ned as the lowest concentration of extract that kills > 99.9% of the initial bacterial population where no visible growth of the bacteria was observed on the NA plates (CLSI, 1999). Th e tests were carried out in triplicate.
Th e in vitro antimicrobial activity of ethanolic wheat, sunfl ower and maize crop extracts and the commercial antimicrobial agent is demonstrated in Table 1. For most of the tested bacteria, the MIC value at the examination of all three crop residue extracts was 320 μg/mL. Th e exception was L. monocytogenes which revealed MIC value of 640 μg/mL MIC for wheat ethanolic extract, while the MIC of sunfl ower ethanolic extract for S. Typhimurium was 160 μg/mL. Th ere was no MBC value for any of the microorganisms at the extract's concentrations used (> 2560 μg/mL).

DISCUSSION
Th e susceptibility of the bacterial strains toward the commercial antibiotic tetracycline used in the present study was in accordance with the data reported in the literature (Musumeci et al., 2003;Purushotham et al., 2010).
Th e chemical characterization of the main constituents of plant parts that are considered crop residues, including those examined in the present study was summarized by Sadh et al. (2018). Slight diff erences in the chemical composition among these three crop residues were detected; the highest content of cellulose (61.2%) and the lowest content of lignin (6.9%) were determined in maize stalk residues, sunfl ower stalks had the highest content of hemicellulose (29.7%), lignin (13.4%) and ash (11.17%), while wheat straw had the lowest content of cellulose (32.9%) and ash (6.  (Alexandrino et al., 2021), while in our study S. Typhimurium was the most sensitive bacteria. Th e total phenolic content of this sunfl ower seed fl our was 4.00 g CGA eq/100 g on a dry basis. Aft er concentration, the ethanolic extract had a total phenolic value of 15.44 g CGA eq/100 g, with 62% of chlorogenic acid as the predominant phenolic compound (Alexandrino et al., 2021). According to previous reports, among the analyzed phenolic compounds, chlorogenic acid was predominant in defatted sunfl ower kernels and shells (Weisz et al., 2009). Th e antibacterial eff ect of sunfl ower extracts was mainly attributed to chlorogenic acid as it can bind to the outer bacteria membrane, increase the permeability of the outer and plasma membrane and lead to its damage with the leakage of intracellular components, fi nally resulting in cell death (Lou et al., 2011). Th e effi ciency of sunfl ower-based extracts depends on the concentration of chlorogenic acid, that is, on the purity of the obtained extracts aft er the extraction process and the presence of other compounds in addition to phenolic components, such as soluble sugars or proteins (Alexandrino et al., 2021). Namely, the inhibition of the growth of various bacterial strains, including S. aureus, Streptococcus pneumonia, B. subtilis, E. coli, Shigella dysenteriae, and P. aeruginosa is achieved at concentrations from 10 to 30 times lower using chlorogenic acid with ≥ 98% purity (Lou et al., 2011;Fu et al., 2017). Martillanes et al. (2020) found that in both aqueous and ethanolic rice bran extract, with trans-ferulic acid, p-coumaric acid, and γ-oryzanol as the main components, the growth of E. coli and L. innocua was inhibited. However, the percentage of inhibition was notably higher in an ethanolic extract with high γ-oryzanol and low phenolic compounds concentration. Contrarily, by examination of methanolic and ethanolic extracts from 20 diff erent agroindustrial wastes, the positive correlation between total phenolic content and antimicrobial activity was confi rmed (Martin et al., 2012). Martin et al. (2012) found that, besides the absence of an inhibitory eff ect against gram-negative bacteria (S. Enteritidis and E. coli), the best antimicrobial activity against S. aureus showed ethanol extract of peanut peel with a MIC value of 0.78 mg/ mL and total phenolic value of 374.5 gallic acid equivalent (GAE)/kg, while L. monocytogenes growth was inhibited by guava bagasse ethanol extract (1.56 mg/mL) with a total phenolic value of 43.1 GAE/kg. Compared with the results of our study, ethanolic wheat, sunfl ower and corn crop residues showed higher antimicrobial potency and inhibited both gram-positive and gram-negative bacteria in concentrations twice lower than those obtained by Martin et al. (2012). Among the compounds with antibacterial activity in agro-industrial waste extracts, Martin et al. (2012) confi rmed the predominant presence of dicarboxylic acids: azelaic and succinic acids, then caff eic, p-coumaric, syringic, gallic, ferulic acids and fl avonoids: epicatechin, myricetin, and quercetin. Earlier studies reported 0.9% of polyphenols in wheat crop residues (Kumar and Goh, 2003) and the following organic acids in the wheat straw water extract: o-dihydroxybenzene, p-hydroxybenzoic acid, ferulic acid, and catechinic acid (Hongzhang and Liying, 2007).
In the present study, 96% ethanol was used for the extraction of polyphenol-rich paste from crop residues, whereas Vijayalaxmi et al. (2015) demonstrated that by using 100% ethanol, the extraction yield was better for wheat bran (3.5%) compared to corn husk (4%) and that the total polyphenols, total tannins and total fl avonoids contents in corn husk extract were 35.80 g GAE/100 g extract, 29.33 g tannic acid equivalents (TAE)/100 g extract and 7.35 g quercetin equivalents (QE)/100 g extract, respectively, while in wheat bran these contents were 40.12 g GAE/100 g extract, 33.35 g TAE/100 g and 5.86 g QE/100 g extract, respectively. In that study, HPLC analysis of corn husk and wheat bran extracts detected two major peaks corresponding to gallic acid and ferulic acid and three minor peaks identifi ed as epicatechin, quercetin and kaempferol.
Th e observed antibacterial activity of our crop residue extracts could be due to the fl avonoids; even though they could be found in small amounts, they exhibit membrane-disrupting activities. Th e mechanism of interaction involves the specifi c binding of fl avonoids with the polar head groups of membrane lipids and non-polar compounds inside the membrane, as well as nonspecifi c interactions of fl avonoids and phospholipids that change the thickness and fl uctuations of the membrane and, therefore, indirectly modulate the distribution and/or function of membrane proteins. In this way, binding to the lipid bilayer and inactivation and inhibition of intracellular and extracellular enzymes synthesis results in bacterial cell membrane damage and increased permeability (Górniak et al., 2019). In addition, tannins found in crop extracts can exhibit an antibacterial eff ect due to interactions with proteins in the bacterial cell wall, formation of stable water-insoluble protein components, and interfering with protein synthesis (Si et al., 2012). Si et al. (2012) found that tannins obtained by ethanolic extraction from agricultural by-products inhibited the growth of several pathogenic bacteria, including L. monocytogenes, E. coli, and Methicillin-and Vancomycin Resistant S. aureus.
In accordance with the present results, Alexandrino et al. (2021) did not report the bactericidal eff ect of the sunfl ower seed fl our ethanolic extract against S. aureus, B. subtilis, E. coli and P. aeruginosa in the maximum concentration used (39.8 mg eq CGA/mL). On the other hand, Martin et al. (2012) showed that out of seven ethanolic extracts of diff erent agro-industrial wastes, six had a bactericidal eff ect against E. coli and fi ve against L. monocytogenes, with the observation that the bactericidal potential was lower than the inhibitory one so that, e.g., the MBC value of the most eff ective extract (guava bagasse) against L. monocytogenes was eight times higher than the MIC value.

CONCLUSION
Th e results of our study indicate that ethanolic extract of wheat, sunfl ower, and maize crop residues possess bacteriostatic activity against some of the most common foodborne pathogens. Th is preliminary investigation suggests that non-edible plant parts biomass, mainly consisting of lignocellulose, has the potential as a low-value renewable source for the extraction of bioactive compounds. However, for a better understanding of the mechanism of action of the examined crop residue extracts, further research is required to precisely determine the chemical composition and identify those phenolic compounds to which antimicrobial activity can be attributed. For the appropriate exploitation of such residues in the production of added value compounds, optimizing the extraction process and applying methods that would allow the subjection of a large amount of agricultural residues to treatment with minimal environmental impact and therefore to obtain more extract in a cost-eff ective way is necessary. In the context of crop residue extracts as potential natural antioxidants and antimicrobial agents that could be alternatives to synthetic additives in foods, especially in meat products, research should be directed towards consumer protection in terms of determining whether such extracts are safe for consumption, and later to develop technological processes for meat product reformulation by using such new additives with a deviation in sensory characteristics of the products that would be acceptable to consumers.

Author's Contribution:
MG and MBC -made equal contributions to conceptualization, data curation, formal analysis, investigation and methodology regarding MIC and MBC analysis and wrote an original draft . NČ carried out validation, visualization, and revised the manuscript. MŽB was involved in supervision, funding acquisition and revised the manuscript critically. JV performed microbiology analysis, revised and edited the draft . SS carried out plant material collection, the extracts preparation, reviewed and edited the manuscript. ZM made contributions to conceptualization, project administration, funding acquisition, supervision, review and editing of the draft .