2.2. Presence of Phytochemicals in AS Samples
The AS extracts were subjected to a phytochemical screening using various well-known standard qualitative methods. The results are shown in
Table 1.
It is important to highlight that alkaloids, flavonoids, phenolic compounds, saponins, steroids, terpenoids, and quinones were present in all three types of extracts. The research showed the absence of anthraquinones, carbohydrates, cardiac glycosides, emodins, phlobatannins, and tannins. Anthocyanins and coumarins were present in the UE and SE extracts, while they were absent in the SFE extract, which can be justified by explaining the polarity of the solvents used in the extraction process. Anthocyanins are known as water-soluble flavonoids [
27] and were not extracted by SFE most likely due to the lower polarity of scCO
2 and EtOH solvent mixtures. Coumarins are well soluble in EtOH and slightly soluble in H
2O, while the solubility of scCO
2 depends on the variables in the extraction process itself (operating pressure and temperature) [
28,
29], and therefore, it is possible that they were not extracted under the applied operating conditions.
No phytochemical screening on SFE avocado seed extracts was detected in the reviewed literature, which means that this study is the first to provide additional insight into a more modern, green, and unconventional technique for extracting phytochemicals from AS. On the other hand, quite a few studies [
30,
31,
32,
33] with a preliminary screening of phytochemicals in AS extracts obtained by conventional extractions have already been published, but the results themselves are difficult to compare due to the different extraction methods, conditions, and solvents used. For example, Rivai et al. [
34] found the presence of tannins and the absence of flavonoids, terpenoids, saponins, and steroids in EtOH and H
2O extracts of AS, which is inconsistent with results in the presented study. On the contrary, Oboh et al. [
10] found the presence of alkaloids, saponins, and terpenoids and the absence of anthraquinones and phlobatannins in the aqueous extract, which is in line with the presented results. Here, it is necessary to emphasize, however, that the chemical profile, the content, and activities of biological compounds from AS may differ due to the influence of the variety, origin, season, maturity, growth, post-harvest, and environmental conditions [
35].
2.3. Content of Total Phenols, Proanthocyanidins, and Total Proteins in AS Samples
As seen in the previous subsection, the presence of phytochemicals in AS extracts, the method, and the solvent used greatly affected both the extraction efficiency and the successful extraction of various phytochemicals. Accordingly, a quantitative investigation of the content of TPC, PAC, and PC of the examined AS extracts was carried out. The results of the study are presented in
Figure 2.
TPC obtained with SFE was the highest among all extracts and amounted to 36.01 mg GAE/g of extract. Compared to SFE, TPC with UE (6.31 mg GAE/g) was almost six times lower, while TPC with SE (15.91 mg GAE/g) was almost two times lower than SFE. In the reviewed literature, the TPC values for AS extracts ranged up to 146.60 mg GAE/g with UE using hydroalcoholic mixtures as solvents [
36], while no TPC values were detected in the reviewed literature using only H
2O as a solvent. Segovia et al. [
37] demonstrated that ultrasonic power and temperature have a huge impact on the extraction of polyphenols with H
2O as solvent, as with the increase of the mentioned parameters, the TPC should also be enhanced. Therefore, it is likely that the UE efficiency of the present study is lower due to the low operating temperature (20 °C), and with more optimal conditions (higher operating temperature), the UE yield of AS extracts could be increased. With SE using EtOH as solvent, the TPC values ranged between 19.87–29.92 mg GAE/g [
21,
38], while with SFE, the TPC values reached up to 51.36 mg GAE/g [
21]. Hence, the lower TPC value for SE compared to SFE is also justified, as SE needs higher operating temperatures to improve solvent solubility and to reduce its viscosity and surface tension, while high temperatures can lead to degradation of certain phenolic compounds and therefore lower TPC. On the other hand, low viscosity and near-zero surface tension allow scCO
2 to easily penetrate the matrix material to extract phenolic compounds. Additionally, Kruskal–Wallis H test showed that there was a statistically significant difference between TPC prepared by different extraction methods. A post hoc Dunn–Bonferroni test revealed that there was statistically higher content of TPC present in extract obtained by SFE compared to SE and UE.
Hereafter, PACs were determined in AS extracts. The experiments showed that the PAC value was the lowest in the SFE extract (2.84 mg/g), followed by the UE extraction (24.67 mg/g). The highest PAC value, 32.13 mg/g, was detected in the AS extract obtained with SE. After a thorough review of the already-published literature, it was found that the PAC value was not quantitatively determined comparatively in AS extracts. Only a study by Noorul et al. [
39] determined PAC using the vanillin-H
2SO
4 protocol in AS extracts obtained by SE. The EtOH extract showed 13.70 μg catechin/mL and the H
2O extract 9.30 μg catechin/mL. PACs are phytochemicals with added value, as their health aspects (e.g., antimicrobial, antioxidant, anticancer, antidiabetic, and neuroprotective activities) have been validated [
40,
41]. The mentioned results of the presented study are important data for potential further applications of AS extracts in biomedicine, pharmaceutical products, cosmetics, functional food, and nutraceuticals. Additionally, PC in AS extracts was examined. The highest PC value is contained in the SFE extract (16.10 mg/g), followed by the UE extract (12.43 mg/g) and finally the SE extract (11.74 mg/g). In the reviewed literature, quantitatively determined PC was not detected in AS extracts, but in some studies [
42,
43,
44], proximate composition analyses were performed, where PC values reached up to 23.0%. The use of scCO
2 affects the AS cells by damaging the cell walls, which results in the release of intracellular materials. Consequently, intracellular proteins contribute to PC [
45], which explains the higher content in the SFE extract. Regarding content of PAC and PC, there was also a statistically significant difference using different extraction methods.
2.4. Content of Certain Phenolic Compounds in AS Samples
A comprehensive analytical characterization of certain phenolic compounds was performed in AS extracts. The HPLC allowed effective and rapid separation and identification of 14 divergent phenolic compounds based on comparison of retention times with corresponding standards. The content of three flavonoids (epicatechin, hesperidin, and quercetin), ten phenolic acids (benzoic acid, 2,3-dihydroxy benzoic acid, 4-hydroxy benzoic acid, caffeic acid, chlorogenic acid, cinnamic acid, p-coumaric acid, ferulic acid, gallic acid, and salicylic acid) and vanillin was determined in UE, SE, and SFE extracts of AS, which are presented in
Table 2.
Regarding the flavonoids in AS extracts, the common fact between the UE and SE extracts is that hesperidin predominated (118.10–226.78 mg/100 g DW), while its content was lower in the SFE extract (9.37 mg/100 g DW). In comparison, hesperidin was also detected by Zaki et al. [
46], but its content was much higher in peel extracts (up to 61.94 mg/100 g DW) than in seed extracts (up to 2.62 mg/100 g DW). However, the presence of quercetin was found in the SFE extract (58.07 mg/100 g DW), while it was not detectable in the remaining extracts. In the reviewed literature, different quercetin contents in AS extracts are reported, ranging from 0.07 to 88.18 mg/100 g [
5,
19,
46,
47,
48]. Since epicatechin is a building block of proanthocyanidins, whose total content was high, especially in the UE extract, it was unambiguously identified in all AS extracts (15.56–39.20 mg/100 g DW) by comparing the retention time with that of the standards. It has also been confirmed many times in similar AS extracts in the reviewed literature [
5,
16], up to a content of 2.91 mg/100 g DW.
When comparing the presence of valuable phenolic acids in AS extracts, 2,3-dihydroxybenzoic acid (also known as pyrocatechuic acid) content was the highest (14.42–106.48 mg/100 g DW) in all extracts (UE > SE > SFE). The same sequence of the AS extracts (UE > SE > SFE) can also be detected in the content of 4-hydroxybenzoic acid, which is also known as
p-hydroxybenzoic acid (3.47–15.41 mg/100 g DW), while benzoic acids were found only in UE extract. All mentioned acids were also identified in the reviewed literature [
5,
47,
49]. Caffeic acid has only been detected in SFE extract at a content of 5.92 mg/100 g DW, while chlorogenic acid appeared in all AS extracts (UE > SE > SFE, 0.73–9.36 mg/100 g DW) as well as cinnamic acid (UE > SFE > SE, 11.33–34.36 mg/100 g DW). The content of caffeic acid (4.18–60.19 mg/100 g), chlorogenic acid (0.05–2.61 mg/100 g), and cinnamic acid (up to 0.09 mg/100 g) was previously determined by other researchers [
5,
19,
46,
48]. Next, the highest content of p-coumaric acid was detected in the SE extract (6.26 mg/100 g DW), followed by the SFE and UE extracts. The literature indicates p-coumaric acid content in AS extracts from 0.62 to 10.23 mg/100 g [
19,
46,
48]. Hereafter, ferulic acid was found in SE and UE extracts (up to 6.86 mg/100 g DW), gallic acid in SFE and UE extracts (up to 3.99 mg/100 g DW), and salicylic acid in SE and SFE extracts (up to 15.15 mg/100 g DW). For comparison, the content values for ferulic acid in the reviewed literature are between 0.09–82.53 mg/100 g, for gallic acid between 1.21–67.38 mg/100 g, and for salicylic acid between 2.57–5.50 mg/100 g [
5,
19,
46,
48]. Finally, vanillin was also previously determined in AS extracts [
47] and is known as an antimicrobially active phenolic compound [
50,
51]. In the presented study, vanillin was identified in all extracts (UE > SE > SFE) and quantified up to a content of 55.02 mg/100 g DW.
Overall, the highest total content of analyzed phenolic compounds was contained in UE extract (393.94 mg/100 g DW), followed by SE extract (351.03 mg/100 g DW) and SFE extract (177.99 mg/100 g DW). In the presented study, compared to the contents of other authors, the high contents of hesperidin, epicatechin, cinnamic acid, and salicylic acid stood out. Additionally, 2,3-dihydroxybenzoic acid, 4-hydroxybenzoic acid, benzoic acid, and vanillin have been identified in already-published research, while their quantification in AS extracts has not yet been detected. Importantly, many studies have examined the content of different phenolic compounds in AS extracts obtained by different extraction methods and under different conditions and with different solvents. Therefore, it must be emphasized that there are certain deviations in the results of various studies, as the chemical profile and the content of biologically active compounds in AS extracts differ due to the previously mentioned different extraction conditions as well as due to the variety and maturity of the fruit used in the studies. What gives great importance to the presented study is the characterization and quantification of selected flavonoids and phenolic acids in SFE extracts since no similar study could be detected in the reviewed published literature heretofore. However, the optimization of important variables (operating conditions) in SFE is also important here, which could further increase the efficiency of the recovery of phenolic compounds. In any case, for the extraction and potential isolation of an individual compound, it would be necessary to carefully study the influence of the operating conditions since the solubility trend changes for certain compounds [
23].
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.
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.
2.7. 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.
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.
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.
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.
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.
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 MIC90 values) than after 24 h incubation time. In general, SFE extract compared to UE and SE extract resulted in lower MIC90 values, which is a great contribution to research in the field of the antimicrobial action of SFE AS extracts.