Enzymatic, Antioxidant, and Antimicrobial Activities of Bioactive Compounds from Avocado (Persea ame

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2.5. Enzyme Activities of AS Samples

Since plants are a valuable source of enzymes [ 52 ], a comparative study of important enzyme activities in AS extracts was carried out. The obtained results of the study are delineated in Table 3 .

Table 3

Enzyme activities of AS extracts obtained by ultrasonic extraction (UE; solvent H ₂ O), Soxhlet extraction (SE; solvent EtOH), and supercritical fluid extraction (SFE; solvent scCO ₂ , co-solvent EtOH).

Enzymes	Activity (U/g ± SD)
Enzymes	UE (H ₂ O)	SE (EtOH)	SFE (scCO ₂ + EtOH)
Cellulase	-	-	30.74 ± 0.11
Lipase	56.30 ± 0.49 ^a	-	24.54 ± 0.20 ^a
Peroxidase	0.01 ± 0.00 ^a	0.01 ± 0.00 ^a	0.01 ± 0.00 ^a
Polyphenol oxidase	4087.50 ± 71.43 ^a	4250.00 ± 84.01 ^a	3451.99 ± 47.17 ^b
Protease	0.10 ± 0.01 ^a	0.01 ± 0.00 ^a	0.33 ± 0.03 ^b
Transglutaminase	0.06 ± 0.01 ^a	0.05 ± 0.00 ^a	0.02 ± 0.00 ^b
Superoxide dismutase	1435.97 ± 21.41 ^a	3123.97 ± 69.05 ^b	638.55 ± 15.99 ^c

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Each value represents mean of three replicates ± SD. Different letters in the same row indicate significant difference ( p < 0.05).

The results of the study showed cellulase activity only in the SFE extract. Cellulases are extremely applicable enzymes in many industries and have recently shown good potential in the fight against antibiotic-resistant bacteria [ 53 ] and in the conversion of agricultural waste into bioethanol and sugar [ 54 ]. Furthermore, the highest lipase activity was demonstrated in the UE extract (56.30 U/g), followed by the activity in the SFE extract (24.54 U/g), while lipase activity was not detected in the SE AS extract. Since lipases catalyze the hydrolysis of ester-carboxylate bonds and release organic alcohols and fatty acids with high efficiency and stability [ 55 ], they are extremely appealing from a commercial point of view for quite a few industries. Many different seeds have already been demonstrated as a potential source for possible lipase exploitation [ 56 ], and with this study, AS have also become an attractive alternative. Thereafter, peroxidases are important antioxidant enzymes that are also applied in medicine, analytics, agriculture, and other fields [ 57 ]. Peroxidase was active in all AS extracts, but compared to other analyzed enzymes, its activity was the lowest. On the contrary, the polyphenol oxidase (PPO) activity was generally the highest and was expressed in all extracts (SE > UE > SFE, up to 4250.00 U/g). The results are not surprising since PPO is the enzyme responsible for the browning. In the presence of oxygen, PPO changes phenolic compounds into various quinones through the oxidation process, which further react and form melanin. Melanin is dark pigment that colors the fruits/seeds brown. When AS are crushed in the presence of air, they soon develop a red-orange color [ 58 ]. Hatzakis et al. [ 59 ] discovered that AS extract contains a pigment called perseorangin, which is a yellow-orange solid and is the result of a PPO-dependent reaction. All AS extracts also resulted in protease (SFE > UE > SE) and transglutaminase activity (UE > SE > SFE). Plant proteases are also actively used in medicine, as they exhibit a wide spectrum of therapeutic actions. Moreover, antimicrobial, antioxidant, anti-inflammatory, and antidiabetic properties of bioactive peptides obtained from plant proteases have also been proven [ 60 ]. Transglutaminase, in addition to other applications (e.g., food additives), may be considered as an innovative category of wound-healing mediators [ 61 , 62 ]. Next, superoxide dismutase (SOD) activity has been evaluated in AS extracts. Specifically, SE and UE AS extracts demonstrated high SOD activity (up to 3123.97 U/g), while the activity in SFE extract was slightly lower. Due to their antioxidant effects, SODs have enormous potential for applications in many industries, including medicine, as an abundant number of studies have reported their physiological importance and therapeutic potential [ 63 ]. Importantly, to the best of our knowledge, only studies containing enzyme-inhibitory potential against specific enzymes [ 64 , 65 ] are found in the reviewed literature, while no similar comparative study covering the activity of selected enzymes in any AS extracts has been published so far. Accordingly, the presented results greatly contribute to the identification of valuable enzymes in AS, which could potentially be a source for their exploitation and further applications in divergent branches and industries. However, it is important to point out that only the SFE extract contains all the tested enzymes in their active form; therefore, the SFE extract is the most suitable source of active enzymes from AS of all tested extracts.

2.6. Antioxidant Activity of AS Samples

Plants contain a large number of bioactive compounds with high antioxidant activity. Studies of the antioxidant activity of various plant species contribute to revealing the value of these species as a source of new antioxidant compounds. Therefore, the antioxidant potential of AS extracts was examined. The results are depicted in Figure 3 .

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Figure 3

Antioxidant activities of AS extracts obtained by ultrasonic extraction (UE; solvent H ₂ O), Soxhlet extraction (SE; solvent EtOH), and supercritical fluid extraction (SFE; solvent scCO ₂ , co-solvent EtOH).

Using the DPPH method, the extracts showed inhibition in the range of 58.50–67.49%. The highest antioxidant activity was detected in SE extract, followed by SFE and UE extract. Health-promoting biologically active compounds (e.g., phenolic compounds) definitely contribute to the antioxidant potential of the extracts. Various studies indicate that some phenolic compounds (epicatechin, quercetin, benzoic acid, caffeic acid, chlorogenic acid, ferulic acid, 4-hydrohibenzoic acid, and p-coumaric acid) also quantified in the presented study have already been proven to be associated with the antioxidant activity of AS extracts [ 66 ].

Nonetheless, the before-mentioned high enzyme activity of the SOD enzyme in AS extracts, which is well known for its antioxidative effects, should be emphasized. Kruskal–Wallis H test showed that there was a statistically significant difference in the antioxidant activity of AS extracts prepared by different extraction methods. For comparison, Weremfo et al. [ 19 ] detected 73.61% inhibition of AS EtOH extract obtained by microwave-assisted extraction (MAE) and 60.56% inhibition of extract obtained by conventional solvent extraction (CSE). In the present study, 67.49% inhibition was achieved with the same EtOH solvent, but the extraction method was different (SE). In comparison, the extraction method has an effect on the antioxidant potential of the extracts, as SE in the present study resulted in a higher I than CSE, while it demonstrated lower I than MAE. Furthermore, Zaki et al. [ 46 ] determined up to 97.84% inhibition (MeOH extracts), while Al-Juhaimi et al. [ 48 ] determined up to 83.70% inhibition using UE (hexane, MeOH/H ₂ O extracts). The importance of the solvent used with the same extraction method can also be detected here, as with other solvents (MeOH, hexane, etc.) AS extracts showed a higher antioxidant potential than the presented UE H ₂ O extract (58.50%). Recently, Kautsar et al. [ 67 ] studied the effect of AS extract storage on antioxidant activity, which decreased from 91.58 to 84.05% after 4 weeks. Differences between studies also arise as a result of the influence of many factors such as the variety, origin, and maturity of the fruit, and as mentioned, additional discrepancy is caused by the use of different methods and solvents for the extraction of biologically active compounds.

2.7. Antimicrobial Activity of AS Samples

The increase in antimicrobial resistance, the decrease in the effectiveness of synthetic drugs, and, at the same time, their increased toxicity has led to an ever-increasing search for alternative biologically active substances. As AS are a by-product of the avocado industry, they have already been investigated as a potential source of antimicrobial compounds. Data in previously published studies indicate that diverse AS extracts inhibit the growth of Candida spp., Cryptococcus neoformans , Malassezia pachydermatis [ 4 ], Corynebacterium ulcerans , Escherichia coli , Staphylococcus aureus , Streptococcus pyogenes , Salmonella typhi [ 30 ], Entamoeba histolytica , Giardia lamblia , Trichomoniasis vaginalis , Mycobacterium tuberculosis [ 68 ], Clostridium sporogenes [ 69 ], Proteus mirabilis , Pseudomonas aeruginosa , Aspergillus niger [ 70 ], Porphyromonas gingivalis [ 71 ], Bacillus cereus , Listeria monocytogenes , Pseudomonas spp., Yarrowia lipolytica [ 72 ], Klebsiella pneumoniae [ 3 ], Staphylococcus epidermidis , Enterococcus faecalis , Salmonella enteritidis , Citrobacter freundii , Enterobacter aerogenes , Zygosaccharomyces bailii , Aspergillus flavus , and Penicillium spp. [ 73 ]. Many studies have investigated the antimicrobial effect of AS extracts obtained by well-known conventional methods on plenty of microorganisms. On the other hand, it should be point out that so far, it is possible to find more than one a recent study, which is by David et al. [ 74 ], that tested the antimicrobial effect of AS extracts obtained with greener SFE. The susceptibility of L. monocytogenes , S. typhimurium , and E. coli to AS extracts obtained by scCO ₂ at different operating temperatures and pressures was studied. Extracts obtained at 40 °C and 30 MPa and at 50 °C and 20 MPa showed only inhibition of L. monocytogenes growth. Hence, the presented study is a major contribution to this field, as it includes a comprehensive and comparative study of the qualitative and quantitative antimicrobial efficacy of UE, SE, and SFE AS extracts on 15 different microorganisms.

Initially, the antimicrobial activity was tested qualitatively using the disc diffusion method. The results are shown in Table 4 .

Table 4

Qualitatively determined antibacterial activity of AS extracts obtained by ultrasonic extraction (UE), Soxhlet extraction (SE), and supercritical fluid extraction (SFE) on selected bacteria and fungi.

Microorganism		Inhibition Zone (mm ± SD)
Microorganism		UE (H ₂ O)	SE (EtOH)	SFE (scCO ₂ + EtOH)
Gram-negative bacteria	E. coli	14 ± 2	17 ± 2	11 ± 1
	P. aeruginosa	11 ± 1	11 ± 1	11 ± 1
	P. fluorescens	15 ± 1	18 ± 2	15 ± 1
Gram-positive bacteria	B. cereus	-	15 ± 1	17 ± 1
	S. aureus	11 ± 1	13 ± 1	12 ± 1
	S. platensis	12 ± 1	11 ± 1	-
	S. pyogenes	-	11 ± 1	-
Fungi	A. brasiliensis	-	-	18 ± 3
	A. flavus	-	-	14 ± 1
	A. fumigatus	12 ± 1	-	22 ± 2
	A. niger	-	-	13 ± 2
	C. albicans	-	-	17 ± 2
	P. cyclopium	10 ± 1	13 ± 1	11 ± 1
	S. cerevisiae	-	-	-
	T. viride	-	-	-

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Promisingly, all AS extracts inhibited the growth of all selected Gram-negative bacteria. The inhibition zone with the addition of AS extracts obtained with different methods on P. aeruginosa was the same, while the addition of SE extract to E. coli and P. fluorescens resulted in the largest inhibition zone. Furthermore, SE extract inhibited the growth of all Gram-positive bacteria, while B. cereus was not susceptible to the addition of UE extract and S. pyogenes to the addition of UE and SFE extracts. The sensitivity of fungi to AS extracts was also examined. The SE extract showed the lowest antifungal performance, as it only inhibited the growth of P. cyclopium . UE extract only inhibited the growth of A. fumigatus and P. cyclopium . On the contrary, the SFE AS extract was very effective as an antifungal agent, as it inhibited the growth of six out of eight selected fungi; only S. cerevisiae and T. viride were not susceptible to its addition. Overall, using the disc diffusion method, SFE AS extract proved to be the most antimicrobially effective, inhibiting the growth of 11 out of 15 microorganisms, followed by SE (8/15) and UE extract (7/15).

Given the promising results of the qualitative study, the antimicrobial effectiveness of AS extracts was quantified using broth microdilution method. The MGIRs were determined for selected Gram-negative and Gram-positive bacteria and fungi at five different concentrations of AS extracts. A comparison of MGIRs after 8 and 24 h was also carried out, which enables a more comprehensive insight of the antimicrobial activity of AS extracts for possible applications in various industries. According to all the reviewed literature, similar studies containing a certain percentage level of growth inhibition for selected microorganisms with any AS extracts have not yet been published. Therefore, Figure 4 shows the results of a quantitative study of the antimicrobial efficacy of AS extracts against selected Gram-negative bacteria, which was carried out by broth microdilution method.

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Figure 4

Microbial growth-inhibition rates (MGIRs) for AS extracts obtained by ultrasonic extraction (UE; solvent H ₂ O), Soxhlet extraction (SE; solvent EtOH), and supercritical fluid extraction (SFE; solvent scCO ₂ , co-solvent EtOH) using 70, 140, 210, 280, and 2780 μg sample per mL of selected Gram-negative bacteria suspension.

After 24 h of incubation with the highest added concentration of inhibitors, all AS extracts completely inhibited growth of Gram-negative E. coli (with 100% MGIR). More specifically, UE extract at the concentration of 2780 μg/mL inhibited MGIR by 82% after 8 h of incubation, while E. coli was then not susceptible to lower concentrations. After 24 h, the addition of the lowest UE extract concentration (70 μg/mL) resulted in 71% MGIR and addition of 210 μg/mL in as much as 98% MGIR. Furthermore, the addition of SE extract at a concentration of 210 μg/mL showed 72% MGIR after 8 h, and after 24 h, more than 50% inhibition of E. coli growth was achieved with 140 μg/mL. At both times, almost complete inhibition (99 and 98% MGIR) was achieved with the addition of 280 μg/mL SE extract as an inhibitor. The SFE extract also proved to be a good inhibitor of the growth of E. coli , which already showed 33% MGIR after 8 h with the concentration of 70 μg/mL as the lowest concentration and as much as 83% MGIR with 140 μg/mL. After 24 h, the concentration of 210 μg/mL SFE extract reached as much as 98% MGIR.

Gram-negative P. aeruginosa was the least susceptible to the addition of UE extract, as only the highest added concentration of the extract achieved its complete growth inhibition. Lower concentrations of UE extract, however, resulted in MGIRs of up to 65%. On the contrary, SE and SFE extracts even with the addition of the lowest concentration, 70 μg/mL, showed MGIRs between 46–64% after 8 and 24 h. In the case of the addition of SE extract as an inhibitor, higher-than-90% MGIRs were achieved with a concentration of 280 μg/mL. However, the SFE extract proved to be exceptionally effective as a growth inhibitor of P. aeruginosa , as it reached 95% MGIR after 8 h of incubation with 140 μg/mL of the added extract and 97% MGIR after 24 h of incubation with 280 μg of SFE extract per mL of suspension.

Exceptional results of antibacterial efficiency were achieved with the addition of UE and SFE extracts as inhibitors on the growth of Gram-negative P. fluorescens . After 8 h of incubation, the addition of 70 μg/mL UE extract resulted in 77% MGIR, while 140 μg/mL and higher concentrations showed complete inhibition of P. aeruginosa growth. After 24 h of incubation, the lowest concentration inhibited its growth with 50% MGIR, and further concentrations resulted in more than 85% MGIRs. After 8 h of incubation of P. aeruginosa with 210 μg/mL SE extract, the result was 50% MGIR and 96% with the addition of 280 μg/mL. The mentioned bacterium was less susceptible to lower concentrations of SE extract after 24 h of incubation, but the highest concentration of 2780 μg/mL still completely inhibited its growth. Importantly, even the lowest added concentrations of SFE extract significantly affected the growth of P. aeruginosa (42 and 49% MGIR after 8 and 24 h), while the addition of 140 μg/mL SFE extract completely inhibited its growth in both time periods.

Figure 5 shows the results of a quantitative study of the antimicrobial efficacy of AS extracts against selected Gram-positive bacteria, which was carried out by BMM.

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Figure 5

Regarding Gram-positive B. cereus , generally higher antibacterial efficiency of AS extracts was observed after 8 h of incubation, while after 24 h, the efficiency decreased except at the highest added concentration. The UE extract has a remarkable impact on the B. cereus , as it completely inhibited (100% MGIR) its growth even at the lowest concentration after 8 h of incubation. The results were similar regarding SFE extract, starting with 91% MGIR at 70 μg/mL. B. cereus was least susceptible to the addition of SE extract. After 8 h of incubation, concentrations of SE extract in the range of 70–280 μg/mL resulted in 37–41% MGIRs, while 2780 μg/mL completely inhibited the growth of B. cereus .

AS extracts were shown to be good growth inhibitors of Gram-positive S. aureus . Similar to B. cereus , generally, after 8 h of incubation even the lowest added concentrations, AS extracts greatly inhibited the growth of S. aureus (79–100% MGIRs). Again, all tested concentrations of UE extract completely inhibited the growth of the mentioned Gram-positive bacteria after 8 h, and after 24 h, the MGIRs increased from 64 to 91%. Moreover, 70 μg/mL of SE extract resulted in 86% MGIR after 8 h, and further concentrations inhibited the growth of S. aureus with 90 and 99% MGIRs. After 24 h, 210 μg/mL of SE extract completely inhibited its growth. Finally, SFE extract inhibited S. aureus growth with 79% MGIR (8 h) when added at 70 μg/mL, and MGIRs increased with a concentration up to 100%. S. aureus was not susceptible to the three lowest concentrations of SFE extract after 24 h of incubation, although 280 and 2780 μg/mL completely inhibited its growth.

The effect of the addition of AS extracts as inhibitors on Gram-positive S. pyogenes was also investigated. In contrast to B. cereus and S. aureus , the greatest antibacterial potential was shown by SE extract, where even lower concentrations resulted in higher MGIRs compared to UE and SFE extracts. After 8 h, 140 μg/mL of SE extract inhibited the growth of S. pyogenes by 90% and after 24 h by 73%, while higher concentrations resulted in MGIRs ranging between 83 and 99%. UE extract showed a higher level of inhibition after 24 h (47–95% MGIRs). In contrast, SFE extract showed a certain level of inhibition (39–72% MGIRs) after 8 h of incubation, while S. pyogenes was not susceptible to the addition of SFE extract after 24 h of incubation, which is in agreement with the findings of the disc diffusion method.

Using the broth microdilution method, it could be estimated that, in general, when lower concentrations of AS extracts were added, Gram-positive bacteria were more sensitive and susceptible to the addition of AS extracts, which is in line with the claims of other authors [ 72 ]. This can be explained by the fact that Gram-negative bacteria are more resistant to the addition of inhibitors due to the presence of an additional protective outer membrane, which Gram-positive bacteria lack [ 75 ].

Furthermore, the antifungal activity of AS extracts was also investigated. Since most fungi form spores, which makes them incompatible with the broth microdilution method, the quantitative antifungal efficacy of UE, SE, and SFE extracts was tested against C. albicans . The results are shown in Figure 6 .

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Figure 6

The results are in accordance with the disc diffusion method because even when using BMM, only the SFE extract showed good antifungal efficiency. UE extract resulted in 12–43% MGIRs after 8 h of incubation, but after 24 h, the fungus was not sensitive to the addition of the extract at all. In the case of the addition of SE extract as an inhibitor, lower concentrations did not affect the growth of C. albicans , and the two highest concentrations (2780 and 280 μg/mL) reached 32–65% MGIRs. On the other hand, the SFE extract was more effective against C. albicans . A higher degree of growth inhibition was achieved after 24 h of incubation, when even 70 μg/mL of SFE extract showed 65% MGIR, and complete inhibition was achieved with the addition of 210 μg/mL.

Antimicrobial activity is attributed to many phytochemicals or biologically active compounds. Other authors [ 76 , 77 , 78 ] mainly cite the antimicrobial effect of phenolic compounds, which change the function of bacterial cell membranes and thereby slow down growth and inhibit bacterial reproduction. Furthermore, the antimicrobial effect of fatty acids (e.g., palmitic acid) and their derivatives acetogenins (e.g., avocadene, persin, persediene, and persenone A, B, and C) obtained from AS is also reported [ 79 , 80 ]. The fluidity, disorganization, and also the disintegration of the cell membranes occur due to disordering of the phospholipid chain, which is the cause of the leakage of intracellular content and, consequently, cell death [ 81 ]. However, the correlation between the identified phenolic compounds in the obtained AS extracts and their antimicrobial effectiveness is important. The antibacterial/antifungal activity of hesperidin [ 82 ], quercetin [ 83 ], benzoic acid [ 84 ], 2,3-dihydroxybenzoic acid [ 85 ], 4-hydroxybenzoic acid [ 86 ], caffeic acid [ 87 ], chlorogenic acid [ 88 ], cinnamic acid [ 89 ], p-coumaric acid [ 90 ], ferulic and gallic acid [ 91 ], salicylic acid [ 92 ], and o-vanillin [ 50 ] has already been demonstrated. It is possible to conclude that the high content of the mentioned phenolic compounds synergistically affects the antimicrobial efficiency of the obtained AS extracts.

For ease of review, MIC ₉₀ values were also determined from the BMM results as the concentrations at which AS extracts inhibited the growth of a particular bacterium/fungus by at least 90% of the MGIR. The results are shown in Table 5 .

Table 5

Minimum inhibitory concentrations (MIC ₉₀ ) for AS extracts obtained by ultrasonic extraction (UE; solvent H ₂ O), Soxhlet extraction (SE; solvent EtOH), and supercritical fluid extraction (SFE; solvent scCO ₂ , co-solvent EtOH) on various microorganisms.

Microorganisms		Incubation Period	MIC ₉₀ (μg/mL)
Microorganisms		Incubation Period	UE (H ₂ O)	SE (EtOH)	SFE (scCO ₂ + EtOH)
Gram-negative bacteria	E. coli	8 h	-	280	280
	E. coli	24 h	210	280	210
	P. aeruginosa	8 h	2780	280	140
	P. aeruginosa	24 h	2780	280	280
	P. fluorescens	8 h	140	280	140
	P. fluorescens	24 h	280	2780	140
Gram-positive bacteria	B. cereus	8 h	70	2780	70
	B. cereus	24 h	280	2780	210
	S. aureus	8 h	280	140	140
	S. aureus	24 h	2780	210	280
	S. pyogenes	8 h	-	140	-
	S. pyogenes	24 h	2780	280	-
Fungi	C. albicans	8 h	-	-	2780
Fungi	C. albicans	24 h	-	-	2780

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Only a small number of studies covering MIC values for AS extracts can be detected in the literature. Nwaoguikpe and colleagues [ 3 ] determined MIC values for E. coli , P. aeruginosa , and S. aureus in the range of 40,000–50,000 μg/mL for aqueous, methanolic, and ethanolic AS extracts. Furthermore, a study by Idris et al. [ 30 ] resulted in MIC values for petroleum ether, chloroform, ethyl acetate, and methanol SE AS extracts in the range of 10,000–50,000 μg/mL for E. coli , P. aeruginosa , S. aureus , S. pyogenes , and C. albicans . Raymond et al. [ 73 ] determined MIC values for P. aeruginosa of 250.0 ± 216.5 μg/mL and for E. coli and S. aureus of greater than 500 μg/mL with ethanolic AS extract (Hass variety). MIC values for Gram-negative P. fluorescens and Gram-positive B. cereus have not been studied in the literature so far. In the presented research, the lowest MIC value was determined for B. cereus after 8 h of incubation in the case of UE and SFE extracts (70 μg/mL) compared to all tested microorganisms. MIC values were also determined for P. fluorescens and were especially promising for SFE extract (140 μg/mL) after 8 and 24 h of incubation. Compared to the previously listed research, our extracts showed incomparably lower MIC values for the remaining microorganisms, which greatly contributes to the current information on the antimicrobial activity of AS extracts.

For further applications in which it is necessary to consider, for example, the release of AS extract as a potential antimicrobial agent and the MIC for microorganisms after a certain incubation, contact time is extremely important. In the presented study, it was demonstrated that microorganisms are differently susceptible to the addition of diverse AS extracts and are variously sensitive to the addition of inhibitors after certain periods of time. For example, S. aureus is more susceptible to the addition of all three studied AS extracts after 8 h (lower MIC ₉₀ values) than after 24 h incubation time. In general, SFE extract compared to UE and SE extract resulted in lower MIC ₉₀ values, which is a great contribution to research in the field of the antimicrobial action of SFE AS extracts.

3. Materials and Methods

3.1. Chemicals, Reagents, and Microorganisms

Chemicals including malt extract, potato dextrose broth, Triton X-100, tryptic soy broth, and tryptone were obtained from Fluka, Buchs, Switzerland. Mueller–Hinton broth (MHB) and potato dextrose agar (PDA) were purchased from Biolife, Milano, Italy. 4-Aminoantipyrine (4-APP), Coomassie Blue G-250, ethanol (EtOH, ≥99.5%), ferric chloride (FeCl ₃ ), hydrochloric acid (HCl, 37.0%), iodine (I ₂ ), meat extract, meat peptone, n-butanol (≥99.5%), 1-napthol (≥99.0%), phosphoric acid (85.0%), potassium dihydrogen phosphate (KH ₂ PO ₄ ), potassium iodide (KI), sodium chloride (NaCl), sodium dihydrogen phosphate monohydrate (NaH ₂ PO ₄ ·H ₂ O), and sodium hydrogen phosphate (Na ₂ HPO ₄ ) were from Merck, Darmstadt, Germany. Calcium chloride (CaCl ₂ ), D-(+)-glucose anhydrous, and ferrous sulfate heptahydrate (Fe(SO ₄ )·7H ₂ O) were purchased from Kemika, Zagreb, Croatia. Carbobenzoxy-L-Glutaminylglycine (CBZ-Gln-Gly) was obtained from Zedira GmbH, Darmstadt, Germany. Acetic acid (glacial, ≥99.7%), acetonitrile (≥99.9%), agar, ammonium hydroxide solution (NH ₄ OH), ascorbic acid, benzoic acid (≥99.5%), bovine serum albumin (BSA), caffeic acid (98.0%), casein, chloroform (CHCl ₃ , ≥99.0%), chlorogenic acid (≥95.0%), cinnamic acid (≥99.0%), 2,3-dihydroxybenzoic acid (99.0%), L-3,4-dihydroxyphenylalanine (L-DOPA, ≥98.0%), 3,5-dinitrosalicylic acid (DNS), 2,2-diphenyl-1-picrylhydrazyl (DPPH, ≥97.0%), ellagic acid (≥95.0%), (-)-epicatechin (≥90.0%), ethylenediaminetetraacetic acid (EDTA, 98.5–101.5%) ferulic acid (99.0%), Folin–Ciocalteu’s phenol reagent (FC), gallic acid (GA, ≥97.5%), glucose assay, hesperidin (≥97.0%), hydrogen peroxide (H ₂ O ₂ ), hydroxylamine hydrochloride (HONH ₂ ·HCl), L-glutamic acid (99.0%), L-glutathione reduced (≥98.0%), maltose, methanol (MeOH, ≥99.9%), o-vanillin (99.0%), yeast extract, peptone from soybean, phenol (C ₆ H ₅ OH), p-coumaric acid (98.0%), p-hydroxy benzoic acid (99.0%), p-nitrophenyl butyrate (p-NPB, ≥98.0%), potassium sodium tartrate tetrahydrate (KNaC ₄ H ₄ O ₆ ·4H ₂ O, 99.0%), 2-propanol (99.9%), pyrogallol (≥99.0%), quercetin (≥95.0%), salicylic acid (≥99.0%), Sigmacell cellulose, sodium acetate (CH ₃ COONa, ≥99.0%), sodium carbonate (Na₂CO₃, ≥99.5%), sodium hydroxide (NaOH, ≥95.0%), starch, sulfuric acid (concentrated, H ₂ SO ₄ ) trichloroacetic acid (TCA, ≥99.0%), and Trizma Base (NH ₂ C(CH ₂ OH) ₃ , ≥99.7%) were purchased from Sigma-Aldrich, St. Louis, USA. Carbon dioxide (CO ₂ , purity 2.5) was obtained from Messer, Ruše, Slovenia.

Selected microorganisms including bacteria ( B. cereus (DSM 345), E. coli (DSM 498), P. aeruginosa (DSM 1128), P. fluorescens (DSM 289), S. aureus (DSM 346), S. platensis (DSM 40041), and S. pyogenes (DSM 11728)) and fungi ( A. brasiliensis (DSM 1988), A. flavus (DSM 818), A. fumigatus (DSM 819), A. niger (DSM 821), C. albicans (DSM 1386), and S. cerevisiae (DSM 1848)) were purchased from DSMZ-German Collection of Microorganisms and Cell Cultures GmbH from Berlin, Germany. The fungi P. cyclopium and T. viride were gifted from the Department of Agricultural Chemical Technology, Budapest University of Technology and Economics, Hungary.

3.2. Plant Material and Preparation of Samples

Avocado fruits (Hass variety) were stored at room temperature until full ripeness. Complete avocado seeds were then manually separated from the ripe avocado fruits and washed under the continuous flow of tap water. Obtained avocado seeds (AS) were then chopped into small pieces in order to accelerate the drying process. The final drying process took place at room temperature to avoid any effect of higher temperature on the content of bioactive compounds. Sliced, dried seeds (including seed coats) were then evenly ground and stored at room temperature and protected from light until their extraction and further analysis.

Extractions of dried AS were performed using different extraction methods in order to compare the content of various phytochemicals and bioactive compounds and to evaluate their bioactivity. First, under UE, the mixture of 20 g of dried AS and 150 mL of water (H ₂ O) as solvent was exposed to ultrasonic irradiation at 40 kHz for 3 h at 20 °C using an ultrasonic bath (Iskra PIO-Sonis 4, Iskra, Šentjernej, Slovenia). After extraction, the mixture was filtered using Büchner funnel and flask to remove solid particles. Furthermore, for SE, about 25 g of dried AS was added into porous cellulose thimble and placed in Soxhlet extractor. A volume of 150 mL EtOH as a solvent was added into flask, attached to extractor and condenser, and heated to reflux. Extractions were conducted for 6 h. SFE of dried AS was performed using a semi-continuous apparatus [ 93 ]. To achieve better extraction of phenolic compound, EtOH was added as a co-solvent. Approximately 20 g of dried AS was placed into the extractor (60 mL), which was placed into a water bath preheated to operating temperature (40 °C). SFEs were performed at an operating pressure of 200 bar. The flow rate of CO ₂ was maintained at a constant of 2 mL/min while EtOH was pumped continuously using a high-pressure pump with a flow rate of 0.5–0.8 mL/min. The extractions took place for 2 h. Samples were collected in the previously weighted glass tubes at ambient conditions. After the conducted extractions, the solvents (H ₂ O and EtOH) and co-solvents (EtOH) were evaporated under reduced pressure at 40 °C using the rotary evaporator (Büchi Rotavapor R-114, Flawil, Switzerland). The final extracts were stored and kept in a freezer at −20 °C until further use.

3.3. Process Efficiency Evaluation

The extraction yields ( η ) are presented as mean values of two conducted extractions and were calculated as recently described by Kupnik et al. [ 23 ] using the equation below.

η [%] = (\frac{m_{e x t r a c t}}{m_{d r y m a t e r i a l}}) \times 100

where

$η$ is extraction yield $(%)$ , $m_{e x t r a c t}$ is mass of extract $(g)$ , and $m_{d r y m a t e r i a l}$ is mass of dry AS $(g)$ .

3.4. Qualitative Determination of Phytochemicals

Phytochemical screening of extracts was conducted in order to qualitatively determine the presence of various phytochemicals and bioactive compounds. Prior to analysis, the extracts were prepared at a concentration of 1 mg/mL. Phytochemicals were determined using standard qualitative tests previously described in the literature [ 94 , 95 , 96 ], with slight modifications. The procedures and observations indicating a positive test are presented in Table 6 . All experiments were carried out in triplicates, and results are presented based on visual observations.

Table 6

Qualitative methods for phytochemical screening [ 94 , 95 , 96 ].

Phytochemicals	(Test) Procedure	Observation
Alkaloids	(Wagner’s reagent test) 1 mL of extract + 3–4 drops of Wagner’s reagent along the sides of test tube	A reddish-brown coloration
Anthocyanins	(NaOH test) 2 mL of extract + 1 mL of 2 N NaOH, heat for 5 min at 100 °C	A bluish-green color
Anthraquinones	(Borntrager’s test) 1 mL of extract + few drops of 10% ( w / v ) ammonia solution	Pink-colored solution
Carbohydrates	(Molish’s test) 2 mL of extracts + few drops of alcoholic α–naphthol + 1 mL conc. H ₂ SO ₄ along the sides of test tube	Violet ring at the interface of the two liquids
Cardiac glycosides	(Keller–Killiani test) 1 mL of extract + 1.5 mL glacial acetic acid + 1 drop of 5% ( w / v ) FeCl ₃ solution + few drops of conc. H ₂ SO ₄ along the side of test tube	A greenish-blue-colored solution
Coumarins	(NaOH test) 1 mL of 10% ( w / v ) NaOH + 1 mL of extract	A yellow color
Emodins	1 mL of extract + 2 mL NH ₄ OH + 3 mL benzene	A red color
Flavonoids	(Alkaline reagent test) 1 mL of extract + 2 mL of 2% ( w / v ) NaOH + few drops of diluted HCl	A yellow color; solution then becomes colorless on addition of diluted acid
Phenolic compounds	(Ferric chloride test) 1 mL of extract + 2 mL of distilled H ₂ O + few drops of 5% ( w / v ) FeCl ₃ solution	A blue/green/black color
Phlobatannins	(HCl test) 1 mL of extract + 2 mL of 1% ( v / v ) HCl	A red precipitate
Saponins	(Foam test) 2 mL of extract + 5 mL distilled H ₂ O; vigorously shake for 15 min	Persistent foam after 10 min
Steroids	(Salkowski test) 2 mL of extract + 2 mL of CHCl ₃ + 2 mL of conc. H ₂ SO ₄	A reddish-brown ring at the junction
Tannins	(Braymer’s test) 1 mL of extract + few drops of 10% ( w / v ) FeCl ₃ solution	A dark blue or greenish-black color
Terpenoids	1 mL of extract + 2 mL of CHCl ₃ + few drops (max. of 0.5 mL) of conc. H ₂ SO ₄ along the side of test tube	A reddish-brown color at the interface
Quinones	(Sulfuric acid test) 1 mL of H ₂ SO ₄ + 1 mL of extract; shake well for 5 min	A red color

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3.5. Total Phenolic Content (TPC) Determination

The colorimetric method with Folin–Ciocalteu’s phenol reagent, accurately described by Leitgeb et al. [ 97 ], was used for determination of TPC. The results are reported as mg of gallic acid equivalents (GAE) per g of extract. Experiments were carried out in triplicates, and results are presented as mean value ± standard deviation (SD).

3.6. Total Proanthocyanidins Content (PAC) Determination

The calorimetric method, where acid hydrolysis occurs using Fe(SO ₄ )·7H ₂ O in a mixture of HCl and n -butanol, accurately described by Leitgeb et al. [ 97 ], was availed for determination of PAC. The results are reported as mg of PAC per g of extract. Experiments were carried out in triplicates, and results are presented as mean value ± SD.

3.7. Total Protein Content (PC) Determination

PC was determined by Bradford method [ 98 ] using BSA as a standard. The results are reported as mg of total proteins per g of extract. Experiments were carried out in triplicates, and results are presented as mean value ± SD.

3.8. Identification and Quantification of the Phenolic Compounds

Prior to analysis, the extracts were prepared at a concentration of 10 mg/mL and filtered using 0.2 μm pore-size cellulose syringe filters. Extracts of dried AS were analyzed using an Agilent 1200 Series HPLC System equipped with a quaternary HPLC high-pressure pump, automatic sampler, column compartment, and variable wavelength detector (VWD). The chromatographic separation was performed with a Zorbax SB-C18 column (4.6 × 150 mm i.d., 5.0 μm particle size) at a flow rate of 2.0 mL/min using an injection volume of 10 μL. The column temperature was maintained at 25 ± 1 °C. The elution gradient consisted of acidified water (0.1% acetic acid, v / v ) and acidified acetonitrile (0.1% acetic acid, v / v ) as mobile phases A and B, respectively. To achieve efficient separation, the following multistep gradient program was used: 5% B (5 min), 10% B (10 min), 11% B (12 min), 18% B (18 min), 42% B (19 min), 50% B (24 min), 60% B (25 min), 70% B (28 min), and 5% B (30 min). Each standard and sample were analyzed in triplicate. The peaks were detected at 280 nm. In order to identify phenolic compounds in the extracts, their retention times were compared with corresponding standards. Additionally, all identified phenolic compounds were quantified using calibration curves. The results are reported as mg of certain phenolic compound per 100 g of DW. All experiments were carried out in triplicates, and results are presented as mean value ± SD.

3.9. Determination of Enzymatic Activity

Specific spectrometric assays (Varian—CARY ^® 50 UV–VIS Spectrophotometer, Varian Inc., The Netherlands) were availed for determination of activities of selected enzymes. Exact procedures for the determination of α-amylase, cellulase, lipase, peroxidase, protease, and transglutaminase activities are precisely described by Leitgeb et al. [ 97 ]. Polyphenol oxidase activity was determined using Creative Enzymes ^® protocol [ 99 ], where the concentration of o-benzoquinone formed from L-DOPA by polyphenol oxidase was measured at 265 nm. Superoxide dismutase activity was determined using a reaction of pyrogallol autoxidation at 325 nm, described by the Creative Enzymes ^® protocol [ 100 ]. The results are reported as units (U) per g of extract. All experiments were carried out in triplicates, and results are presented as mean value ± SD.

3.10. Determination of Antioxidant Activity

DPPH free radical method, based on electronic transfer that produces a purple-colored solution in EtOH and accurately described by Leitgeb et al. [ 97 ], was used for determination of antioxidant activity. The results are reported as a percentage of inhibition ( I ) relative to reference solution. Experiments were carried out in triplicates, and results are presented as mean value ± SD.

3.11. Determination of Antimicrobial Activity

First, the disc diffusion method, previously described by Kupnik et al. [ 101 , 102 ], was used to qualitatively assess the antimicrobial activity of the analyzed extracts. Tests were carried out at selected concentrations of microorganisms (1–5 × 10 ⁶ CFU/mL). Deionized H ₂ O and 5% DMSO were used as negative controls, while 30 μg of amoxicillin, nystatin, or vancomycin per disc was used as positive control. The results are reported as mm of inhibition zone. All experiments were carried out in triplicates, and results are presented as mean value ± SD.

Furthermore, the broth microdilution method [ 101 , 102 ] was used to quantitatively assess the antimicrobial activity of the analyzed extracts. Tests were carried out at selected concentrations of microorganisms (1–5 × 10 ⁶ CFU/mL) using MHB as a universal nutrient medium for all microbial strains. As a negative control, 5% DMSO was used. MGIRs were determined after 8 and 24 h of incubation under optimal conditions for the growth of each microbial strain for five concentrations of added extract (2780, 280, 210, 140, and 70 μg extract/mL of suspension). Additionally, the concentration at which the extract inhibited the growth of the microorganism with at least 90% MGIR was defined as MIC ₉₀ . All experiments were carried out in triplicates, and results are presented as mean value ± SD.

3.12. Statistical Analysis

All statistical data analyses were performed using IBM ^® SPSS ^® Statistics. Statistical data analysis was performed to study differences between extraction methods. The normality of the distribution of data was tested using Shapiro–Wilk’s test. The homogeneity of variances was determine using Levene’s test. Differences between extractions were determined with one-way analysis of variances (ANOVA) followed by Tukey’s post hoc test (for normally distributed data) and with nonparametric Kruskal–Wallis H test, followed by pairwise comparison using the Dunn–Bonferroni post hoc method (for abnormally distributed data).

4. Conclusions

Humanity is increasingly striving for a broader strategy for the shift and transition from a linear to a circular economy, including through a number of initiatives to reduce the inexhaustible waste generated annually. Food wastes present a renewable resource that can be collected and subsequently converted into value-added products, while reducing the volume of waste gathered in landfills and at the same time expanding the economic market share of new sustainable products. With this aim, avocado seeds were investigated as a potential source of biologically active compounds in extracts obtained by different methods. It was found that different solvents and extraction techniques affect both the extraction yield itself and the content of the selected biologically active compounds. Due to the different content of biologically active compounds, the enzymatic, antioxidant, and antimicrobial activities of AS extracts also differ. Importantly, compared to already known data, an extremely high content of hesperidin was found in SE (226.78 mg/100 g DW) and UE (118.10 mg/100 g DW) extracts. For the first time, the high content of 2,3-dihydroxybenzoic acid in the UE extract (106.48 mg/100 g DW) and vanillin (up to 55.02 mg/100 g DW) in all AS extracts was quantified. All three phenolic compounds are well known for their antimicrobial effects, so they most likely contributed to the good antimicrobial potential of the tested AS extracts. Furthermore, the high activity of the well-known antioxidant enzyme superoxide dismutase (up to 3123.97 U/g extract) most likely contributed to the antioxidant potential of AS extracts. However, AS extracts had a significant effect on various microorganisms, as they inhibited as many as 13 out of 15 tested fungi and bacteria. Only the fungi S. cerevisiae and T. viride were not susceptible to the addition of any of the AS extracts. Additionally, compared to the literature, extremely low MIC ₉₀ values were determined, starting with the lowest of 70 μg/mL for UE and SFE extract against the Gram-positive bacterium B. cereus . For this reason, the obtained AS extracts could be good alternative to synthetic antimicrobial agents. Overall, it is necessary to accentuate the AS extract obtained with a sustainable, greener, and modern SFE. The comprehensive study of the SFE extract in the presented research, which offers a comparison with the more well-known and conventional UE and SE, provides a great deal of new information. The SFE extract resulted in the highest content of total phenols and total proteins. It contained 12 of the 14 selected phenolic compounds and was the only one to show the activity of all the studied enzymes. It also emerged as the most promising antimicrobial agent, as it preliminarily inhibited as many as 11 out of 15 selected microorganisms and generally showed the lowest MIC ₉₀ values for 6 out of 7 investigated microorganisms.

Despite many published studies involving the phytochemistry and bioactivity of avocado seed extracts, the presented study provides additional knowledge and the contribution of important new information. The significant results of SFE provide additional insight into the extraction of bioactive ingredients from AS, while the results in the field of enzymatic activity and antimicrobial efficiency of AS extracts prove their extraordinary potential for further applications. Therefore, it would definitely make sense to exploit AS as a food waste for the recovery and production of value-added biologically active compounds, which could be further utilized in biomedicine, pharmacy, cosmetics, or other industries.

Funding Statement

This research was supported by the Slovenian Research Agency (ARRS) within the frame of program P2-0046 (Separation Processes and Production Design), P2-0424 (Design of novel nano/material properties & applications), project No. J2-3037 (Bionanotechnology as a tool for stabilization and applications of bioactive substances from natural sources), L2-4430 (Production, Isolation and Formulation of Health Beneficial Substances from Helichrysum italicum for Applications in Cosmetic Industry), and young researcher ARRS fellowship Contract No. 2187/FS-2019.

Author Contributions

Conceptualization, M.L. and M.P.; methodology, M.L., M.P. and K.K.; formal analysis, K.K.; data curation, K.K.; writing—original draft preparation, K.K.; writing—review, M.L., M.P. and V.K.; writing—editing, K.K.; visualization, K.K.; supervision, M.L., M.P. and V.K.; funding acquisition, M.L., V.K. and Ž.K. All authors have read and agreed to the published version of the manuscript.

Data Availability Statement

The data presented in this study are available in article.

Conflicts of Interest

The authors declare no conflict of interest.

Footnotes

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