Antibacterial Activity Potential of Industrial Food Production Waste Extracts against Pathogenic Bacteria: Comparative Analysis and Characterization

The Food and Agricultural Organization estimates a 17% loss in the food production chain, making it imperative to adopt scientific and technological approaches to address this issue for sustainability. Industrial food production waste and its value-added applications, particularly in relation to a wide variety of pathogenic microorganisms and the health-related effects have not been thoroughly investigated. This study explores the potential of food production waste extracts—lemon peel (LP), hot trub (HT), and coffee silverskin (CSS) as sources of bioactive compounds. Extraction was conducted using hydro-methanolic extraction with yields in LP (482 mg/1 g) > HT (332 mg/1 g) > CSS (20 mg/1 g). The agar diffusion assay revealed the substantial antibacterial activity of all three extracts against Erwinia Amylovora, Escherichia coli, Bacillus subtilis, and Bacillus aquimaris. All extracts demonstrated activity against Gram-positive and Gram-negative bacteria, displaying minimum inhibitory concentrations effective against pathogenic bacteria like Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus, and Salmonella enterica. Total phenolic content (TPC in mg GAE/1g) was 100, 20, and 100 for CSS, HT, and LP, respectively. Antioxidant activity by ABTS indicated IC50 of 3.09, 13.09, and 2.61 for LP, HT, and CSS, respectively. Also, the antioxidant activity of the extracts was further confirmed by DPPH assay with the best activity in CSS (9.84 GAEg−1) and LP (9.77 mg of GAEg−1) rather than in HT (1.45 GAEg−1). No adverse cytotoxic effects on HaCaT cells were observed. Pancreatic amylase inhibition demonstrated antidiabetic potential, with LP showing the highest levels (92%). LC-MS characterization identified polyphenols as the main compounds in CSS, prenylated compounds in HT, and flavanols in LP. The findings imply the potential sustainable use of food production waste in industry.


Introduction
Globally, 1.3 billion tons of food is wasted annually, translating into 940 billion US dollars and contributing 10% of greenhouse gases.Food production waste, generated across the supply chain, comprises agricultural, industrial, and distribution processes that have a global environmental and economic impact.Household activities contribute 42%, and the food industry and services sectors contribute 39% and 14%, respectively, with 5% lost during distribution [1,2].Disposal of food production waste in landfills releases methane, contributing to climate change.Extracting bioactive compounds, fibers, and proteins from food production waste is crucial for addressing environmental and economic impacts.The sustainable recovery of bioactive compounds from plant waste is essential for enhancing food availability and reducing reliance on cash crops, contributing to industrial food sustainability [3].

Food Production Waste
Coffee silverkin (CSS) was generously donated by Gollücke & Rothfos GmbH Bremen, Germany, for the testing of waste management and its reuse for innovative applications.CSS consists of a thin envelope directly attached to a coffee bean.During roasting, coffee silverskin is recovered after moisture loss [8].Hot trub (HT) from the Bremen beer brewery company, Germany (Becks GmbH & Co., KG, AnBev, Bremen, Germany), was used for analysis of bioactive compounds.HT is a beer slurry waste generated in the brewery industry during beer production.It is the most abundant waste in the industry after spent grain waste.The most common components of hot trub are the hop particles, unstable colloidal protein particles, and carbohydrates.HT is composed of raw material waste from barley, hops, and other sources [16].Lemon peel (LP) is waste generated in the beverage industry and was purchased from Henrich Klenk GmbH and Co. KG (Schwebheim, Bavaria, Germany).LP contains several bioactive compounds such as polyphenols and lipids, making it a potential recyclable material for industrial dietary food applications [17].

Extraction Procedure
The crude phytoextracts from the three food production wastes were assessed by the ultrasonication and maceration methods simultaneously in a stepwise manner while agitating the mixture with a magnetic stirrer as previously reported [18] with modifications.Briefly, 1 g of food production waste was weighed and 10 mL of 70% MeOH solution was added.The mixture was then sonicated in a water bath for 15 min.The mixture was stirred using a magnetic stirrer for 1 h.The extract was filtered using a filter paper filter over a funnel.The extraction procedure was repeated three times.The extract solvent was evaporated in a rotary evaporator, frozen at −80 • C, and lyophilized into powder.The dried weight was quantified to obtain the crude extract yield.This process was repeated in triplicates, the averages and standard deviations were calculated, and their yield was compared.The obtained phytoextract was stored at −20 • C for further analysis.

Antibacterial Screening of Phytoextract 2.4.1. Agar Diffusion Assay
The agar diffusion method is well known for determining inhibition zones on agar plates.This method was used for screening the antibacterial activities of the crude phytoextract as described earlier [5], with some modifications.The sterilized LB agar was prepared as previously described [19], 20 mL was poured into each sterilized plate and allowed to solidify in a flow chamber.The LB agar plates were stored at −20 • C until use.Phytoextracts were screened against the following non-pathogenic organisms: Erwinia amylovora (EA); Escherichia coli (EC), Bacillus subtilis (BS), and Bacillus aquimaris (BA) using 100 µL of a defined organism.An optical density of 0.1 of the bacteria solution spread over an agar plate using sterile glass beads.Bacteria-covered agar plates were prepared with wells filled with 100 µL of extract solution.Controls included water, 70% MeOH, or ampicillin in the wells.After overnight incubation, zones of inhibition were assessed, reflecting the phytoextract's bioactivity against microorganisms.The antibacterial activity test used in this experiment explored two solvents: an aqueous solvent and a 70% methanol solvent for dissolving the extracts.

Minimum Inhibitory Concentration (MIC) Assay
The MIC analysis was performed against seven pathogenic organisms: Listeria monocytogenes, Vibrio parahaemolyticus, Chronobacter sakazakii, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumoniae, and Staphylococcus aureus.The serial dilution method was used to determine the MIC of the phytoextract in a sterilized 96-well plate as described previously [19], with modifications.The pathogenic organisms were pre-cultured overnight using a single colony of bacteria.LB broth was used to dilute the pre-culture to attain an O.D. of 0.1 at 600 nm wavelength, measured using a spectrophotometer.The O.D. of 0.1 was further diluted to achieve 1 × 10 5 bacterial cells/well before analysis.Phytoextracts at Foods 2024, 13,1902 4 of 21 known concentrations were added to a series of dilutions of the microorganisms mentioned above.The microorganisms had an approximate concentration of 1 × 10 5 CFU/mL and were placed in the growth medium in the wells.Each well contained 200 µL of the mixture and the experiments were performed in duplicate.The mixture was then incubated overnight.Microbial growth was observed after the overnight incubation.Phytoextract MICs were compared to experimental controls (ampicillin, only, media, and only water/70% MeOH) performed simultaneously with phytoextracts.The minimum inhibitory concentration of each phytoextract was defined as the minimum concentration at which no bacterial growth was observed.Resazurin is a fluorometric indicator, whose color change aids in determining the MICs of colored extracts by staining [20].Rezasurin dye solution was prepared at a concentration of 0.2 mg/mL for bacterial staining to determine its MIC.After overnight incubation of the bacterial and extract mixtures on MIC plates, 50 µL of the prepared resazurin solution was added.Ampicillin and distilled water were used as controls and the treatments were observed after 3 h for color changes.Suspensions with surviving bacteria had a color like that of the control (media only).

Lactate Dehydrogenase (LDH) Keratinocyte Cytotoxicity Assay
The human immortalized and non-tumorigenic keratinocyte cell line (HaCaT) was kindly supplied from Prof. Dr. Klaudia Brix's laboratory (Constructor University).The cytotoxicity of the phytoextracts was tested using a CzQUANTTM LDH Cytotoxicity Assay kit.

UHPLC-ESI-QTOF-MS Characterization
The obtained phytoextracts were characterized using the UHPLC-ESI-QTOF-MS/MS method described before [18] on an Agilent 1260 (Karlsruhe, Germany) UHPLC system in a negative ion mode.The system coupled to a Bruker Daltonics (Bremen, Germany) Impact QTOF MS with an ESI source in the negative ionization mode was used for identifying the secondary metabolites.The Agilent C-18 Poroshell column (0.5 × 250 mm and pore size 2.7 µm) was connected to the guard column (2.1 × 5 mm and pore size 2.7 µm) for the chromatography separation.The mobile phase constituted of 0.01 v/v formic acid in water (A) and (0.01 v/v formic acid in acetonitrile (B)).The gradient elution started with 5% B moving up linearly over 25% B in 35 min, this was followed by a 10 min linear increase to 45% B and a 5 min linear increase to 85% B with a total run time of 50 min.To equilibrate the chromatographic conditions before injecting the next sample, each run was followed by a 10 min post run.The samples were injected at a volume of 2 µL and an elution flow rate of 0.350 µL/min.The MS conditions were operated as follows: capillary temperature, 200 • C; drying gas flow rate, 9 L/min; nebulizer pressure, 1.8 bars; funnel 1RF, 300 Vpp; funnel 2 RF, 450 Vpp; hexapole RF of 50, Vpp; in-source CID energy, 0 V; transfer time, 80.0 µs; and pre-pulse storage, 7.0 µs.The eluted compounds were detected in the mass range of m/z 80-2000 Da.The tandem mass spectra were obtained in auto-MS n mode (smart fragmentation) by ramping up the collision energy.In the first minute of each chromatographic run, internal calibration was performed using 5 mL of 0.1 M sodium formate solution injected through the six-port valve.The MS data calibration mode used was High precision Calibration (HPC).MS data acquisition was performed using data analysis software 4.2 (Bruker Daltonics, Bremen, Germany).Three processes were employed for the characterization: a literature search of compounds using the Metaboanlyst 6.0 software from BruckerDaltonics, deduction of fragmentation ions from the data, and dereplication.The MS/MS data from the QTOF MS were converted into mzML using proteowizard software version 3.0 (MS convert) and uploaded into an array of software for matching compounds.The extract run underwent matching of the peaks through the Global Natural Products Social Molecular Networking (GNPS), Metlin, and Sirius 5.8.2-win64msi software.The MS data were assigned to compounds in the databases based on high-resolution m/z values and tandem MS fragment spectra.The assigned compounds were further confirmed using literature references.Compound assignment relied on the Foods 2024, 13, 1902 5 of 21 identity within experimental variations in high-resolution m/z values and the similarity of the tandem MS fragments ions as described [21].

Spectrophotometric Analysis 2.7.1. Total Phenolic Content (TPC)
Total phenolic content was determined using a spectrophotometer as described previously [18] with modifications.Briefly, 30 µL of the sample or standard (gallic acid) was mixed with 150 µL of Folin reagent for 5 min at room temperature, and 120 µL of sodium bicarbonate was added.After 90 min of reaction, absorbance was measured using a spectrophotometer at 725 nm.2.7.2. 2,2 ′ -Azino-bis (3-ethylbenzothiazoline-6-sulfonic Acid (ABTS)) Radical Scavenging Activities The antioxidant activities of the phytoextracts were measured using a spectrophotometer following previous work [22] with some modifications.Briefly, 10 µL of each sample was serially diluted in a 96-well plate to determine the IC 50 values of the phytoextracts.The ABTS working solution was prepared and 200 µL was added at a concentration of 0.5 mM.The solution was incubated for 55 min at room temperature and the absorbance was measured at 734 nm of wavelength.IC 50 was quantified and expressed as mg/mL of gallic acid.The IC 50 value defines the concentration required by the phytoextract to scavenge 50% of the ABTS initial radicals.The lower the IC 50 value, the more potent the phytoextract.

2,2-Diphenyl-1-picrylhydrazyl (DPPH) Radical Scavenging Activity
The DPPH radical scavenging of the extracts was determined as described previously [22] with some changes using a 96-well plate wet chemistry assay.Briefly, 20 µL of Phytoextract solution or gallic acid as a standard was pipetted into a 96-well plate (in triplicate).A DPPH solution of 0.5 mM (200 µL) was added to each well and the mixture was gently shaken.The mixture was incubated in the dark for 30 min and the absorbance was measured at 517 nm.

α-Amylase Enzyme Inhibition Activity
The α-amylase inhibitory activity of crude extracts of the food production waste were performed according to the methods described [23], with slight modification.It involved reagents preparation, α-amylase assay optimization, and bioassay of the food waste phytoextract described below.

Preparation of α-Amylase Reagents
The Starch solution (1% w/v) was prepared by boiling 0.5 g of potato starch in 50 mL of milliQ water for 15 min.Dinitrosalicylic acid (DNSA) reagent was prepared by mixing 20 mL of 96 mM DNSA in milliQ water with 5.31 M of sodium potassium tartrate in 2 M of sodium hydroxide (8 mL) and 12 mL of deionized water.

α-Amylase Assay
Experiments were performed to determine the optimum concentration using a concentration series of enzymes ranging from 1 unit/mL-10 units/mL under laboratory conditions.α-amylase (Porcine pancreas, Type VI-B, 23 U/mg of protein) was dissolved in 20 mM of phosphate buffer (pH 6.9) containing 6.7 mM of NaCl to prepare a concentration series.Each enzyme solution (100 µL) was mixed with 100 µL of Milli-Q water in a semi-microcentrifuge tube, incubated at 37 • C for 30 min, and then 100 µL of this mixture was drawn out and mixed with 100 µL of starch solution in another semi-microcentrifuge tube.The samples were incubated again at 37 • C for 10 min and 100 µL of the DNSA reagent was added to the solution and incubated at 85 • C in a heating blot for 15 min.The mixture was diluted with 900 µL of milli-Q water.When preparing blanks for each enzyme concentration, the color reagent solution was added prior to the addition of starch solution to denature the enzyme kept in 85 • C water bath for 15 min, and then diluted with 900 µL of distilled water as explained above.The absorbance of each test solution was measured at 540 nm against the blank and control.Absorbance was determined at each enzyme concentration and the optimum enzyme concentration was determined.
α-Amylase Enzyme Bioassay with Food Production Waste Extracts Each food production waste phytoextract was dissolved in milli-Q water to obtain a concentration of 50 mg/mL.Each food production extract (100 µL) and 100 µL of enzyme solution with an optimum concentration (8 U/mL) were mixed in a semi-micro centrifuge tube and incubated at 37 • C for 30 min.Subsequently, the same procedure as described above was employed.The same procedure was followed to prepare the blanks, but the DNSA reagent was added before the starch.Negative controls were conducted equivalently by replacing the food production waste extracts with 100 µL of milli-Q water.
Percentage inhibition of α-amylase activity = Abs negative control − Abs sample A negative control × 100% (1) where, Abs negative control : absorbance of the negative control at 540 nm; and Abs sample : absorbance of the sample at 540 nm.Statistical analysis: The extraction yield, antibacterial activities, antioxidant activity assays, and α-amylase inhibition were performed in triplicate with MICs in duplicate.All the results were presented as means ± SD.One-way analysis of variance (ANOVA) was performed to compare the different food production waste (p-value) using OriginPro 2018 64-bit software.

Results
An initial screening of three food production waste extracts obtained from food production sites in Northern Germany was performed using an antibacterial assay against standard model organisms.The model organisms considered were EA = Erwinia amylovora (Gram-negative), EC = Escherichia coli (E.coli) (Gram-negative), BS = B. subtilis (Grampositive), and B. aquimaris (Gram-positive).
Based on the experimentally observed properties, which included analysis results for solubility and sensory parameters, the HT, CSS, and LP extracts were chosen for further detailed evaluation.The evaluation includes an agar diffusion assay on model bacterial strains, MIC determination including relevant pathogenic bacteria, and chemical characterization of extracts including some parameters of detailed LC-MS profiling.

Antibacterial Activities and Minimum Inhibitory Concentration
Phytoextracts obtained from three food production wastes were assayed for EA, EC, BS, and BA using various concentrations of lyophilized extract powder (Figure 1).All tested phytoextracts exhibited some level of inhibition against the considered microorganisms.Lyophilized extract powder dissolved at 50 and 100 mg/mL showed good solubility at both concentrations and inhibited the growth of Gram-positive (BS and BA) and Gram-negative (EA and EC) bacteria.The bioactivity of these phytoextracts makes them potential candidates for various applications, such as additives to food, enhancing polyphenol content, and serving as an antioxidant.Comparatively, the overall inhibition of various bacteria by the extract against the selected microbes was lower for HT than for CSS and LP (Figure 1).
The inhibition of the three extracts varied according to their source and concentration.The CSS extract demonstrated high potency against EA, EC, BS, and BA at 100 and 50 mg/mL in water and methanol, respectively.EC growth was more compromised by the CSS extract compared to its effects on EA, BA, and BS at both 100 and 50 mg/mL in methanol and water.Except for ampicillin (positive control), which exhibited a higher inhibition rate than CSS, 70% methanol and water (negative control) showed poor or no inhibition rate (Figure 1A).
Prior studies had not compared the activities of industrial food production wastes under the same conditions for their possible use economically and sustainably such as considering different concentrations and solvents as was carried out in this study.However, a study found that the antibacterial activities of hot trub with different fractions showed some level of bioactivity against both, Gram-positive and Gram-negative bacteria [16].This analysis does not compare the most economical raw materials for industrial application.Therefore, a deeper analysis of various food production wastes under the same conditions is necessary for their realistic industrial application.Moreover, the inhibition of HT varied according to concentrations of 100 mg/mL and 50 mg/mL in water and methanol, respectively.While 100 mg/mL HT highly inhibited the growth of EC, EA, BA, and BS, 50 mg/mL HT was more effective against EC compared to other concentrations (Figure 1B).The marginal inhibition rate of the control group was similar to that observed in Figure 1A.
Similarly, the rate of inhibition of LP against the four model organisms showed effective inhibition against BA at 100 mg/mL in 70% methanol, compared to the rest of the concentrations in all the organisms, but was less active than the positive antibiotic control.It was also observed that 50 mg/mL of LP inhibition by EA and BA was almost the same but at a lower rate than the positive control and significantly higher than the controls with 70% methanol and water only, respectively (Figure 1C).These results are consistent with those of previous studies on coffee extracts [24,25].
Prior studies had not compared the activities of industrial food production wastes under the same conditions for their possible use economically and sustainably such as considering different concentrations and solvents as was carried out in this study.However, a study found that the antibacterial activities of hot trub with different fractions showed some level of bioactivity against both, Gram-positive and Gram-negative bacteria [16].This analysis does not compare the most economical raw materials for industrial application.Therefore, a deeper analysis of various food production wastes under the same conditions is necessary for their realistic industrial application.
Both Gram-positive and Gram-negative pathogens were considered in the current study for determining the MICs of CSS, HT, and LP extracts.The tested pathogens were Listeria monocytogenes, Vibrio parahaemolyticus, Cronobacter sakazakii, Pseudomonas aeruginosa, Salmonella enterica, Klebsiella pneumonia, and Staphylococcus aureus.Following the quantitative serial dilution assay in a 96-well microtiter plate, the MIC values of each extract was determined (Table 1).Antibacterial activity against the two Gram-positive bacteria (Listeria monocytogenes and Staphylococcus aureus) were assessed and MIC values were determined (Table 1).The 70% MeOH-dissolved CSS extract had the lowest MIC (3.1 mg/mL), making it the strongest inhibitor of growth for the two pathogens with slightly less efficiency than the waterdissolved CSS extract.The latter showed MIC values against Listeria monocytogenes and Staphylococcus aureus, with values of 6.3 mg/mL and 12.5 mg/mL, respectively (Table 1).This suggested that water-dissolved CSS extract contained different or lower concentrated antibacterial compounds suggesting a differential extract efficiency for both solvents, which is not surprising.Considering the susceptibility of Gram-negative pathogens to CSS extracts, Vibrio parahaemolyticus had the lowest MIC (Table 1).
Similarly, the LP extract showed the highest potency against Listeria monocytogenes with MICs of 3.2 mg/mL and 6.3 mg/mL for the 70% MeOH-and water-dissolved extracts, respectively.Except for Chronobacter sakazakii and Salmonella enterica with the same MIC susceptibility for LP between 70% MeOH and water extracts, the rest of the MICs for all the pathogens increased two-fold compared to 70% MeOH and water, respectively.This observation could be expected since the composition of the compounds in the dissolved extracts with 70% methanol might differ from that in water and might actually be higher in amount or diversity in each solvent.
In addition, the best MIC exhibited by HT was at 0.4 mg/mL for 70% MeOH dissolved extracts and water-dissolved extracts against Listeria monocytogenes and Pseudomonas aeruginosa, respectively (Table 1).These two bacteria recorded the highest susceptibility towards HT extract in both water and 70% MeOH.Vibrio parahaemolyticus had MICs of 1.6 mg/mL, 6.3 mg/mL, and 1.6 mg/mL for the 70% MeOH-dissolved phytoextract in CSS, LP and HT, respectively.The phytoextracts dissolved in water performed with 6.3 mg/mL, 12.5 mg/mL, and 6.3 mg/mL for CSS, LP, and HT, respectively.The performance of Vibrio parahaemolyticus gives the crude extract more relevance for serving as antibacterial activity towards hygiene product formulation as a means of waste valorization principle.Vibrio parahaemolyticus, though a Gram-negative bacterium, performed better than the inhibition performance of Listeria monocytogenes in terms of MICs (Table 1).Similarly, Cronobacter sakazakii performed weakly when compared with the other pathogens considering the 6.3 mg/mL MIC for CSS and HT phytoextract dissolved in 70% MeOH with the rest being 12.5 mg/mL for the phytoextract (Table 1).Similarly, there was a comparable performance of Cronobacter sakazakii MICs to Kblebsiella pneumoniae MICs, except CSS phytoextract with a 3.1 mg/mL MIC and a 6.5 mg/mL MIC for the LP phytoextract, the rest having 12.50 mg/mL (Table 1).Both 70% MeOH and water for the CSS, LP, and HT phytoextracts showed the lowest MICs against Salmonella enterica with 12.50 mg/mL (Table 1).
In addition, their antibacterial activity as crude materials gives them the advantage of being used as active agents for natural products in cosmetics [8].The above-reported MICs imply possible synergistic effects of various potentially bioactive compounds in the Foods 2024, 13,1902 9 of 21 tested extracts contributing to the bioactivity that has been previously reported [26].The latter study focused on plant-borne phenolics and their antibacterial activities.The results confirmed previous studies, which analyzed the bioactivity of similar food production wastes in terms of MIC determination.In this study, the achieved MIC values were lower than those reported for the total crude extracts.MICs of 37.5 mg/mL and 75 mg/mL were reported for Staphylococcus aureus and Pseudomonas aeruginosa, respectively, from coffee aqueous extracts and, in another study, showing MICs > 200 and suggesting the MIC value for coffee silverskin to be above 512 mg/mL [10].Additionally, the organisms considered in this study were mainly food pathogens, including Listeria monocytogenes, Vibrio parahaemolyticus, Salmonella enterica, and Cronobacter sakazakii.
Even though the mechanisms of plant-based extracts are not well studied, several pieces of evidence point to the mechanisms of polyphenols as antibacterial agents against microorganisms.Flavonoids, such as quercetin, are known to inhibit DNA gyrase, catechins inhibit the cytoplasmic membrane function of microorganisms, and chalcones (such as xanthohumol) inhibit the energy metabolism of microorganisms, leading to cell death [27][28][29].The numerous bioactive compounds characterized in each phytoextract (CSS, HT, LP) point to the efficient antibacterial activities observed in each extract against the seven pathogenic organisms.However, the antimicrobial effect cannot be attributed to a single compound in the extract but a concomitant effect with multiple mechanisms participating in the killing of the bacteria.Polyphenols inhibit microbial enzymes by reacting with sulfhydryl groups or through nonspecific protein interactions [30].Another mechanism of polyphenol compounds against microbes is to react with protein sulfhydryl groups, generating phenolic toxicity and hindering microbial growth [31].

Antioxidant Sum Parameters of Extracts
The extraction yields of the food production waste were successful, with high amounts obtained from each extract: CSS (20 mg/1 g), HT (331 mg/1 g), and LP (482 mg/1 g) (Figure 2A).Additionally, the extraction yields indicated significant differences between the means, with p < 0.05.
Polyphenolic compounds are vital plants secondary metabolites which have important functional properties such as antioxidants or antibacterial properties.Spectrophotometric determination using the Folin-Ciocalteau, DPPH, and ABTS assays constitute useful parameters for estimating total polyphenol content (TPC) and antioxidant capacities.Using gallic acid equivalent (GAE) as a proxy, the assay was employed on the three bioactive phytoextracts.
The extraction yields of the food production waste were successful, with high amounts obtained from each extract: CSS (20 mg/1 g), HT (331 mg/1 g), and LP (482 mg/1 g) (Figure 2A).Additionally, the extraction yields indicated significant differences between the means, with p < 0.05.
The TPC content obtained included 140 mg GAE g −1 , 100 mg GAE g −1 , and 20 mg of GAE g −1 CSS, LP, and HT, respectively (Figure 2B).Among the three samples, the TPC was higher in CSS if compared to LP. HT showed the lowest TPC values the least.The different food production waste was independent of the TPC with p < 0.05.
The ABTS antioxidant activity of the extracts of each waste material was defined by the IC 50 value.The amount of each extract activity showed that CSS recorded the highest radical scavenging ability of 1.88 mg/mL as against LP with 3.08 mg/mL, and HT with the lowest of 12.93 mg/mL IC 50 , respectively.However, comparing the statistical means revealed that the extract's ABTS activities were significantly different from each other with p < 0.05.
The results revealed a higher difference among the phytoextracts, such as HT compared to CSS and LP.As shown in Figure 2C,D, CSS had the highest radical scavenging of 9.94 g-GAE/kg followed by LP with 9.77 g-GAE/kg and HT with the lowest level at 1.45 g-GAE/kg (Figure 2D).In addition, the mean of the three industrial wastes shows them to be significantly different from each other with p < 0.05.

In Vitro α-Amylase Inhibition Activity
A common property of plant polyphenols is their ability to non-selectively bind to proteins through H-bonds and π-interactions.This phenomenon often leads to the loss of enzyme functions.As a model, the enzyme α-amylase was chosen to further characterize the extracts under investigation, serving as a powerful proxy for the protein-binding properties of the extracts.The polyphenols with antibacterial and antioxidant potential exhibited by the food production waste extract demonstrated relevance for their enzyme inhibition.
The percentage of α-amylase inhibitory activity was analyzed for the three extracts (Figure 3) using α-amylase.Comparing the three industrial waste extracts, they exhibited difference significance with p ≤ 0.05.Using 50 mg/mL of the LP extract exhibited the highest inhibition rate of 92.14%, while CSS and HT displayed almost the same inhibition effect with mean percentages of 60.12% and 61.01%, respectively.
Controlling the release of glucose from dietary carbohydrates is one way to reduce blood glucose levels in patients with type II diabetes mellitus.This may be achieved by inhibiting carbohydrate hydrolyzing enzymes in the digestive organs such as α-amylase, which catalyzes the hydrolysis of starch into maltose and glucose [33].Synthetic α-amylase inhibitors such as acarbose control postprandial hyperglycemia [34].The higher the total phenolic content in the extract, the higher its corresponding radical scavenging activity in both ABTS and DPPH, suggesting that the most active class of compounds responsible for the extracts could be phenolic compounds.ABTS and DPPH are among the most used in vitro assays for antioxidant sum parameters, based on the neutralization of radicals by antioxidant compounds.ABTS works as an antioxidant via a reaction with an organic radical.ABTS is a good method for the measurement of bioactive compounds such as polyphenols and flavonoids [18,32].DPPH has an intense deep purple color.In assays, the DPPH radical is neutralized by accepting either a hydrogen atom or an electron from an antioxidant species, converting it into a reduced form (DPPH or DPPH-H).The TPC obtained in the three-food production wastes correlates with the antioxidant activity of ABTS and DPPH (Figure 2B-D).It was also observed that the higher the polyphenolic content, the higher the antioxidant activity.The ABTS cationic radical generated is soluble in both organic and aqueous media, unlike DPPH, which dissolves only in organic media.Consequently, ABTS can screen both hydrophilic and lipophilic compounds, unlike DPPH, as observed in the results, confirming the compounds participating in the bioactivity of the extract are polyphenols from the extracts.

In Vitro α-Amylase Inhibition Activity
A common property of plant polyphenols is their ability to non-selectively bind to proteins through H-bonds and π-interactions.This phenomenon often leads to the loss of enzyme functions.As a model, the enzyme α-amylase was chosen to further characterize the extracts under investigation, serving as a powerful proxy for the protein-binding properties of the extracts.The polyphenols with antibacterial and antioxidant potential exhibited by the food production waste extract demonstrated relevance for their enzyme inhibition.
The percentage of α-amylase inhibitory activity was analyzed for the three extracts (Figure 3) using α-amylase.Comparing the three industrial waste extracts, they exhibited difference significance with p ≤ 0.05.Using 50 mg/mL of the LP extract exhibited the highest inhibition rate of 92.14%, while CSS and HT displayed almost the same inhibition effect with mean percentages of 60.12% and 61.01%, respectively.

UHPLC-ESI-QTOF-MS Characterization of Compounds in Phytoextracts of the Three Production Wastes
The database search revealed 30 compounds identified in CSS, 22 in HT, and LP.Figures 4-6 shows structures of some selected identified compounds from the ex Figure 7 shows a representative base of peak chromatograms from the extracts pounds with errors above 5 ppm were not considered.

Characterization of Coffee Silverskin Phytoextract
The 30 compounds were identified in CSS following database matching (Figu Some of the main compounds are chlorogenic acid eluted at 20.5 min with m/z of 353 (C16H18O7).This peak was identified as chlorogenic acid (5-caffeoylquinic acid) owi the matching fragment ions at the base peak at 191.0542, the other peaks include 85 87.0071, and 93.0327.Similarly, 1, 3, 7-trimethyluric acid was tentatively assigned t parent ion at 209.0667 (C7H13O7) with its base peak fragment ion occurring at 137.0222 other peaks at 79.9583 and 179.0168 occurring at 13.6 min.Hyperoxide appeared a beginning of the chromatogram at 4.4 min with a main peak at 463.0882 (C21H19O12 with a base peak at 300.0258 and other fragment ions at 271.0236 and 301.0312.Ta provides detailed information on all the compounds identified in the CSS extract Chlorogenic acids predominantly identified in coffee silverskin have been shown in Controlling the release of glucose from dietary carbohydrates is one way to reduce blood glucose levels in patients with type II diabetes mellitus.This may be achieved by inhibiting carbohydrate hydrolyzing enzymes in the digestive organs such as α-amylase, which catalyzes the hydrolysis of starch into maltose and glucose [33].Synthetic α-amylase inhibitors such as acarbose control postprandial hyperglycemia [34].

UHPLC-ESI-QTOF-MS Characterization of Compounds in Phytoextracts of the Three Food Production Wastes
The database search revealed 30 compounds identified in CSS, 22 in HT, and 34 in LP.Figures 4-6 shows structures of some selected identified compounds from the extracts Figure 7 shows a representative base of peak chromatograms from the extracts Compounds with errors above 5 ppm were not considered.without losing their biological properties.CSS is a byproduct of coffee production, which has been suggested as an antibacterial agent [10].2.  2.
the hops.The chromatogram confirmed the presence of most of these compounds data analysis.A typical example is Xanthohumol with an m/z peak at 353.1384 (C2 and fragment ions at 119.0498 (base peak), 175.0032, and 163.0025, which occur min.Cohulupone with an m/z peak at 317.1758 (C19H25O4) occurred towards the end chromatogram at 49.9 min and characteristic fragment ions at 205.0863 (base 203.0449, and 133.0652.Similarly, the prenylated compound 8-Prenylnarigenin wa tified with an m/z peak at 339.1238 (C20H19O5) and its characteristic peaks at 119.049 peak), 133.0652, 219.0630, and 93.0333.Most of these compounds have already bee tified in hot trub [16].Hop compounds have been investigated for their antibacte tivities.However, there is limited research on their broad spectrum of antibacter antifungal activities, as well as the yield of these extracts compared to other waste Table 3 indicates the detailed information of each fragmentation pattern pea total number of compounds identified and characterized in HT was 22 (Table 3).Th tified compounds were predominantly α and ß-acids of hop, which was expected the partial insolubility of bitter acids during beer production [16].It has been estab that the alpha acid present in beer production usually absorbs yeast cells, trub, and materials causing it to undergo different chemical changes to form several comp [38].The extraction procedure requires a controlled manner to recover the majo these compounds in HT.Of the 22 compounds identified in HT, most of them hav reported to be bioactive and nontoxic to several bacteria and fungi.HT extract shown nontoxicity against various cell lines making them valuable resources for cular economy [16].Structures of some of the identified compounds are presented ure 5.  3.  3.  [39].The structures of some of the compounds identifi in the LP extract are shown in Figure 6.  4.  4.

Characterization of Coffee Silverskin Phytoextract
The 30 compounds were identified in CSS following database matching (Figure 4).Some of the main compounds are chlorogenic acid eluted at 20.5 min with m/z of 353.0851 (C 16 H 18 O 7 ).This peak was identified as chlorogenic acid (5-caffeoylquinic acid) owing to the matching fragment ions at the base peak at 191.0542, the other peaks include 85.0289, 87.0071, and 93.0327.Similarly, 1, 3, 7-trimethyluric acid was tentatively assigned to the parent ion at 209.0667 (C 7 H 13 O 7 ) with its base peak fragment ion occurring at 137.0222 with other peaks at 79.9583 and 179.0168 occurring at 13.6 min.Hyperoxide appeared at the beginning of the chromatogram at 4.4 min with a main peak at 463.0882 (C 21 H 19 O 12 ) and with a base peak at 300.0258 and other fragment ions at 271.0236 and 301.0312.Table 2 provides detailed information on all the compounds identified in the CSS extract [35].Chlorogenic acids predominantly identified in coffee silverskin have been shown in previous work to possess antiviral activity [36].
The derivatives of chlorogenic acids such as caffeoylquinic acids: 3-caffeolyquinic acid, 5-caffeolyquinic acid, 3,5-dicaffeolyquinic acid, 3,4-dicaffeoylquinic acid, 3,5-di caffeoylquinic acid, and 4,5-di-caffeoylquinic acid are known bioactive compounds locked in CSS and require recovery and potential application [10,37].The presence of these compounds in CSS confirms the possible synergistic phytoextract bioactivity against microorganisms.It also implies the extraction method used sustained the bioactive compounds without losing their biological properties.CSS is a byproduct of coffee production, which has been suggested as an antibacterial agent [10].
Foods 2024, 13,1902 pounds are indicated in Figures 4-6.The presence of these compounds in the phytoextracts demonstrates the retention of several bioactive compounds after the extraction process.The chromatogram represents the intensity and retention time of the identified compounds in each extract including the coffee silverskin base peak chromatogram (Figure 7A), hot trub phytoextract base peak chromatogram (Figure 7B), and lemon peel base peak chromatogram (Figure 7C).133.0652, 219.0630, and 93.0333.Most of these compounds have already been identified in hot trub [16].Hop compounds have been investigated for their antibacterial activities.However, there is limited research on their broad spectrum of antibacterial and antifungal activities, as well as the yield of these extracts compared to other waste.
Table 3 indicates the detailed information of each fragmentation pattern peak.The total number of compounds identified and characterized in HT was 22 (Table 3).The identified compounds were predominantly α and ß-acids of hop, which was expected due to the partial insolubility of bitter acids during beer production [16].It has been established that the alpha acid present in beer production usually absorbs yeast cells, trub, and other materials causing it to undergo different chemical changes to form several compounds [38].The extraction procedure requires a controlled manner to recover the majority of these compounds in HT.Of the 22 compounds identified in HT, most of them have been reported to be bioactive and nontoxic to several bacteria and fungi.HT extracts have shown nontoxicity against various cell lines making them valuable resources for the circular economy [16].Structures of some of the identified compounds are presented in Figure 5.

Characterization of Lemon Peel Phytoextract
Herein, 34 compounds were identified using LC-ESI-QTOF-MS (Table 4) with literature and database matching.Characterization of the compounds indicated that Vicenin-2, which was previously identified in LP, eluted at 7 min with a major m/z mass of 593.1512 (C 27 H 29 O 15 ) and its fragment ions at 353.0637 (base peak), 383.0748, and 325.0689.In addition, Lacitrin-O-rutinoside was identified in LP with a main m/z of 639.1569 (C 28 H 31 O 17 ) and 315.0127 (base peak fragment), and other fragment ions at 330.0362, 316.0184, and 331.0452 at 31.3 min of the chromatogram.Eriodictyol was eluted at 44.2 min with an m/z peak at 287.0561 (C 15 H 11 O 6 ) and its characteristic fragment ions were 135.0435 (base peak), 134.0365, 83.0117, and 151.0054 [39].The structures of some of the compounds identified in the LP extract are shown in Figure 6.
Figure 7 indicates the base peak chromatogram of the three food waste valorizations considered in this study.The chromatographic diagram shows several peaks of high intensity and has been characterized to identify the compounds in these phytoextracts.Details of the compounds are listed in Tables 2-4.The structures of selected identified compounds are indicated in Figures 4-6.The presence of these compounds in the phytoextracts demonstrates the retention of several bioactive compounds after the extraction process.The chromatogram represents the intensity and retention time of the identified compounds in each extract including the coffee silverskin base peak chromatogram (Figure 7A), hot trub phytoextract base peak chromatogram (Figure 7B), and lemon peel base peak chromatogram (Figure 7C).

Cell LDH Cytotoxicity Activity of Phytoextracts
The safety of a product for target users is crucial for preventing negative side effects.The first point of contact of the product is the skin, which needs assessment of its safety; hence the need for this cytotoxicity testing.HaCaT cells are a model cell line regarded as suitable for in vitro testing of the cytotoxicity of substances or products intended for dermatological applications.The LDH assay was used to measure cell damage and the release of oxidative stress to increase the cell membrane permeability.As represented in the Supplementary Figure S1, none of the three extracts showed nontoxicity when compared to the control.In this study, cell cytotoxicity assessment was conducted using an LDH assay against the keratinocyte cell line and HaCaT cells, due to the bioactivity of all the extracts (CSS, HT and LP, Table 1).The positive control used had a higher absorbance at 0.78 nm (results shown in Supplementary Information Figure S1).The cytotoxicity assays of the HaCaT cells at varying concentrations of the extracts indicated that none of the extracts' tested concentrations exceeded the positive control when assessing their toxicity towards HaCaT cells.The varying concentrations of the cytotoxicity testing were considered due to differences in MIC values at different concentrations of extracts and solvents (Table 1).Since the food production extracts are nontoxic to the skin cells (HaCaT cells), we suggest their possible application as disinfectants or for hygiene applications in the food industry.Furthermore, their nontoxicity shows their possible application as food ingredients and ultimately for the isolation of bioactive compounds for pharmaceutical application.It was previously established that extracts from food production waste are nontoxic due to their origin from food sources, such as hot trub [16].

Conclusions
In conclusion, this research demonstrates that extracts of food production waste materials such as coffee silverskin, lemon peel, and hot trub possess antibacterial activities against both Gram-positive and Gram-negative bacteria.A basic LC-MS-based chemical characterization reveals chlorogenic acids in CSS, flavonoids in LP, and terpenes in HT as contributors to antibacterial activities.Such activities could also be demonstrated against relevant pathogenic bacteria such as Listeria monocytogenes, Staphylococcus aureus, Vibrio parahaemolyticus or Salmonella enterica.Experimentally determined MIC values suggest that such extracts could be employed in multiple applications including hygiene applications in the food industry or public spaces such as hospitals or public transport, cosmetic applications in deodorants, or applications in crop protection.

Figure
Figure 2B-D, respectively, illustrates TPC, ABTS, and DPPH scavenging activity.The higher the total phenolic content in the extract, the higher its corresponding radical scavenging activity in both ABTS and DPPH, suggesting that the most active class of compounds responsible for the extracts could be phenolic compounds.ABTS and DPPH are among the most used in vitro assays for antioxidant sum parameters, based on the neutralization of radicals by antioxidant compounds.ABTS works as an antioxidant via a reaction with an organic radical.ABTS is a good method for the measurement of bioactive compounds such as polyphenols and flavonoids[18,32].DPPH has an intense deep purple color.In assays, the DPPH radical is neutralized by accepting either a hydrogen atom or an electron from an antioxidant species, converting it into a reduced form (DPPH or DPPH-H).The TPC obtained in the three-food production wastes correlates with the antioxidant activity of ABTS and DPPH (Figure2B-D).It was also observed that the higher the polyphenolic content, the higher the antioxidant activity.The ABTS cationic radical generated is soluble in both organic and aqueous media, unlike DPPH, which dissolves only in organic media.Consequently, ABTS can screen both hydrophilic and lipophilic compounds, unlike DPPH, as observed in the results, confirming the compounds participating in the bioactivity of the extract are polyphenols from the extracts.

Figure 4 .
Figure 4. Compounds identified in coffee silverskin phytoextracts by LC-ESI-QTOF-MS.Detailed information can be found in Table 2.Figure 4. Compounds identified in coffee silverskin phytoextracts by LC-ESI-QTOF-MS.Detailed information can be found in Table2.

Figure 4 .
Figure 4. Compounds identified in coffee silverskin phytoextracts by LC-ESI-QTOF-MS.Detailed information can be found in Table 2.Figure 4. Compounds identified in coffee silverskin phytoextracts by LC-ESI-QTOF-MS.Detailed information can be found in Table2.

Figure 7 .
Figure 7. LC-ESI-QTOF-MS base peak chromatograms of phytoextracts in negative ion mode: (A) coffee silverskin with details in Table 2, (B) hot trub with details in Table 3, and (C) lemon peel.All plots represent intensity versus retention time.Details of identified compounds are in Tables 2-4.

3. 4 . 2 .
Characterization of Hot Trub Phytoextract In hot trubs, the most known compounds are usually the remaining bitter acids from the hops.The chromatogram confirmed the presence of most of these compounds in the data analysis.A typical example is Xanthohumol with an m/z peak at 353.1384 (C 21 H 21 O 5 ) and fragment ions at 119.0498 (base peak), 175.0032, and 163.0025, which occur at 50.7 min.Cohulupone with an m/z peak at 317.1758 (C 19 H 25 O 4 ) occurred towards the end of the chromatogram at 49.9 min and characteristic fragment ions at 205.0863 (base peak), 203.0449, and 133.0652.Similarly, the prenylated compound 8-Prenylnarigenin was identified with an m/z peak at 339.1238 (C 20 H 19 O 5 ) and its characteristic peaks at 119.0496 (base peak),

Table 1 .
Minimum inhibitory concentration (MIC) of food production waste phytoextracts against seven pathogenic microorganisms.

Table 2 .
Database identification of compounds from coffee silverskin methanolic extract using LC-ESI-QTOF-MS with their MS fragments in negative ion mode.

Table 3 .
Comprehensive identification of Hot Trub compounds using LC-ESI-QTOF-MS with their MS fragments in negative ion mode.

Table 4 .
Comprehensive identification of lemon peel compounds using LC-ESI-QTOF-MS with their MS fragments in negative ion mode.Foods 2024, 13, 1902 and 315.0127 (base peak fragment), and other fragment ions at 330.0362, 316.0184, a 331.0452 at 31.3 min of the chromatogram.Eriodictyol was eluted at 44.2 min with an m peak at 287.0561 (C15H11O6) and its characteristic fragment ions were 135.0435 (base pea 134.0365, 83.0117, and 151.0054