Extraction method for blackberry fruit pomace and its active compounds: Antioxidant activity and antimicrobial effects on human oral flora isolates

Abstract

Background

Blackberry fruit processing results in biowaste in the form of blackberry fruit pomace (BFP), which retains many valuable biologically active compounds (BAM).

Objective

This study aimed to investigate the chemical constituents of BFP from Montenegro, as well as its antioxidant (a.a.) and antimicrobial (a.m.) activities against human oral flora and food isolates, depending on the extraction method used, either conventional maceration (MAC) or sonication/ultrasonically assisted extraction (UAE).

Methods

The phenolic profile and total phenol (TPC), flavonoid (TFC), anthocyanin (TAC), and proanthocyanidin (PC) contents were determined by HPLC and spectrophotometry. The a.a. was determined using the DPPH-based and FRAP methods, while the antimicrobial activity was analyzed using two appropriate assays. A battery of qualitative pharmacognosy tests was performed. The data were processed using principal component analysis (PCA).

Results

The most abundant secondary metabolite (SM) was ellagic acid (122.50/208.20 μg/mL) in both sample types. The UAE sample yielded a higher concentration of SM/BAM than the MAC sample, with the notable exceptions of quercetin and chlorogenic acid. The highest a.a. in the DPPH assay was measured as 1.27 μL/mL (IC₅₀) and 1045.45 μmol FRAP/L (in the FRAP assay). P. aeruginosa was the most sensitive strain (human pathogenic oral flora isolates) closely followed by S. aureus. B. cereus, among the food isolate microbes, was the most sensitive strain, requiring 3.125 mg/mL as the MIC of the UAE sample.

Conclusion

BFP is rich in phytochemicals with antioxidative and antimicrobial potential, whose concentration in extracts can be increased by sonication and possibly used as antioxidants and adjuvants in food, or novel antimicrobial agents for treatments in human oral cavity.

Keywords

blackberry pomace antioxidant activity antimicrobial activity oral pathogens phytochemistry

Introduction

Berry fruits (and supplements made from their extracts) are among the most antioxidant-rich foods. Use of these (as food, or supplements) has been persistently correlated with the prevention and attenuation of age-related and oxidative stress-related conditions (such as Parkinson's disease, dementia and sarcopenia to name just a few). These findings are, of course, very much expected, given that they exhibit strong antioxidant, antimicrobial, anti-inflammatory and anticancer properties, to name a few.^1–5 Despite this, berry fruits are infrequently consumed raw but are rather processed instead. Valuable, edible and phytochemical-rich parts are being discarded in the process, simultaneously becoming a challenge in the area of waste management.^6,7 Blackberry fruit (Rubus fruticosus L., Fam. Rosaceae) is one such example - being rich in beneficial secondary metabolites that affect its shelf life and human health favourably, yet it's still mostly consumed processed, in the form of fruits, jams, sweets and other processed forms.⁸

Considering the growing issue of antimicrobial resistance in pharmacy, agriculture, and aquaculture, correlating with the lack of novel, effective, and safe antimicrobial agents, persisting for decades now,^9–12 blackberry fruit and its associated bio-waste—such as pulp and blackberry fruit pomace (BFP)—represent a potentially valuable source of novel antimicrobial compounds and therefore warrant further scientific attention rather than being overlooked. Additionally considering the increased shift in consumers’ consciousness towards the more “natural” alternatives to synthetic food additives, dyes and even synthetic antibiotics, blackberry fruit & BFP warrant more research, especially in vivo, considering they are a safe source of valuable biologically active molecules (BAM).^13–15

In vitro screening and detailed phytochemical characterization of fruits and their bio-waste are essential for understanding the biochemical/molecular interactions of BAM and the corresponding signaling cascades in human cells. Blackberry fruits have been getting the research spotlight lately, as galocatechin has proven itself as a powerful antimicrobial agent against Streptococcus mutans and Porphyromonas gingivalis. Additionally, the fruit and its extracts have shown selective antimicrobial activity against pathogenic oral flora in humans, simultaneously acting as prebiotics for commensals.^16,17

Blackberry fruit is also interesting as a source of anthocyanins, which have demonstrated biological activity against several cancer cell line proliferation, as well as anti-inflammatory activity both in vitro and in vivo.¹⁵ Interestingly many of the compounds that are responsible for its antioxidant activity (such as ellagic acid, cyanidine-3-glucoside, catechins and anthocyanins) are also considered responsible for its antimicrobial effects.^18–20 This supports the need for parallel testing of these two types of biological activities, as was done in this study.

Considering that extraction methods can widely affect the yield of antioxidant and antimicrobial compounds in berry fruits,^21,22 our study aimed to evaluate the effect of sonication and solvent acidulation on the extraction of targeted groups of chemical compounds in BFP, in comparison to the conventional maceration extraction method, with the same but non-acidulated solvent. Additionally, another objective of the study was to assess whether the two extraction protocols would result in different in vitro antioxidant and antimicrobial activities of the BFP extracts, particularly on human oral flora isolates. This in turn, was done to shed some light on the biotechnological aspects of optimizing the extraction of BFP.

Materials and methods

Samples

The BFP used was obtained from the village of Crljenice (43.3569°N 19.3922°E), Pljevlja municipality, and country of Montenegro, where it was considered a waste product in the process of juice extraction via a destoning-press apparatus. The blackberries used were grown commercially. Immediately after the process of juice extraction, the bio-waste was packaged in sterile plastic containers and sent to a laboratory in Podgorica, where it was kept at −4 °C until use.

Prior to the analyses, two series of samples were prepared, differing in the solvent type and method of extraction. The aim was to assess how ultrasound extraction of BFP compares to conventional maceration in terms of SM yield and a.a. and a.m. activity.

For this purpose, the samples were prepared as follows, aiming to optimize the yield of BAM when extracting them from berry fruit, as discussed by Hidalgo & Almajano²¹:

Conventional maceration (MAC) sample: Freshly thawed 2 g of BFP was covered with aqueous methanol solvent (80% methanol) and agitated and pressed for 2 h at room temperature.

Ultrasonically assisted extraction (UAE) sample was obtained by covering the freshly thawed 2 g of BFP with an acidulated (1% HCOOH) aqueous methanol solvent (80% methanol) and sonicating the mixture at 50 kHz, 50 °C for 2 h in an ultrasonic bath (Vims Elektrik, Serbia).

Following the end of reaction time, both samples were centrifuged at 4000 rpm for 20 min. Supernatants, once carefully decanted, were filtered through a Whatman grade 597 filter paper and into clean glass vials. Some of the final extracts/samples were then used in analyses, while the remaining were kept in a fridge at −4 °C until further analysis.

Microbial lines and fungi

The microbial cultures used in the disc diffusion screening were isolates obtained from the oral cavities of adult and child patients, courtesy of a local private microbiological laboratory. These included the Gram-negative strains: Pseudomonas aeruginosa, Escherichia coli and Klebsiella pneumoniae, as well as the Gram-positive strains: Staphylococcus aureus, methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus agalactiae. Additionally, Candida spp. culture isolated from the oral cavity of a patient with oral candidiasis was used.

The microbial lines used in the broth dilution MIC assay were food isolates (ATTC cultures), namely, Escherichia coli ATCC (25922), Pseudomonas aeruginosa ATCC (27853), Staphylococcus aureus ATCC (6538), Listeria monocytogenes ATCC (13932), and Bacillus cereus ATCC (11778).

HPLC-DAD phytochemical characterization, determination of total phenolic, flavonoid, anthocyanin and proanthocyanidin content and phytochemical screening

The chemical characterization of the examined extracts alongside quantification of the selected compounds was performed after hydrolysis using an Agilent Technologies 1100 liquid chromatographer equipped with a diode-array detector (Agilent Technologies, Santa Clara, CA, USA). Gallic, p-coumaric, caffeic, quercitrin, and chlorogenic acids (obtained from Sigma Aldrich (St Louis, Missouri, USA)) were analyzed using a validated high-performance liquid chromatography (HPLC) method, as described by Salaj et al..²³

Separation was accomplished using a reversed-phase Nucleosil C18 column (250 mm × 4.6 mm, 5 μm particle size; Agilent Technologies) held at 30 °C after injecting 10 µL of the sample. The mobile phase consisted of two solvents, namely, A (0.1% (v/v) aqueous HCOOH with 10 mmol CH3COONH4) and B, which was pure methanol. The mobile phase was delivered in gradient mode (0 min 10% B, 10 min 25% B, 20 min 45% B, 35 min 70% B, 40 min 100% B, 46 min 10% B). using a variable flow rate (0–10 min, 1 mL·min-1; 10–20 min, 0.8 mL·min-1; 20–30 min, 0.7 mL·min-1; 30–46 min 1 mL/min. The total runtime was 48 min. The quantity of ellagic acid was determined using a Zobax SBC18 column (250 × 4.6 mm i.d.; 5 μm), detected at 254 nm, using a constant flow rate of 1.0 mL/min at 30°C. The mobile phase was made up with methanol, ethyl acetate and potassium dihydrogen phosphate: phosphoric acid (both at 0.05 M) in the 34:2:64 volume ratio.

The TPC, TFC, and TAC of the BFP, UAE, and MAC samples were determined spectrophotometrically using a series of appropriate assays. The TPC content was determined using the Folin–Ciocalteu method as described by Singleton and Rossi²⁴ and expressed as mg of gallic acid equivalents (GAE)/L. The TPC was calculated from the calibration curve (y = 0.0057x + 1.208, R2 = 0.9909) of the gallic acid solution (serial dilution 10–50 mg/mL), by means of linear regression in the Microsoft's Excell software.

Flavonoids were quantified by the aluminium chloride colorimetric method as described in a paper by Chandra et al.²⁵, expressed as mg of quercetin equivalents (QE)/L. Quercetin was dissolved in methanol (initially 5.0 mg in 1 mL of methanol, and then from this/stock solution serial dilutions were made in the 5–200 µg/mL range), and used as standard to obtain the calibration curve (y = 0.119x + 0.9456, R2 = 0.9026). From the calibration curve, the TFC was calculated via the linear regression in the Microsoft's Excell software.

The TAC was determined using the classical pH differential method pioneered by Giusti and Wrolstad²⁶, and the results were quantified as mg cyanidine-3-glucoside equivalents (C3G EQ)/L. The results were calculated using the following formula:

A x MW x DF x 103 / ɛ x l

where the A = (A520 nm - A700Nnm) pH = 1,0 – (A520 nm – A700 nm) pH = 4,5,

the MW represents the molecular weight for C3G (449.2 g/mol),

DF is the dilution factor,

l is the pathlength in cm (cuvette width = 1 cm) and the ɛ stands for the molar extinction coefficient (26 900 in this case).

Finally, the proanthocyanidin (PC) content was determined using the Bates-Smith method, as detailed by Cáceres-Mella et al.²⁷. The assay required two tubes (reagent and blank tubes), in which the sample was diluted with deionized water and reacted with hydrochloric acid. The reagent tube was incubated (in a heated bath at 100 °C), whereas the blank tube was simply left at room temperature in the dark. After the reaction time, the absorbance of the tube contents was measured at 550 nm. Then the absorbance difference was multiplied by the factor 19.33, and the concentration of proanthocyanidins was expressed as g/L catechin equivalents (CE) units.

The extraction was performed once, and all measurements/determinations were performed in triplicate. The values were expressed as mean ± standard deviation (SD).

The BFP extracts were also subjected to a battery of qualitative tests for the presence of secondary metabolites, according to the typical pharmacognosy procedures described by Trease and Evans’ “Pharmacognosy”²⁸. The samples were tested against positive controls (extracts of plants known to possess high quantities of the compound that was being screened for).

Antioxidant assays

The antioxidant activity (a.a.) assay, with the 1,1-diphenyl-2-picrylhydrazyl (DPPH), was conducted according to Brand-Williams et al.²⁹ method and expressed as both Vitamin C equivalents and as IC₅₀ (μLQ/mL). The a.a. expressed in μmol/mL Vit. C EQ was calculated from the Vitamin C calibration curve made with serial dilutions of Vit C. solution (concentration: 0, 5, 10, 25, 50, 125, and 250 µmol) via linear regression (in Microsoft's Excell software).

The reducing power was determined using the Ferric Reducing Ability of Plasma (FRAP) assay, according to the original method described by Benzie and Strain.³⁰ As the standard a Fe2 + ions solution of a known concentration was used (as suggested per authors: 1000 µmol/L). The reducing power of the samples (a.a.) was calculated using the following formula:

The results were additionally calculated per 1 g of dry sample, for easier comparison with the literature data.

All measurements were performed in triplicate, and the results are expressed as mean ± standard deviation (SD).

Antimicrobial assays

Antimicrobial screening via the disc diffusion method was performed on Mueller–Hinton agar, according to the American Society of Microbiology protocol.³¹ Several antibiotics were used as positive controls; Amoxiclav, Chloramphenicol, Erythromycin, Clindamycin and others (See Results 3.5. and Table 5. For a detailed list). The results were expressed as zone of inhibition (ZOI) radii and mm of length, according to which the organisms were marked as either R (resistant) or S (sensitive).

The broth microdilution method was done according to the method Perovic et al.³² used, and the results were expressed as MIC (minimal inhibitory concentration) in mg/mL.

Statistical analysis

The data obtained experimentally (the results) were subjected to statistical analysis via the Statistica v. 12.5 (StatSoft, Tulsa, OK, USA). The input data were analyzed using the multivariate statistical method of hierarchical cluster analysis (HCA), performed on squared Mahalanobis distances.

Results

HPLC phytochemical characterization, determination of total phenolic, flavonoid, anthocyanin and proanthocyanidin content and phytochemical screening

Phenolic profile and major constituents

The phenolic secondary metabolites, evaluated using HPLC, varied depending on the extraction method used during sample preparation. Generally, all compounds other than quercetin and chlorogenic acid were detected in higher amounts in the UAE sample (Table 1).

Table 1.

The content of polyphenols of the blackberry fruit pomace determined by HPLC – DAD method.

µg/g extract
Type of extraction	Ellagic acid		Caffeic acid		p-coumaric acid		Quercetin		Chlorogenic acid		Gallic acid
Type of extraction	x*	u**	x	u	x	u	x	u	x	u	x	u
UAE	208.20	8.33	56.65	2.52	2.78	0.28	7.54	0.53	31.42	1.69	46.37	6.96
MAC	122.50	4.90	50.32	2.83	2.26	0.23	10.60	0.74	33.84	1.57	34.91	5.24

*x - quantified value;

**u - expanded measurement uncertainly calculated using coverage factor k = 2

The most abundantly present compounds were ellagic acid (as expected for the fruits of the plants in the Rubus genus) and caffeic acid, with 208.20 and 122.50 μg/g for UAE and MAC samples, respectively, that is with 56.63 and 50.32 μg/g in case of the caffeic acid.

Conversely, quercetin content was considerably higher in the MAC sample with 10.60 μg/g, compared to the 7.54 μg/g measured in the UAE sample. Chlorogenic acid, on the other hand, while indeed found in a higher amount in the MAC sample, was still relatively close to the value measured for the UAE sample (33.84; 31.42, respectively).

Total phenolic, flavonoid, anthocyanin and proanthocyanid content

The total phenolic (TPC), flavonoid (TFC), and anthocyanin (TAC) contents in the samples varied from 19.2 to 73.40 mg GAE/L (TPC), 2.99 to 6.52 mg QE/L (TFC), and 18.67 to 23.90 mg C3G EQ/L (TAC) (Table 2). The proanthocyanidin content, determined by the Bate-Smith method, was 5.91 g CE/L and 0.097 g CE/L catechin equivalents for UAE and MAC samples, respectively.

Table 2.

Total phenolic (TPC), total flavonoid (TFC) and total anthocyanin (TAC) content of the BFC, along with proanthocyanidines content.

Polyphenolic compounds content, total
Sample type	TPC(mg GAE/L)	TFC(mg QE/L)	TAC(mg C3G EQ/L)	Proanthocyanidines(g CE/L)
UAE	73.40 ± 1.75	6.52 ± 0.25	23.90 ± 1.05	5.91 ± 0.34
MAC	19.02 ± 0.92	2.99 ± 0.29	18.67 ± 0.85	0.97 ± 0.81

Values represent mean ± SD of three measurements (n = 3). GAE - gallic acid equivalent; QE - quercetin equivalent; C3G - cyanidine-3-glucoside, CE - catechin equivalent.

Phytochemical screening

Screening the BFP confirmed the presence of tannins, terpenoids, catechin, chlorogenic acid and cyanogenic glycosides, while anthraquinones, saponins, and alkaloids were not detected, as shown in table 3. The BFP was compared against a series of known positive controls, and its content was assessed both organoleptically and comparatively. Based on this evaluation, the compounds’ content was categorized as high (++), low (+), or absent (−).

Table 3.

BFP phytochemical screening results.

	Anthraquinones	Sapponins	Tanins	Terpenoids	Catechin	Chlorogenic acid	Alkaloids	Cyanogenic glucosides
BFP	-	-	++	+	++	++	-	++

(++) – High content; (+) – Low content; (-) - Absent

In vitro antioxidant activity

The conducted in vitro antioxidant assays (DPPH and FRAP assays) have detected arguably very strong antioxidant potential in both blackberry samples (MAC and UAE). The results are summarized in Table 4.

Table 4.

The antioxidant activity of the BFP in DPPH and FRAP assays.

Antioxidant activity
Sample type	DPPH AAE(μmol Vit. C EQ/ml)	DPPH (IC₅₀)( μL/mL)	FRAP**(μmol FRAP/L)	FRAP*** (μmol FRAP/g)
UAE	345.86 ± 4.21*	1.27 ± 0.04	1045.45 ± 11.15	525.7
MAC	326.57 ± 5.05*	2.94 ± 0.05	977.28 ± 10.08	488.64

*Sample conc. 20 μL; **micromoles FRAP/L - a unit suggested by the method authors; *** micromoles of FRAP/g of dry pomace matter – converted and adjusted. Values represent mean ± SD of three measurements (n = 3).

The UAE sample, as hypothesized (we hypothesized that sonication, due to the cell wall cavitation effect, will cause a higher phenolic content in the sample and also higher in vitro antioxidant activity), exhibited markedly stronger antioxidant potential (345.86 μmol ml g Vit C. EQ/mL, IC₅₀ 1.27 µL/mL) in the DPPH assay and 1045.45 μmol FRAP/L (in the FRAP assay) than that of the other sample obtained via conventional maceration (326.57 μmol Vit C. EQ/mL IC₅₀ 2.94 µL/mL) in DPPH assay, and 977.28 μmol FRAP/L (in the FRAP assay). The difference was more noticeable in the DPPH assay results, which may be attributable to the different nature of the radical molecules used and consequently, the different methods of measuring the antioxidant/reductive potential in the two assays.

It's noteworthy that these results are in good accordance with the TPC, TFC and TAC content of the samples, considering that all three groups of analyzed bioactive compounds were found in higher quantities in the UAE sample (see Table 2.).

Antimicrobial assays

Antimicrobial screening on human oral flora isolates

The extracts analyzed in this study showed to a.m. activity against the tested bacterial isolates and C. albicans (Table 5). The UAE BFP extract demonstrated greater AM in the disc-diffusion screening in comparison to that of the MAC, with zones of inhibition (ZOI) ranging from 13 to 17 mm. Considering the ZOI, the UAE extract exhibited the following hierarchy of a.m. activity concerning the tested strains: P. aeruginosa > E. coli = C. albicans > S. aureus = K. pneumoniae > MRSA = S. agalactiae. The AM activity of the MAC extract its AM activity the tested strains followed a similar, but not identical, trend: C. albicans > S. aureus > S. aeruginosa = E. coli > MRSA. The K. pneumoniae and S. agalactiae isolates used in our study have proven to be resistant to the MAC extract, although not to the UAE one.

Table 5.

Results of the antimicrobial screening via the disc diffusion methods using commercially available antibiotics for comparison.

Microorganism	BFP UAE	BFP MAC	Amikacin 30 μg	Penicillin 10 μg	Ceftriaxone 30 μg	Gentamicin 10 μg	Meropnem 10 μg	Trimethoprim/sulpha-metho-xazole 1.25/23.75μg (Bactrim^®)	Ciprofloxacin 5 μg	Cefixime 5 μg	Ampicillin 10 μg	Clindamycin 2 μg	Erythromycin 15 μg	Chloramphenicol30 μg	Amoxicillin/Clavulanic acid20/10 μg
Pseudomonas aeruginosa	17 mm	8 mm	20 mm S	N/A	18 mm R	17 mm S	31 mm S	N/A	30 mm S	N/A	N/A	N/A	N/A	N/A	N/A
Staphylococcus aureus	15 mm	9 mm	N/A	6 mm R	N/A	25 mm S	N/A	24 mm S	23 mm S	N/A	N/A	N/A	33 mm S	N/A	N/A
MRSA*	13 mm	4 mm	28 mm S	R	N/A	N/A	N/A	N/A	N/A	N/A	R	26 mm S	R	N/A	R
Escherichia coli	16 mm	8 mm	19 mm S	N/A	N/A	18 mm S	N/A	N/A	32 mm S	23 mm S	N/A	N/A	N/A	N/A	N/A
Klebsiella pneumoniae	15 mm	R	20 mm S	N/A	R	N/A	30 mm S	R	N/A	N/A	R	N/A	N/A	N/A	R
Streptococcus agalactiae	13 mm	R	N/A	30 mm S	N/A	N/A	N/A	N/A	N/A	N/A	N/A	N/A	33 mm S	23 mm S	N/A
Candida spp.	16 mm	11 mm	/	/	/	/	/	/	/	/	/	/	/	/	/

*MRSA – Methicillin – resistant Staphylococcus aureus; N/A = Not applicable – The antibiotic was not tested againist bacterial strain; (/) – Not tested - denotes Candida spp., for which antibiotic susceptibility testing is not applicable due to methodological limitations;

Antimicrobial testing; minimal inhibitory concentration (MIC)

Tested against the five bacterial food isolates, blackberry extracts analyzed in this study showed a certain specter of antimicrobial activity, comparable to that of the control (Ceftriaxone 100 mg/mL).

BFP extract obtained by sonication, which was tested for antimicrobial activity, exhibited the strongest inhibitory activity towards B. cereus, with MIC of 3.125 mg/mL (Table 6). The following, as a most sensitive, out of the bacterial strains tested, was the L. monocytogenes, while the Gram-negative bacteria tested (E. coli, P. aerusginosa and S. aureus) all required equal, and in comparison; higher MIC of the UAE sample (25 mg/mL). These results therefore imply that the Gram-negative bacteria (such as E. coli and P. aeruginosa) appear to be more resistant to the BFP extracts.

Table 6.

Antimicrobial activity of the BFP UAE extract, on selected, referent bacterial cell lines, in comparison to the ceftriaxone^© antibiotic.

Bacterial strain	BFP UAE extractMIC (mg/mL)	Ceftriaxone^® (QC)(100 mg/mL)
E. coli ATCC (25922)	25	6.25
P. aeruginosa ATCC (27853)	25	1.5625
S. aureus ATCC (6538)	25	50
L. monocytogens ATCC (13932)	12.5	12.5
B. cereus ATCC (11778)	3.125	12.5

QC – quality control

By testing the UAE extract (which was hypothesized to exhibit a higher degree of a.m. activity, both by observing higher antioxidant content and larger ZOI in the disc diffusion method) on the food isolates, as well as human oral cavity isolates, we were able to observe (and compare) a promising a.m. activity of BFP, with possible application in both the field of oral care/health and food technology.

Discussion

HPLC phytochemical characterization, determination of total phenolic, flavonoid, anthocyanin and proanthocyanidin content and phytochemical screening

HPLC phytochemical characterization

Selected polyphenolic compounds were quantified in our BFP samples using target HPLC, out of which the ellagic acid content was markedly higher than that of the other compounds. This was also appreciably higher than the gallic acid content, which is a similar finding to that reported by Guedes et al.,³³ although the results obtained in our study were higher for both compounds. Similarly, the ellagic acid content measured in both UAE and MAC samples was higher than that reported by Gil-Martínez et al. reported.³⁴ The quercetin content we have measured in our samples is in good accordance with that which Ungureanu et al.³⁵ have reported analyzing Romanian blackberry fruit and its byproducts (they found quercetin in the 0.58–2.96 mg/100 g d.s range).

Notably, quercetin and chlorogenic acid were higher in our MAC than in our UAE samples, and the opposite was true for ellagic, p-coumaric, gallic, and caffeic acids. Possibly the conventional maceration may somehow increase the yield of the quercetin and chlorogenic acid in extracts of the BFP, or the sonication process may degrade some of these molecules.

Determination of total phenolic, flavonoid, anthocyanin and proanthocyanidin content

The BFP analyzed in our study was found to contain phenolic content in amounts of the same order as those commonly reported in the literature,^36–44 for various blackberry fruit extracts, juices, and byproducts. The UAE sample of BFP showed higher amounts of TPC, TFC, and TAC than those reported by Vulić et al..³⁶ Conversely, the MAC sample analyzed in our study was found to contain a lower quantity of total phenols and flavonoids, although still higher amounts of anthocyanins. The TPC and TAC measured in the BFP samples in this study (both the UAE and MAC samples) were higher than those reported for the BFP of two different blackberry varieties, as analyzed by Četojević-Simin.³⁸

The relatively scarce literature data regarding the phenolic content of whole blackberry fruits suggest that their TPC, TFC, and TAC values vary over a broad range. This is probably greatly dependent on several factors, such as the methods of extraction, the solvent, and the edaphoclimatic conditions where the source plants were grown.²¹ It then comes as no surprise that the TPC, TFC and TAC determined in this study, for the BFP UAE sample, are both lower than those reported for the whole fruit (or their extracts); for example, those reported by Guerrero C. et al.,⁴⁵ while simultaneously higher or comparable to those reported in other studies for the whole blackberry fruits, and their extracts.^4,39,41 Finally, the content of these groups of bioactive molecules may also vary greatly depending on the blackberry fruit ripeness and developmental stage, as demonstrated by Huang et al.,⁴³ who have reported lower total phenolic and flavonoid content in whole green, unripe blackberry fruits, than those we measured in our work for both of the samples. These results suggest that ripe(r) blackberry fruits and their pomace are likely to be a richer source of phenolic compounds, particularly anthocyanins.

The TPC, TFC, and TAC of the UAE sample from this study are, interestingly, higher than those found in blackberry jams³⁹ and fresh juice obtained by microfiltration.³⁷ This is illustrative of the potential of BFP as a source of bioactive molecules otherwise not found in processed blackberry products, particularly the non-extractable polyphenolic compounds bound to the fruit fiber, which are missing in the juice.^46,47

The PC content found in the UAE sample was several times higher than that measured in the MAC sample. This could be explained by the fact that the sonication and higher temperatures seem to optimize the extraction of proanthocyanidins from plant material like grapes and medicinal plant leaves.^48–50 The PC content found in the MAC sample was comparable to that reported by Cáceres-Mella et al.²⁷ for vine spirits and higher than that reported for several types of juices by Kelm Ma and associates.⁵¹ Comparatively, the PC content of the MAC sample in this work was only higher than the one they reported for the apple juice.

BFP phytochemical screening

Phytochemical screening of BFP revealed the presence of compounds expected to be found in fresh fruit as well.

Tannins are routinely found in, and even quantified and characterized in, fresh or frozen blackberry fruit (as well as in its close relative raspberry, strawberry, and even blueberry fruits), as evidenced by Subbiah and associates.⁵² Similarly, terpenes are also found abundantly in fresh fruit (alongside other volatile organic compounds abbrev. VOC) contributing to the flavor and aroma of the fruit, at least six of which have been identified and quantified in Rubus fruticosus.⁵³ Catechin and chlorogenic acid were screened for, and quantified in several varieties of blackberry fruit grown in Anatolia by a group of scientists,⁵⁴ and they found the both compounds varied significantly among the selected cultivars. Catechins could in fact be the dominant polyphenolic compound group in blackberry fruits, at least in those grown at higher altitudes in tropical climates.⁵⁵

Most plants of the Rosaceae family contain cyanogenic glucosides in their fruit⁵⁶ at varying concentrations. In the phytochemical screening of BFP during our own tests, we found that it also tested positive for the presence of cyanogenic glucosides.

While saponins can be found in the roots of some Rubus species (ex. Rubus parvifolius and Rubus elipticus),^57,58 the BFP extract analyzed in this study did not test positive for their presence. Likewise, alkaloids and anthraquinones were not present in the BFP extracts. Similarly, neither of these are typically found in fresh fruits.

Antioxidant activity (DPPH and FRAP assay)

The antioxidant potential of fruits, their juices, and extracts, which is dependent on the bioactive molecules present in them, is often a good indicator of their antioxidant activity in vivo. For example, polyphenols have been shown to scavenge free radicals and quench ROS in experimental animals such as mouse,⁵⁹ but experience has taught us that few complementary in vivo, and in vitro assaying methods/tests are far more telling than any single one on its own. Additionally, the extraction methods and solvents used are routinely and persistently shown to elicit different scopes of antioxidant activity in samples, particularly those of berry fruits.^21,60

The higher in vitro antioxidant activity of the UAE extract compared to that obtained by conventional maceration in our study is most likely due to the increased yield of polyphenolic compounds. This is common and expected when extracting fruit and vegetable biowaste in particular, as the sonication creates cavitations in the cell wall structure, as well as causing plant cell vacuoles to burst, which results in a higher amount of BAM extracted in the solvent.^7,8,22 This was found to be the case with proanthocyanidins in some fruit extracts,^48,50 which then naturally contributed to the increased a.a. of these same extracts. Additionally, mild acidulation of the organic solvents (such as methanol) helps extract BAM from the fruits, particularly the red fruits and berries, causing phenols in particular, to be more readily extractable into the organic solvent, while also potentially preventing their denaturation and denaturing cell membranes and facilitating their initial release.^7,21,22

Despite the scarce data in the literature concerning the antioxidant activity of the BFP specifically, we made the effort to compare the data we have obtained to the relevant, similar data concerning blackberry fruit and products made from it (juices, jams, for example), as well as byproducts, available in the literature.^{36,39,43,44,61–66}

The IC₅₀ (μL/mL) (representing the concentration of the tested substance in the sample/extract required to inhibit/neutralize 50% of the DPPH molecules present in the reaction mixture) calculated for the UAE and MAC samples in this study was lower than that found by researchers studying wild and cultivated blackberry fruit pomace from Serbia.⁶¹ Both IC₅₀ (μL/mL) values in our work, were however higher (implying therefore lower antioxidant activity) than those Vulić et al.³⁶ have measured evaluating a.a. for the blackberry pomace while researching the pomace of the 4 berry fruits from Serbia.

This value, however, (IC₅₀ (μL/mL) in DPPH assay) for both the UAE and MAC extracts (in our work) was lower than those in literature which reported this type of result for whole blackberry fruits or their extracts.^39,43^62–64 The fact that the fruit pomace extract demonstrates higher antioxidant activity than that of the whole fruit (and also its juice) in the DPPH assay has been reported before among the Rosaceae family fruits, such as sweet rowanberry⁶⁷ and various apple cultivars.⁶⁸

It would be wise to note here that, despite our best attempts to compare the data obtained to those typically reported in the literature, we were precluded from achieving ideal comparisons due to several factors. The methods applied to test a.a. in vitro vary abundantly in literature, as do the units by which the a.a. is reported.^60,69,70 This, in turn, makes it utterly difficult to form completely satisfactory comparisons. Additionally, the a.a. of berry fruits, by itself and naturally, presents within a wide spectrum of value variations (even in the same type of samples, from the same species), which is a result of biochemical (and genetic) polymorphism and environmental factors such as growing location, cultivation methods, fruit maturity, processing, and storage conditions.⁷¹

Antimicrobial activity

Blackberry fruit pomace has, in recent years, been studied as an inexpensive source of antimicrobial compounds against Campylobacter jejuni from poultry, and as a key ingredient in an edible antioxidant and antimicrobial food coating.^72,73 A few studies, however, have dealt with determining the in vitro, antimicrobial potential of the BFP, as the whole fruit extract, and processed byproducts (such as juice etc.) are more often used.

In our study, the UAE BFP extract showed markedly higher antimicrobial activity against both Gram-positive and Gram-negative strains using both methods. This could be due to almost twice the content of ellagic acid, and higher content of gallic acid in the UAE sample, both of which are well-established as antimicrobial agents.

Our extracts, particularly the UAE, arguably showed more pronounced activity against gram-positive strains than gram-negative strains. A similar finding was reported by Gil-Martínez and her colleagues when they tested blackberry fruits’ antimicrobial activity.³⁴

One of the few studies that also tested the antimicrobial activity of BFP using the broth microdilution method³⁸ reported results that were almost diametrically opposed to ours. They found that the most sensitive were S. aureus and P. aeruginosa (MIC respective ranges for the two being: 0.39–3.125 mg/mL and 0.39–6.25 mg/mL), while the most resistant microorganisms were Bacillus cereus, and Candida albicans (MIC for B. cereus being above the tested range; > 25 mg/mL and MFC for C. albicans being in the same range).

The disc diffusion method in our study showed that P. aeruginosa and oral pathogenic strain human isolates were sensitive to the UAE BFP extract, as opposed to the results obtained in the MIC broth microdilution assay, which used food isolates. However, even the same strains may react differently in the disc diffusion and broth microdilution assays, seemingly contradicting one another in terms of results, as reported in a study on whole blackberry fruits from Serbia.⁷⁴ Our ZOI in the disc diffusion method was larger than those reported by these authors for E. coli and S. aureus in the aforementioned study.⁷⁴

It is also worth mentioning that our disc diffusion screening of the antimicrobial potential of the BFP showed that C. albicans was sensitive to the UAE sample, which is of interest considering that it was a human oral pathogen isolate, the same type typically responsible for the appearance of oral thrush.

The statistical analysis (PCA analysis)

The application of principal component analysis (PCA) on the obtained data concerning the phenolic profile and the antioxidant potential of the studied samples (blackberry fruit pomace extracts) showed that the first two principal components (PCs) described more than 94% of the samples’ variability, as shown in Figure 1.

The variability of PC1 is mostly dependent on the quantified amounts of p-coumaric, ellagic, caffeic, and gallic acids, the quantified level of total anthocyanins, and the antioxidant potential as measured in the in vitro assays performed. The variability shape, as determined by PC2, correlated with the quantified levels of the quercetin found in the examined samples.

The position of the examined samples in the space defined by the first two PC axes revealed the localization of sample 1 centroid in the negative part of PCA1, mostly as a result of lower quantities of specific phenolic acids, as well as weaker radical scavenging capacity. In contrast, Sample 2 was localized in the positive part of PCA1 because these extracts exhibited stronger antioxidant potential and were generally more abundant in quantified secondary metabolites (except quercetin and its glycosides).

UAE of blackberry maximized the yield of extraction in terms of total phenolic, flavonoid, and anthocyanin compounds, caffeic, ellagic, p-coumaric, and gallic acids, and the extracts exhibited higher antioxidant potential concerning the MAC. On the other hand, it seems the maceration maximized the yield of quercetin extracted from blackberries.

Conclusion

The BFP we’ve analyzed has proven to be a rich source of valuable BAM, such as ellagic acid, gallic acid, quercetin, catechins, chlorogenic acid, tannins, and nonextractable fibers missing from the processed products. The concentrations we’ve measured are even higher than those reported for whole fresh or freeze-dried fruit, which could be influenced by edaphoclimatic factors and genetic polymorphism, which are some of the factors reported to affect the yield of SM. Many BAM in BFP, in addition to having antiradical potential, also have antimicrobial potential, especially against human oral pathogen isolates, and may also “spare” the useful oral flora, as reported in the literature, warranting further research in the direction of the use of BFP extracts as potential adjuvants to human oral health. Finally, the sonication process, alongside the mild acidulation of the extracting solution, seems to appreciably increase the yield of extracted BAM from this type of biowaste, signaling a possible direction in sustainable green biotechnology to maximize the extraction of the BFP, even if the sonication may reduce the quercetin concentration specifically. It should be noted that due to the solvent difference used in our study, we cannot positively determine whether the yield of these compounds was greater in the UAE sample solely due to sonication, due to acidulation of the sample, or as a result of the synergism of the two; however, we believe it is likely due to synergism. Also, the notably different values in terms of SM yield and a.a. and a.m activity have not been statistically tested for correlation this time, as the dataset was simply not large enough. Nevertheless, the results obtained imply that BFP is a good source of chemicals for the nutraceutical and food industries and a promising source of novel antimicrobials to combat the ever-growing antimicrobial resistance issue.

Supplemental Material

sj-docx-1-ber-10.1177_18785093251400043 - Supplemental material for Extraction method for blackberry fruit pomace and its active compounds: Antioxidant activity and antimicrobial effects on human oral flora isolates

Supplemental material, sj-docx-1-ber-10.1177_18785093251400043 for Extraction method for blackberry fruit pomace and its active compounds: Antioxidant activity and antimicrobial effects on human oral flora isolates by Milorad Vlaović, Kenan Preljević, Svetlana Perović, Biljana Damjanović-Vratnica, Andrej Perović and Slađana Krivokapić in Journal of Berry Research

Footnotes

Acknowledgements

The authors wish to express our utmost gratitude to Emma Devaney and Anita Šabotić, for all the help with making the document far more readable and polished.

ORCID iDs

Milorad Vlaović

Kenan Preljević

Svetlana Perović

Biljana Damjanović-Vratnica

Andrej Perović

Slađana Krivokapić

Al disclosure

The authors declare that generative artificial intelligence (Paperpal's AI) was used solely to improve the readability and language fluency of the manuscript. The authors take full responsibility for the content and the scientific conclusions of this work.

Funding

This research was supported by the Montenegrin Ministry of Science (Project SCIMPLANT S!12689).

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Supplemental material

Supplemental material for this article is available online.

Correction (December 2025):

This article has been updated to correct the unit from µg/ml to µg/g in a few instances.

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