Phenolic Profiling of Berries Waste and Determination of Their Antioxidant Potential

Berries waste is a major issue in Australia’s annual food wastage, which can reach 7.3 million tonnes. This study assessed the phenolic content and antioxidant potential of four fruit berry wastes, including blueberries ( Vaccinium corymbosum ), blackberries ( Rubus spp.), raspberries ( Rubus idaeus ), and strawberries ( Fragaria spp.), followed by their characterization and quantiﬁcation. Blueberry wastes were high in phenolic content (total phenolic content: 1.97 ± 0.16mg GAE/g F.W ; total ﬂavonoid content: 220.43 ± 13.15 μ g QE/g F.W ; total tannins content: 16.47 ± 0.98 μ g CE/g F.W ), and antioxidant potentials are 2,2 ′ -diphenyl-1-pic-rylhydrazyl: 2.23 ± 0.17mg AAE/g F.W ; 2,2 ′ -azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid): 1.79 ± 0.09mg AAE/g F.W ; ferric reducing antioxidant power: 68.71 ± 11.11 μ g AAE/g F.W (total antioxidant capacity: 1.22 ± 0.03mg AAE/g F.W ). The LC-ESI-QTOF-MS/MS analysis identiﬁed 87 compounds from blueberry (57), strawberry (40), raspberry (47), and blackberry wastes (27). Indicated by HPLC quantiﬁcation, blueberry wastes had higher levels of phenolic acid (syringic acid and coumaric acid) and ﬂavonoid (kaempferol and kaempfero l-3-glucoside). Our study reported that phenolics from berry wastes could be utilized in diﬀerent food, feed, pharmaceutical, and nutraceutical industries.


Introduction
Berries have grown in popularity over the last decade and are still available for consumption in fresh or processed forms (such as juices, jams, and frozen berries) [1]. Blueberries (Vaccinium corymbosum L.), strawberries (Fragaria spp.), raspberries (Rubus idaeus L.), and blackberries (Rubus spp.) are the most consumed berries [2]. Consumers desire healthy and nutritious food to help them avoid health risks and enhance their health conditions. As a result, customers are more interested in nutritional and functional foods [3]. e annual harvest of berries in Australia is around 109,000 tons [4], and meanwhile, the annual food waste has reached 7.3 million tonnes [5]. Berries wastage occurs at every level of the food supply chain, including the harvest, transportation, storage, and retail [6]. Berries are likely to get soft or mushy due to inappropriate handling, thus leading to economic loss [7]. Berries contain rich phenolic compounds such as anthocyanins, kaempferol, catechins, myricetin, quercetin, and epicatechins [8].
Phenolic compounds are secondary metabolites that have aromatic rings and hydroxyl groups [9]. Individual or in combination, phenolic compounds have a high antioxidant ability [10]. e structure of dietary phenolic compounds ranges from monomers, oligomers to polymers [11]. Phenolic compounds are classified on the basis of their chemical structure and biological role into phenolic acids, flavonoids, and tannins [12]. e positive benefits of phenolic compounds are their robust antioxidant activity, which has the capability of scavenging oxygen radicals and other harmful substances [13]. Phenolic compounds have antiinflammatory properties and the ability to prevent 2.2. Sample Preparation. Four different Australian-grown berries including blueberries (Northern highbush var.), strawberries (Albion var.), raspberries (Chilcotin var.), and blackberries (Driscoll's Victoria blackberries var.) were collected from local markets in Melbourne, Victoria, Australia. Samples were blended into a slurry by using a 1.5 L electric blender (Russell Hobbs Classic, model DZ-1613, Melbourne, VIC, Australia) and stored at -20°C for further analysis.

Extraction of Phenolic Compounds.
Phenolic compounds of the berries waste were extracted using 5 g of the sample in 20 mL 80% ethanol by modifying the protocol of Gu et al. [21]. Slurry samples were homogenized by using Ultra-Turrax ® T25 Homogenizer (IKA, Staufen, Germany) at 10,000 rpm for 20 s followed by incubation in a shaking incubator (ZWYR-240, Labwit Scientific, Melbourne, Victoria, Australia) at 120 rpm, 4°C for 12 hours. Samples were centrifugated (Hettich ROTINA 380R, Melbourne, Victoria, Australia) at 10,000 rpm for 10 min at 10°C. e extracts were filtered through a 0.45 μm syringe filter ( ermo Fisher Scientific Inc., Waltham, MA, USA) for HPLC-PDA and LC-MS/MS analysis.

Estimation of Polyphenols and Antioxidant Assays.
e estimation of phenolics (TPC, TFC, and TTC) and the determination of total antioxidant capacity (DPPH, FRAP, ABTS, and TAC) were performed according to previously published papers by Tang et al. [22]. Absorption data was attained using the Multiskan ® Go microplate photometer ( ermo Fisher Scientific, Waltham, MA, USA).

Determination of Total Phenolic Content (TPC).
Total phenolic content was determined by modifying the spectrophotometric method of Peng et al. [23]. Twenty-five microliters of Folin-Ciocalteu reagent (1 : 3 diluted with water), 25 μL of extracts, and 200 μL water were added to the 96-well plate (Costar, Corning, NY, USA) and performed in triplicate. e reaction mixture was incubated for 5 min at room temperature (∼25°C) and was protected from light. Twenty-five microliters of 10% (w/w) sodium carbonate was added, and the reaction mixture was protected from light for 1 hour at room temperature. Absorbance was measured at 765 nm using a spectrophotometer ( ermo Fisher Scientific, Waltham, MA, USA). e gallic acid standard was used to calculate the calibration curve, with concentrations ranging from 0 to 200 μg/mL. e TPC of the sample was expressed in mg of gallic acid equivalents per gram of the sample (mg GAE/g F.W ).

Determination of Total Flavonoid Content (TFC).
Total flavonoid content was estimated by modifying the aluminum chloride method of Feng et al. [24]. Eighty microliters of the sample, 120 μL of sodium acetate (50 mg/ mL), and 80 μL of 2% (w/v) aluminum chloride were added to the 96-well plate. e absorbance of the reaction mixture was measured at 440 nm after incubation for 2.5 hours at room temperature and was protected from light. TFC was expressed in quercetin equivalent (μg QE/g F.W ), calculated by the quercetin standard curve ranging from 0 to 50 μg/mL.

Determination of Total Tannins Content (TTC).
Total tannins content was determined by modifying the method of Haile and Kang [25]. One hundred and fifty microliters of 4% (w/v) methanolic vanillin solution was added to 25 μL of sample and 25 μL of 32% methanolic sulfuric acid in the 96-well plate. e absorbance of the mixture was measured at 500 nm after the incubation for 15 min. TTC was estimated using a catechin calibration curve with concentrations ranging from 0 to 1000 g/mL and expressed in mg catechin equivalents (CE) per g of sample weight (μg CE/g F.W ).

2,2′
Diphenyl-1-picrylhydrazyl (DPPH) Assay. By adapting the approach of Ouyang et al. [26], the DPPH method was utilized to estimate the berries' free radical scavenging activity. Forty microliters of sample and 260 μL of 0.1 mM DPPH methanolic solution were added to the 96well plate. e absorbance was measured at 517 nm after vigorously shaking the reaction mixture in the dark for 30 min at room temperature. e DPPH radical scavenging activity was determined using an ascorbic acid standard curve with concentrations ranging from 0 to 50 g/mL and expressed in mg of ascorbic acid equivalent per gram (mg AAE/g F.W ) of the sample.

Ferric Reducing Antioxidant Power (FRAP) Assay.
e FRAP assay determines the antioxidant capacity by reducing Fe 3+ to Fe 3+ -TPTZ complex (ferric-2,4,6-tripyridyl-s-triazine) into Fe 2+ -TPTZ. e ferric reducing power of the samples was estimated by modifying the method of Rajurkar and Hande [27]. ree hundred millimolar sodium acetate solution, 10 mM TPTZ solution, and 20 mM Fe [III] solution (ratio 10 : 1:1) were mixed to prepare the FRAP solution. In a 96-well plate, 20 μL of the extract and 280 μL of prepared dye solution were added and incubated for 10 minutes at 37°C. At 593 nm, the absorbance was measured. e results were expressed as μg ascorbic acid equivalents per g (μg AAE/g F.W ), based on the ascorbic acid standard curve ranging from 0 to 150 μg/mL.

2,2′-Azino-bis-(3-ethylbenzothiazoline-6-sulfonic Acid)
(ABTS) Assay. Free radical scavenging activity was estimated by the method of [27] with slight modification. e ABTS + stock solution was prepared by adding 5 mL of 7 mM ABTS and 88 μL of 140 mM potassium persulfate and incubated for 16 hours shielded from light. Ten microliters of sample and 290 μL of ABTS dye solution were added to the 96-well plate incubated for 6 min at room temperature and absorbance was measured at 734 nm. e results were presented in mg ascorbic acid equivalent per gram of sample (mg AAE/g F.W ) with ascorbic acid concentration ranging from 0 to 150 μg/mL for the standard curve.

Determination of Total Antioxidant Capacity (TAC).
As reported in Prieto et al. [28], the phosphomolybdate technique was employed to determine total antioxidant capacity. e antioxidant dye was prepared by the addition of sulfuric acid (0.6 M), 0.028 M sodium phosphate, and 4 mM ammonium molybdate at the ratio of 1 : 1:1. After filling the 96-well plate with 40 μL of sample and 260 μL of antioxidant dye, it was incubated at 95°C for 10 mins. e absorbance at 695 nm was measured after the mixture had been cooled to room temperature. TAC was calculated using the ascorbic acid standard curve at concentrations ranging from 0 to 200 g/mL and expressed in mg ascorbic acid equivalents (AAE) per g of fresh sample weight.

Characterization of Phenolic Compounds by LC-ESI-QTOF-MS/MS Analysis.
Phenolic compounds identification and characterization were performed by using Agilent 1200 series HPLC (Agilent Technologies, Santa Clara, CA, USA) equipped with an Agilent 6530 Accurate-Mass Quadrupole Time-of-Flight (Q-TOF) LC/MS (Agilent Technologies, Santa Clara, CA, USA), and the method was followed as described by Suleria, Barrow, and Dunshea [18]. e flow rate was set at 0.8 mL/min, and the injection volume of the sample was 6 μL. e separation of the phenolic compounds was carried out by using an LC column 250 × 4.6 mm, 4 μm (Phenomenex, Torrance, CA, USA) (column temperature at 25°C, sample temperature at 10°C). Mobile phase A: water and acetic acid (98 : 2), mobile phase B: acetonitrile, water, and acetic acid (50 : 49.5 : 0.5). e condition set for the program was as follows: 0 min with 10% B, 20 min with 25% B, 30 min with 35% B, 40 min with 40% B, 70 min with 55% B, 75 min with 80% B, 77 min with 100% B, 79 min with 100% B, and 82-85 min with isocratic 10% B. Positive and negative modes were used for peak identification. Nitrogen gas was employed as a nebulizer and drying gas at 300°C, with a flow rate of 5 L/min at 45 psi. Capillary and nozzle voltage was placed at 3.5 kV and 500 V, respectively, and the mass spectra were obtained at the range of 50-1300 amu. Data analyses were performed using Agilent LC-ESI-QTOF-MS/MS Mass Hunter workstation software (Qualitative Analysis, version B.03.01, Agilent).

HPLC-PDA Analysis.
e Agilent 1200 series HPLC (Agilent Technologies, CA, USA), equipped with a photodiode array (PDA) detector, was used to quantify the targeted phenolic components in waste from blueberries, strawberries, raspberries, and blackberries. e method is the sightly modification of Feng et al. [29]. e column and conditions were the same as described above in LC-ESI-QTOF-MS/MS procedure, but the sample injected was 20 μL. Phenolic compounds in the samples were evaluated at three different wavelengths (280 nm, 320 nm, and 370 nm). Agilent LC-ESI-QTOF-MS/MS Mass Hunter Data workstation software (Qualitative analysis, version B.03.01, Agilent) was used to gather and analyze data.

Statistical Analysis.
e trials were carried out in triplicate, and the data were provided as the mean ± standard deviation (n � 3). To test for differences in mean values across samples, a one-way analysis of variance (ANOVA) was employed, followed by Tukey's honestly significant differences (HSD) multiple rank test at p < 0.05. Minitab Program (Windows version 18.0; Minitab, LLC, State College, PA, USA) was used to do ANOVA. e significant difference was set at p < 0.05, followed by Pearson's correlation coefficients.

Phenolic Compound Estimation (TPC, TFC, and TTC).
Berries are famous as nutrient-rich fruits with high antioxidant potential [30]. e berries' waste extracts were used to analyze and estimate the phenolic compounds by TPC, TFC, and TTC assays.
In our study, the blueberry waste possessed statistically (p < 0.05) higher phenolic content (1.97 ± 0.16 mg GAE/g F.W ) than strawberry, blackberry, and raspberry waste. Previously, Castrica et al.'s [31] study showed 1253.6 mg TAE/100 g in strawberry waste. Upon comparing with the fresh berries, the blueberries had 2.93 mg GAE/g [32], strawberries had 649.3 to 2123.8 μg GAE/g [33], blackberries had from 22.1 (fresh) to 126.3 mg GAE/g (dehydrated) [34], and raspberry juice ranged between 1879 and 2465 μg GAE/g F.W [35]. e fresh berries have higher TPC than the waste berries. Considering that phenolic compounds can be further characterized by an analytical methodology, the information can be used for various food, nutraceutical, and pharmaceutical industries. e highest concentration of TFC was observed in blueberry waste with a concentration of 220.43 ± 13.15 μg QE/g, which was a similar result to TPC, followed by raspberry, blackberry, and strawberry waste. Bunea et al. [36] reported that TFC values of different varieties of fresh blueberries extracted by acidified methanol ranged between 84.33 and 112.50 mg QE/100g F.W whereas Okan et al. [37] reported that TFC values ranged between 0.40 and 0.50 mg QE/g in fresh blueberries among several cultivars such as Berkeley, Blueray, Darrow, and Misty. e results are also consistent with Basu and Maier [38], in which the TFC value of blueberries was higher than red raspberries, blackberries, and strawberries with 90% ethanol. e waste blueberries have a higher value than the fresh berries; this might be due to the chemical composition and content of the berries, which differ depending on variety, growing area, ambient circumstances, maturation stage at harvest, and storage conditions. [19].
In the TTC assay, the blueberry waste had the highest value, 16.47 ± 0.98 μg CE/g, which is a significantly higher value than the other three samples. Diaconeasa et al. [39] reported that blueberry and raspberry contain significantly higher (p < 0.05) TTC content compared to strawberry and blackberry, which is inconsistent with the results of this study. According to Subbiah et al.'s [32] study, Australiangrown fresh blueberries were 7.41 mg CE/g. e fresh berries exhibited higher values than waste berries which might be attributed to the extraction using various organic solvents. Depending on the type of extraction, solvent selection, and conditions, the antioxidant capacity may also be different. [16].

Antioxidant Activities (DPPH, FRAP, ABTS, and TAC).
As a consequence of the complexity of bioactive compounds in food, it is common to utilize a mix of analytical procedures to measure the antioxidant capacity of food samples utilizing multiple processes [40]. is study determined the antioxidant capacity of four different berries' waste by DPPH, FRAP, ABTS, and TAC assays.
DPPH assay is commonly used to determine antioxidant capacity. Blueberry waste has the highest radical scavenging activity with 2.23 ± 0.17 mg AAE/g, followed by raspberry, strawberry, and blackberry waste. Previously, fresh berries results indicated that blueberry and raspberry had the highest antioxidant activity, followed by blackberry and strawberry with a reasonable degree of antioxidant activity [41]. Subbiah et al. [32] again showed that blueberries followed by raspberries had the highest radical scavenging capacity whereas the blackberry leaves indicated the presence of 111.5 mg AAE/g d.w. [42]. e difference in the antioxidant potential might be due to the fact that the chemical composition and content of the berries are based on their variety, growing location, environmental conditions, maturity stage at the time of harvest, and storage conditions [19].
In FRAP assay, our study reported that blueberry waste had the highest FRAP content with 68.71 ± 11.11 μg AAE/g, followed by blackberry, raspberry, and strawberry waste. Previously, the processing waste of blueberry peel of fatsoluble fraction was 62.56 μmol Fe (II)/g and the watersoluble fraction was 41.99 μmol Fe (II)/g [43]. Another study showed that fresh blueberries (30.0 μmol of Fe 2+ /g) had higher antioxidant activity than raspberries (27.7 μmol of Fe 2+ /g) [44]. A study by Subbiah et al. [32] of Australiangrown berries showed that the blueberries had the highest antioxidant capacity followed by blackberries.
In the ABTS assay, the antioxidant capacity is estimated by the reaction of the extracts with ABTS + radical cation generated in the system [36]. All berries were found with the ABTS radical scavenging activity: blueberry waste possesses the highest value with 1.79 ± 0.09 mg AAE/g F.W , followed by blackberry waste which is significantly higher (p < 0.05) than strawberry and raspberry waste. e antioxidant ability of fresh raspberries was higher than blackberry, strawberry, and blueberry [38]. Blueberries grown in Korea had higher antioxidant activity than strawberries [45]. According to Subbiah et al. [32], strawberries had a higher antioxidant capacity than blueberries. e difference in results may be due to the method of extraction, solvent selection, and conditions [16].
In the TAC assay, the results obtained in our study showed that blueberry waste has a significantly higher (p < 0.05) TAC value than the other three berries waste samples, which had no significant difference among them. Shan et al. [46] reported that the fresh raspberry extract with acetone contains a lower TAC level. In a previous study led by Huang et al. [47], the total antioxidant capacity in the methanolic extract of blueberries was 14.98 mmol Trolox/100 g; that of blackberries was 11.48 mmol Trolox/100 g, and that of strawberries was 4.44 ± 0.45 mmol Trolox/100 g D.W . e fresh blackberry and blueberry TAC were recorded as 6125.7 and 4814.6 mg AAE/100 g D.W , respectively, by Lee et al. [48]. In Subbiah et al.'s [32] study, fresh blueberries followed by raspberries had higher antioxidant activity. Antioxidant potential can be estimated using various mechanisms since no single method has been developed to estimate overall antioxidant potential accurately due to the complex nature of phenolic compounds. Hence, the characterization of phenolic compounds utilizing the analytical approach can compute total phenolic compounds and their antioxidant potential. Blueberry waste had significantly (p < 0.05) higher phenolic concentration and antioxidant activity than other berries waste. Table 1 shows the estimation of phenolic content and antioxidant activity present in berries waste.

Correlation of Phenolics and Antioxidant Activities.
Pearson's correlation test used Graphpad Prism 8 to determine the correlation between TPC, TFC, TTC, and antioxidant assays (DPPH, FRAP, ABTS, and TAC). TAC was significantly correlated with TTC (r � 0.987, p < 0.05), indicating that tannins significantly contributed to the total antioxidant capacity. e positive correlation suggests that tannins are the primary antioxidants in blueberries, strawberries, raspberries, and blackberries. Because tannins include a variety of phenolic hydroxyl groups, phenolic compounds indicate a strong antioxidant potential and free radical scavenging activity [47].
FRAP had reducing capability measured by the ferric ions, also appeared to have a significant correlation with TTC (r � 0.965, p < 0.05), indicating that tannins content in the waste berries samples had an outstanding contribution to ferric reducing ability. e TTC value of blueberries was highly correlated with FRAP [49]. Previous studies reported that the expression of antioxidant activity was due to the synergistic effect between different phenolic compounds and could not be explicitly attributed to one component. us, antioxidant activity is determined not only by concentration but also by the structure and interaction between the antioxidants [50].
is research's correlation coefficient revealed a nonsignificant relationship between TFC and TPC. Previous research has found that TFC and TPC in berries were unrelated as well, considering that total flavonoid content accounts for only 2.4-4.0% of total soluble phenol content in berry fruits [51]. Pearson's correlation coefficients for the relationship between antioxidant determination assays are shown in Table 2.

LC-ESI-QTOF-MS/MS Analysis of Phenolic Compounds in Berry
Waste. LC-ESI-QTOF-MS/MS is an effective tool for preliminary identification and characterization of phenolic compounds in plants by positive (ESI + ) and negative (ESI − ) ionization modes. Based on their m/z and MS spectra, the phenolic compounds were tentatively identified using Agilent LC-MS mass hunter qualitative software and the Personal Compounds Database and Library (PCDL). e mass error <5 ppm and a PCDL score of more than 80 were used to select compounds for further analysis, and compounds were identified using MS/MS identification and m/z characterization (Table 3). In this study, a total of 87 phenolic compounds were characterized in berries waste samples, including phenolic acids (30), flavonoids (50), other polyphenols (5), and lignans (3).
4-Hydroxybenzoic acid 4-O-glucoside (compound 5, m/z 299.0767) and protocatechuic acid 4-O-glucoside (compound 6, m/z 315.0723) were tentatively characterized, and the fragmentation peaks at m/z 137 and m/z 153, respectively, confirmed the compounds due to the corresponding loss of hexosyl moiety (162 Da) from the parent ions [52]. In our study, compound 5 was detected in strawberry and raspberry waste, whereas compound 6 was identified in strawberry, raspberry, and blueberry waste. Compound 5 was previously detected in spices and herbs including anise, caraway, coriander, fennel, and star anise [64]. Fruits and vegetables have a low percentage of 4hydroxybenzoic acid 4-O-glucoside, according to Lafay and Gil-Izquierdo [65]. Other studies detected the presence of compound 5 in blackberries, red raspberries, strawberries Journal of Food Quality [64], blackcurrant, gooseberry, and blueberries [66]. Compound 6 was previously detected in blackberry, blackcurrant, gooseberry, highbush blueberry, and redcurrant [66].

Hydroxycinnamic Acids and Other Phenolic Acid Derivatives.
Sinapic acid (Compound 24, [M-H] − m/z 223.0612) was detected in raspberry and blueberry waste samples, and the compound was confirmed by the fragments at m/z 205, m/z 179, and m/z 163, due to the corresponding loss of H 2 O, CO 2 , and two CH 2 O units from the precursor ion, respectively [67]. Sinapic acid has been previously reported to have pharmacological properties, including antimicrobial, anti-inflammatory, antidiabetes, antimutagenicity, and antioxidant activity, prevents neurodegeneration [68], and increases hair growth [69]. Previously, this compound was detected in strawberries, raspberries, lemon, broccoli, orange, cabbage, and radish [70]. e compound was reported in spices and herbs as well such as anise, basil, nutmeg, and thyme [71].
m-Coumaric acid (compound 23 at m/z 163.0407) was detected in all four samples, and the compound was confirmed by the fragmentation peaks at m/z 119, due to the consecutive loss of CO 2 [72]. Previously, this compound was detected in blueberries, strawberries, red raspberries [73], and extra virgin olive oil [74]. A previous study showed that coumaric acid had significant potential to exhibit anti-inflammatory and antioxidant properties [75].  is compound was confirmed in MS/MS analysis by losing phloroglucinol from the precursor molecule [76]. Previously, this compound was detected in cocoa powder [77], blackberry, strawberry, apricot, peach, plum, pomegranate [78], and apple [79]. Compound 36 ([M+H] + m/z 611.1418) was identified as Prodelphinidin dimer B3 upon MS/MS analysis with the product ions at m/z 469, due to the heterocyclic ring fission followed by removal of phloroglucinol. e product ions at m/z 311 were attributed to the reduction into monomers through quinone methide fission cleavage [80]. e product ions at m/z 291 were due to the loss of the -OH group from gallocatechin [80]. e compound was detected in raspberry and blueberry waste samples in our study. Previously, this compound was identified in whole-grain barley flour [81], blackberry, grape, banana, and broad bean pod [78]. In MS/MS analysis, further confirmation was achieved by the fragment peak at 317 due to the loss of rhamnoside [82]. Previously, it was found in highbush blueberry [83], white myrtle berries [84], and lentils [85]. 3′-Hydroxygenistein (compound 62, precursor ion [M + H] + m/z 285.0538) was identified by the product ions at m/z 269 (loss of H 2 O) and m/z 259 (loss of CO) [86]. is compound was detected in all four berry waste samples.  3); lettering ( a,b,c,d ) indicated the significant difference in the means (p < 0.05) using a one-way analysis of variance (ANOVA) and Tukey's HSD test. GAE: gallic acid equivalents; QE: quercetin equivalents; CE: catechin equivalents; AAE: ascorbic acid equivalents; TPC: Total phenolic content; TFC: total fl usingd content; TTC: total tannin content; DPPH: 2,2′-diphenyl-1-picrylhydrazyl; FRAP: ferric reducing antioxidant power, ABTS: 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid; TAC: total antioxidant content.     "STB", raspberry waste "RAB", blueberry waste "BLB'', and blackberry waste "BKB".
Anthocyanins are powerful natural antioxidants, and anthocyanidins are abundant in berries [87]. e presence of a significant concentration of anthocyanidins in the skin of the berries has been reported by Olas [55]. Anthocyanins are responsible for colors including blue, purple, and red and are used as natural pigments in the food industries [48].

Lignans.
Schisantherin A (compound 86) was identified in positive ionization mode at m/z 537.2123 and the compound had product ions at m/z 519, m/z 415, m/z 385, and m/z 371 and was characterized as schisantherin A due to the loss of H 2 O, C 6 H 5 COOH, C 6 H 5 COOH-CH 2 O, and C 6 H 5 COOH-C 2 H 4 O [94]. In our study, this compound was detected in raspberry, blackberry, and strawberry waste. Previously, this compound was detected in Schisandra rubriflora [95] and Schisandra chinensis [96].
e LC-ESI-QTOF-MS/MS characterization of the phenolic compounds present in four waste berries has remarkable antioxidant potential. Phenolic acids and flavonoids have vigorous radical scavenging activity, and thus, waste berries can be a valuable source of natural antioxidants in various industries, including food, nutraceuticals, and pharmaceuticals.

Venn Diagram Analysis of Phenolic Compounds
Distribution. Berry wastes have a wide range of phenolic compounds and researchers have shown a keen interest. Venn diagrams have been generated for total phenolic compounds, phenolic acids, flavonoids, and other polyphenols according to the berries waste samples in blueberries (blue), strawberries (red), raspberries (yellow), and blackberries waste (green) in Figure 1.
In the total phenolic Venn diagram, the unique compounds present were 37 in blueberry, 21 in raspberry, 27 in blackberry, and 16 in strawberry waste. 36 compounds were overlapping in all four samples and 30 compounds overlapped in strawberry, raspberry, and blueberry waste. Four compounds were the lowest overlapped in strawberry and blackberry waste. According to Rodrigues et al. [97], fresh blueberries have higher total phenolic compounds than raspberries, strawberries, and blackberries.
In the phenolic acids' Venn diagram, the unique compounds present in blueberry, raspberry, blackberry, and strawberry waste were 6, 3, 3, and 3, respectively. e maximum compound overlapped was 12 in strawberry, blueberry, and raspberry waste. e lowest overlapped compound was 1 in strawberry and raspberry waste. Blueberries have high phenolic acid higher phenolic acids than red raspberries [98].
In the flavonoids' Venn diagram, the maximum unique compounds were in blueberry (20) followed by blackberry waste (17). 17 compounds overlapped among all four samples and the lowest overlapped were 2 in strawberry and raspberry waste. Flavonoids were higher in blueberries and blackberries than in raspberries and strawberries [8]. In other polyphenols, the unique compounds were 12, 9, 8, and 6 in blueberry, raspberry, blackberry, and strawberry waste, respectively. e maximum overlapped compounds were 8 in blueberry, strawberry, and raspberry waste. e lowest overlapped compound was in strawberry and blackberry waste.

Heat Map Analysis of Phenolic Compounds.
A heat map and hierarchical clustering diagram were constructed for the quantitative analysis of phenolic compounds in fruit berries by HPLC-PDA. Ten phenolic compounds, including 5 phenolic acids and 5 flavonoids, were identified. e phenolic components of waste or discarded berries such as blackberries, raspberries, blueberries, and strawberries were created in a hierarchically clustered heat map. e samples and the phenolic compounds were present at the axis of the map. e branching pattern showed their similarity, and each branch point showed a divergence. e lighter green color had higher content, while the blue color included less. Hence, the color distinction indicated the difference between the berries. e phenolic compounds were divided into four groups including PC-1, PC-2, PC-3, and PC4, whereas the berries waste groups into BW-1, BW-2, BW-3, and BW-4. e phenolic acids (chlorogenic acid and protocatechuic acid) and flavonoids (quercetin and kaempferol) showed great similarity. e dissimilarity between phenolic acids (gallic acid, syringic acid) and flavonoids (kaempferol-3-glucoside, quercetin) is striking.
Blueberry waste has shown high phenolic acids (syringic acid, p-coumaric acid, protocatechuic acid) and flavonoids (kaempferol, kaempferol-3-glucoside, catechin, quercetin) in the heat map followed by the strawberries. Raspberry waste was noted for its high gallic acid content, followed by strawberry waste. A similar result was observed in Subbiah, Zhong, Nawaz, Barrow, Dunshea, and Suleria [32] where gallic acid was high in raspberries. Huang et al. [99] recorded the presence of gallic acid in strawberries. p-coumaric acid and catechin were present in the blueberry processing waste [100]. Figure 2 shows that the blueberry and blackberry waste were rich in protocatechuic acid, quercetin, and   kaempferol, which agrees with Aaby et al. [101]. Previously, strawberries (gallic acid and catechin), blueberries (gallic acid, chlorogenic acid, quercetin, and catechin), and blackberries were rich in gallic acid [102].

Conclusion
In conclusion, four waste berries were Australian grown with higher phenolic contents and antioxidant potential. Blueberry waste had the highest phenolic acids and antioxidant content. Eighty-seven phenolic compounds in the four berries were identified by LC-ESI-QTOF-MS/MS. Phenolic compounds present in the berry waste were quantified using HPLC. e produced outcomes of berry waste can be beneficial and employed in the food and nutraceutical industries. Further research on bioavailability, bioaccessibility, and toxicology can be conducted to commercialize the components.

Data Availability
e data used to support the findings of this study are available from the corresponding authors upon request.

Conflicts of Interest
e authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript.
is includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.