Centesimal Composition, Bioactive Compounds, and Antimicrobial Properties of White (Hylocereus undulatus) and Red (Hylocereus polyrhizus) Pitayas

TL;DR

This study compares the nutritional and antimicrobial properties of red and white pitaya pulps, finding both rich in bioactive compounds with antioxidant and antimicrobial potential.

Contribution

The study identifies specific bioactive compounds and evaluates antimicrobial and anti-quorum sensing properties in red and white pitaya pulps.

Findings

Red pitaya pulp has higher total phenolic content and contains betalains like phyllocactin.

Both pulps show antioxidant activity, with red pitaya performing better in ABTS and beta-carotene assays.

Extracts from both pulps exhibit mild antimicrobial activity and anti-quorum sensing effects.

Abstract

This study investigated the centesimal composition, physicochemical properties, bioactive compounds, antimicrobial activity, and anti‐quorum sensing (QS) potential of red pulp (RP; Hylocereus polyrhizus) and white pulp (WP; Hylocereus undulatus) pitayas. Both pulps exhibited high moisture content and low protein levels, with ash contents of 0.78% (RP) and 0.70% (WP), titratable acidity of 0.26% for both, and pH values of 5.75 (RP) and 5.12 (WP). RP pitaya presented a higher total phenolic content and contained 3.15 mg/100 g of betalains. Antioxidant capacity differed according to the assay employed: RP showed higher activity in the ABTS•+ and β‐carotene/linoleic acid methods, whereas WP exhibited greater radical scavenging capacity in the DPPH• assay. LC–MS/MS analysis revealed phyllocactin as the major betalain in RP pitaya, along with 4′‐O‐malonyl‐betanin and isophyllocactin. Four flavonoids were identified, with quercetin 3‐rutinoside as the predominant compound. Both extracts displayed mild antimicrobial activity against foodborne pathogens and inhibited violacein production by Chromobacterium violaceum, indicating anti‐QS activity. However, no inhibitory effects were observed on swarming motility or biofilm formation under subinhibitory concentration. Overall, the results highlight pitaya pulps as sources of bioactive compounds with antioxidant, antimicrobial, and anti‐QS properties, supporting their potential application in food systems and as natural ingredients for microbial control strategies.

Figures

HPLC profile of betalains from the RP pitaya extract recorded at 530 nm. Peaks identities are shown in Table 3 under betacyanins. Peak 1: betanin; Peak 2: (iso) betanin; Peak 3: phylocactin; Peak 4: 4′‐O‐malonyl‐betanin; and Peak 5: isophyllocactin.

Inhibition of violacein production in Chromobacterium violaceum by WP and RP pitaya extracts. C: control; E: extract.

Percentage of inhibition of violacein production by red (A) and white (B) pulp pitaya extract at different concentrations (mg GAE/mL of extract).

Biofilm formation in the presence of white (A) and red (B) pitaya pulp extract. White pitaya extracts at 50%, 25%, and 12.5% correspond to 9.6, 4.8, and 2.4 mg GAE/mL, respectively. Red pitaya extracts at 25% and 12.5% correspond to 5.3 and 2.6 mg GAE/mL, respectively.

Tables

TABLE 1. Proximal composition and physicochemical characteristics of pitayas.

Analysis	Pitaya pulp
	Red pulp (RP)	White pulp (WP)
Proteins (%)	1.23 ± 0.10 a	0.62 ± 0.15 b
Moisture (%)	85.47 ± 0.80 a	88.31 ± 0.55 b
Ashes (%)	0.78 ± 0.09 a	0.70 ± 0.27 a
pH	5.75 ± 0.00 a	5.12 ± 0.00 b
Acidity (%) (g citric acid/100 g)	0.26 ± 0.06 a	0.26 ± 0.04 a

TABLE 2. Content of phenolic compounds, betalains, and antioxidant capacity found on pitaya extracts.

Analysis	Pitaya extract
	Red pulp (RP)	White pulp (WP)
Total phenolic compounds (mg GAE/g)	4.69 ± 0.03 a	4.36 ± 0.22 b
Antioxidant capacity DPPH (EC50 g fruit/g DPPH)	38,018.74 ± 1941.73 a	19,692.21 ± 499.51 b
Antioxidant capacity ABTS (mM Trolox/g)	19.66 ± 0.04 a	0.18 ± 0.01 b
Antioxidant capacity β‐carotene/linoleic acid (% of protection)	35.52 ± 2.97 a	28.48 ± 4.09 b
Betalain (mg betalain/100 g)	3.15 ± 0.15	—

TABLE 3. Mass spectra and amount of betalains identified by LC‐qTOF‐MS/MS in red pitaya extract.

RT (min)	λ max (nm)	[M + H]+ (m/z)	MS2 (m/z)	Compound	Ref	(mg/mL)
Betaxanthins
3.3	481	309.0576	263.0598/217.0607	Isocaxanthin	1	—
3.6	481	309.0572	263.0596/217.0603	Indicaxanthin	1	—
Betacyanins
3.4	533	551.0643	389.0358	Betanina	2	0.495 ± 0.03
4.1	531	551.0636	389.0358	(Iso)betanina	2	0.0489 ± 0.0003
4.9	536	637.0529	593.0701/389.0368	Phyllocactin	1, 2	0.864 ± 0.006
5.1	531	637.0518	389.0365	4′‐O‐malonyl‐betanin	1, 2	0.0734 ± 0.005
5.4	531	637.0521	593.0680/389.0358	Isophyllocactin	1, 2	0.0869 ± 0.0006
				Total		1.569 ± 0.005

TABLE 4. Mass spectra and amount of flavonoids identified by LC‐qToF‐MS/MS of WP and RP pitaya extracts.

RT (min)	[M − H]− (m/z)	MS2	Flavonoid	Pitaya extracts
				RP	WP	WP (µg/mL)
8.8	449.2459	287.1800	Eriodictyol hexoxide	✓	nd	—
10.1	465.0297	303.0006	Taxifolin hexoside	✓	nd	—
10.2	609.2039	300.0565/151.0154	Quercetin 3‐rutinosidea	✓	✓	10.61 ± 0.01
12.3	623.2270	517.3453/315.0815	Isorhamnetin hexoside	nd	✓	—

TABLE 5. Inhibition potential (IP) obtained for different concentrations of pitaya extract against the indicated microorganisms.

Microorganisms	RP pitaya		WP pitaya
	mg GAE/mLa	IPb	mg GAE/mLc	IPb
Escherichia coli	10.6	0.8	9.6	0.8
	5.3	0.4	4.8	0.4
	2.6	0.2	2.4	0.2
Listeria monocytogenes	10.6	1.1	9.6	0.3
	5.3	0.7	4.8	0.2
	2.6	0.6	2.4	0.1
Pseudomonas aeruginosa	10.6	2	9.6	0.7
	5.3	1.4	4.8	0.6
	2.6	0.7	2.4	0.1
Salmonella spp.	10.6	1.5	9.6	0.8
	5.3	1.4	4.8	0.6
	2.6	0.7	2.4	0.5
Staphylococcus aureus	10.6	1.1	9.6	2.4
	5.3	1	4.8	1.3
	2.6	0.6	2.4	0.7

TABLE 6. MIC (%) of extracts against the tested microorganisms.

Microorganisms	Red pitaya MIC		White pitaya MIC
	% of extract	mg GAE/mL of extract	% of extract	mg GAE/mL of extract
Chromobacterium violaceum ATCC 12472	>50	>10.6	>50	>9.6
Aeromonas hydrophila IOC/FDA 110‐36	>50	>10.6	>50	>9.6
Serratia marcescens RM1	>50	>10.6	>50	>9.6
Hafnia alvei	>50	>10.6	>50	>9.6

Full text

Introduction

1

Pitaya is a fruit native to Central America and Mexico that belongs to the Cactaceae family and is grown in different parts of the world. There are several species called “pitayas,” among which can be cited Hylocereus polyrhizus, with a red pulp (RP), and Hylocereus undatus, with a white pulp (WP) (Junqueira et al. 2010; Huang et al. 2021; Attar et al. 2022). Depending on the variety, it has a crimson or yellow rind covered with scales, from which the name dragon fruit is derived. Its pulp is creamy, slightly sweet, red, white, or yellow, depending on the variety. The pulp is filled with small black seeds (de Mello et al. 2014; Ferreira et al. 2023).

Pitaya has attracted attention for its economic value, attractive appearance due to its colors and shapes and its potential benefits to human health (Le 2021). It can be used as an ingredient with functional claims and in nutraceutical products (Wichienchot et al. 2010), as a plant‐based food matrix for the development of functional nondairy probiotic products (Usaga et al. 2022) as well as a source of bioactive compounds (Usaga et al. 2022; Zulkifli et al. 2020).

The color of RP pitaya is mainly due to the presence of betalain, a class of pigments that generate attractive colors, in particular betacyanin (Mahayothee et al. 2019). Among its functional properties, betalains have the ability to neutralize free radicals, delay lipid oxidation, and inhibit microbial growth, in addition to combating inflammatory processes (Luiza Koop et al. 2022). Some studies also detected anthocyanins in Hylocereus sp., including cyanidin 3‐glucoside and cyanidin 3‐rutinoside in red and WP (Fan et al. 2020).

Phenolic compounds are secondary metabolites related to the defense systems of plants against ultraviolet radiation or the aggression of insects or pathogens (Nawaz et al. 2024; Özkan et al. 2025). In humans, they play an important protective role as antioxidant agents, capable of delaying the oxidation of various substrates (Zulkifli et al. 2020; Rahman et al. 2021). In foods, they are responsible for color, astringency, bitterness, and aroma, in addition to conferring oxidative stability to products of plant origin (Albuquerque et al. 2021). Recently, studies have also demonstrated the use of phenolics in extending the shelf life of products and controlling pathogenic bacteria, as demonstrated by Carvalho et al. (2022) and Lima et al. (2022) using phenolic‐rich extract in edible coating against foodborne pathogens.

The antioxidant capacity of bioactive compounds refers to their ability to delay damage caused by reactive oxygen species (ROS) or to prevent the generation of these species through the sequestration of free radicals and electron or hydrogen atom donation (Gulcin 2025; Barbosa et al. 2010). Recently, the search for antioxidant compounds from plants and foods is increasing (Tamfu, Ceylan, Fru, et al. 2020).

Regarding antimicrobial activity of phenolic compounds, several mechanisms can be linked, such as destabilization and/or permeabilization of the cytoplasmic membrane, direct actions on microbial metabolism, inhibition of extracellular microbial enzymes, deprivation of essential substrates, and inhibition of extracellular microbial enzymes (Puupponen‐Pimiä et al. 2005; De Rossi et al. 2025; Takó et al. 2020).

In addition, recent studies have shown that phenolic compounds have quorum sensing (QS) inhibition properties in bacteria (Santos et al. 2020; Lima et al. 2025; Quecán et al. 2026). QS is a communication system between microorganisms that is dependent on population density. In bacteria, this system depends on the production of low‐molecular‐weight signaling molecules known as autoinducers (AIs), which are released into the environment; when a threshold concentration of these compounds is reached, bacteria respond by activating or repressing certain genes that modulate bacterial behavior (Lima et al. 2023; Bassler 2002).

Because QS plays a central role in regulating coordinated microbial activities, recent studies on bacterial QS communication have focused on elucidating its involvement in microbial interactions, biofilm formation, production of bacteriocin, synthesis of virulence factors, and its broader relevance in the field of food microbiology (Grandclément et al. 2016; Lima et al. 2026; Ruan et al. 2026; Machado et al. 2020).

This study aimed to evaluate the centesimal composition and physicochemical characteristics of RP (H. polyrhizus) and WP (Hylocereus undulatus) pitayas, as well as the characteristics of their bioactive compounds and the antimicrobial and anti‐QS activities of the crude extracts obtained from these fruits.

Materials and Methods

2

Plant Material Preparation

2.1

Samples of RP and WP of pitayas were obtained from a local market in the city of Ouro Preto‐MG, Brazil. According to vendors, the fruits were produced in the surrounding region, without detailed data regarding the place of production or cultivation conditions. The fruits were sanitized with 50 mg/L sodium hypochlorite solution for 15 min and then washed again with water to remove residual chlorine. Afterwards, the peels were removed, and the pulps were manually separated and individually homogenized in a domestic multiprocessor and stored at −20°C.

Proximal Composition and Physical–Chemical Analysis of Pitaya Pulps

2.2

The proximal composition of the pitayas pulp was determined according to the methodology proposed by the Association of Official Analytical Chemists (AOAC 1998) for moisture, proteins, and ash. pH and acidity were determined according to the methodology proposed by the Adolfo Lutz Institute (Adolfo Lutz Institute 2008).

Evaluation of Bioactive Compounds

2.3

Preparation of Pitayas Pulp Extracts

2.3.1

Crude extracts were obtained according to the methodology described by Bertoldi (2009), with adaptations detailed by Santos et al. (2020). The pulps of the pitayas were thawed, weighed, and mixed in a blender with an extraction solution containing acetone, ethanol, and methanol at a ratio of 1:1:1 (v/v/v). Subsequently, the samples were transferred to amber tubes, refrigerated, and stirred in a shaker (ACBLabor, Brazil) at 150 rpm for 2 h. Afterwards, the samples were filtered through Whatman no. 1 paper, and the solvents were evaporated in a rotary evaporator (Büchi, Switzerland) at 40°C. The crude extracts were stored in falcon tubes, protected from light, and stored at −20°C until analysis.

Content of Total Phenolic Compounds

2.3.2

The total phenolic compound (TPC) content in the extracts was determined using the Folin–Ciocalteu reagent assay according to the methodology described by Waterhouse (2001). A 0.5 mL aliquot of the crude extracts from the RP and WP pitayas was added to a tube containing 2.5 mL of Folin–Ciocalteu determined at 750 nm using absorption spectrophotometry in the UV–visible region (Global Trade Technology, Brazil), with ethanol used as a blank. Total phenolic content was determined using a gallic acid standard curve (0–200 mg/L). The results were expressed as mg gallic acid equivalent per gram of sample (mg GAE/g sample). The experiment was performed in triplicate.

Content of Total Betalain

2.3.3

Betalain was quantified as described by Tang and Norziah (2007). One gram of red pitaya pulp was homogenized in distilled water and transferred to a volumetric flask to make up the volume to 100 mL. The sample was then filtered through Whatman no. 1 paper, and the filtrate was analyzed using a spectrophotometer (Global Trade Technology, Brazil) at 536 nm. Betalain was quantified using the following equation:

$$[eqn]$$

where Bc is the betanin equivalent (mg/L); A is the mean abs at 536 nm; (0.344); MW is the molecular weight of betanin (550 g/mol); Ɛ is the molar extinction coefficient of betanin (60,000 L/mol cm); I is the cuvette width (1 cm); and 1000 is the dilution factor.

Identification and Quantification of Betalains and Flavonoids

2.3.4

White and red pitaya extracts were cleaned up by solid‐phase extraction using a polyamide column (CC6, Macherey‐Nagel GmbH & Co. KG; Düren, Germany). Betalains and flavonoids were analyzes by LC‐qToF‐MS/MS as described by Fraga et al. (2021) using a liquid chromatography (Prominence, Shimadzu, Japan) coupled to the mass spectrometer type qTOF, model Compact (Bruker Daltonics, Germany). The column used was a Poroshell 120 C18 (2.7 µm particle, 100 × 3.0 mm) (Agilent, CA, USA), with a flow rate of 0.50 mL/min and running temperature of 25°C. The mobile phases used were (A) 0.5% formic acid in water and (B) acetonitrile. The eluates were monitored at 270 and 370 nm for flavonoids and 530 nm for betalains by a diode array detector (DAD). The mass spectrometer was operated in negative mode for flavonoids and positive mode for betalains (source voltage 3500 V, cone temperature 350°C, cone gas flow 20 L/min, heated probe temperature 350°C, probe gas flow 40 U, and nebulizer gas flow 50 U).

Betalains were identified according to combined information provided by mass spectra, retention time (red beet extract from Sigma), and data from literature. All peaks of betalains were quantified using the calibration curve of betanin (red beet extract) from Sigma (901266) and expressed as betanidin 5‐β‐d‐glucoside at 530 nm. Flavonoid was quantified using a calibration curve of quercetin‐3‐rutinoside at 370 nm.

Determination of the Antioxidant Capacity

2.4

The antioxidant capacity was evaluated using three methods: DPPH• (2,2‐dIphenyl‐1‐picrylhydrazyl) radical capture assay according to Brand‐Williams et al. (2005), with some modifications; ABTS•^+^ (2,2′‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid)) radical capture assay according to Rufino (2007), and β‐carotene assay according to Marco (1968). All analyzes were performed in the dark and in triplicate.

For DPPH• assay, briefly, a 0.1 mL aliquot of each dilution of the extracts was allowed to react with 3.9 mL of the DPPH radical solution for 120 min in the dark. The absorbance was determined at 515 nm by absorption spectrophotometry in the UV–visible region (Global Trade Technology, Brazil) after 2 h of rest, protected from light, with methanol as a blank. The antioxidant capacity was determined using the DPPH• standard curve. From the absorbance values, the equation of the straight line was determined, from which the EC50 (g fruit/g DPPH•) was calculated, which corresponds to the concentration of extract needed to reduce the initial concentration of the DPPH• radical by 50% (Brand‐Williams et al. 2005). All analyses were performed in triplicates.

For ABTS•^+^ assay, in a test tube, 5 mL of a previously prepared aqueous solution of 7 mM ABTS•^+^ and 88 µL of 140 mM potassium persulfate were added. The mixture was then allowed to stand for 16 h. Next, the ABTS•^+^ radical cation was generated by diluting 1.0 mL of this mixture in ethanol until an absorbance of 0.70 ± 0.05 nm was obtained at 734 nm. The RP and WP pitaya extracts (30 µL) or 30 µL of the reference compound (Trolox) were reacted with 3 mL of blue–green ABTS•^+^ radical solution. The reduction in absorbance at 734 nm was measured after 6 min. Ethanolic solutions with known concentrations of Trolox were used to construct a standard curve. The results were expressed as micromoles of Trolox equivalents (TE) per gram of pitaya pulp (µmol/g pitaya pulp) (Rufino 2007). The entire experiment was performed in the dark and in triplicate.

For β‐carotene assay, an aliquot (50 µL) of the chloroform solution of β‐carotene (20 mg/mL) was added to a flask containing 40 µL linoleic acid, 1.0 mL chloroform, and 530 µL Tween 40. Chloroform was evaporated using an oxygenator. After the evaporation process, oxygenated distilled water (approximately 100 mL) was added to obtain an absorbance of 0.65 ± 0,1 at 470 nm. An aliquot (0.4 mL) of Trolox solution (200 mg/mL) or pitaya pulp extract (200 mg/mL) was added to 5.0 mL of β‐carotene solution and incubated in a water bath at 40°C. Measurements were performed after 2 and 120 min at an absorbance of 470 nm using a spectrophotometer (Global Trade Technology, Brazil) calibrated with water (Marco 1968). The results are expressed as % of protection.

Determination of Antimicrobial Activity

2.5

Antimicrobial activity was evaluated by determining the inhibition potential (IP) in liquid medium according to the methodology proposed by Nohynek et al. (2006) and Alvarez et al. (2012), with modifications, against the bacteria Staphylococcus aureus ATCC 6538 and Salmonella spp. ATCC 14028, Listeria monocytogenes ATCC764, Escherichia coli ATCC 10536, and Pseudomonas aeruginosa ATCC 15442.

Volumes of 300 µL of Luria Bertani (LB) broth containing different concentrations of red and white pitaya extract (0%, 12.5%, 25%, and 50%) were inoculated into sterile eppendorf tubes. Subsequently, each microtube was inoculated with 3 µL of the test microorganism grown overnight. The samples were incubated at 37°C for 24 h. After incubation, serial dilutions of the samples were performed and plated (surface) on LB agar. The plates were incubated under the same conditions as described above, and the colony forming units (CFUs) were counted. The results were expressed as the IP, calculated according to the following equation:

$$[eqn]$$

where IP is the inhibition potential, N0 is the colony‐forming units on control plates, and N is the colony‐forming units on plates with different concentrations of extracts.

Determination of Anti‐QS Activity of Pitaya Pulp Extracts

2.6

The anti‐QS effect of crude extract of white and red pitayas was evaluated against a panel of microorganisms from the Food Microbiology Laboratory at UFOP, including Aeromonas hydrophila IOC/FDA 110‐36, Chromobacterium violaceum ATCC 12472, Serratia marcescens RM1, and Hafnia alvei. These QS assays were performed at concentrations that do not interfere with bacterial growth (i.e., sub‐MIC concentrations), a premise for QS assays (Lima et al. 2023).

Effect on Violacein Production by C. violaceum

2.6.1

The test in solid medium was performed according to the methodology described by Salawu et al. (2011) with some modifications. A 20 µL volume of molten LB agar containing 10^5^ CFU/mL of C. violaceum ATCC 12472 was poured into pre‐sterilized Petri dishes. After solidification, 5 mm holes (wells) were drilled using sterile 1 mL tips. Then, 20 µL aliquots of the crude extract of the RP and WP pits were added to each of the holes. The plates were refrigerated overnight and incubated at 28°C for 24 h. The inhibitory activity of the extracts was verified by the formation of clear zones around the wells (halos) compared to the control (sterile water). The inhibitory activity was evaluated as the mean of two repetitions, and the results were expressed as the mean diameter of the inhibition halos.

The test was also performed in liquid medium according to the methodology proposed by Tan et al. (2013), with some modifications. Tubes containing 1.5 mL of LB broth with different concentrations (50%, 25%, 12.5%, and 6.5%) of sub‐MIC of crude extract from the RP or WP pitayas were inoculated with 100 µL of a 10^8^ CFU/mL culture of C. violaceum grown overnight. The tubes were incubated at 28°C for 24 h. Subsequently, centrifugation at 13,000 rpm for 10 min was performed to precipitate the violacein pigment. The pellet was solubilized in 1 mL of DMSO (dimethylsulfoxide) and agitated on a vortex‐type shaker. Further centrifugation was performed under the same conditions as described above to separate the cellular debris from violacein. Violacein pigmentation was evaluated at a wavelength of 585 nm using a microplate spectrophotometer (Epoch, BioTek, USA). The negative control was prepared in LB broth without adding extracts, and the positive control of the QS system was prepared by adding 10 mg/mL of furanone ((Z‐)‐4‐Bromo‐5‐(bromomethylene)‐2(5h)‐furanone)‐C30 (Sigma‐Aldrich, USA).

The percentage of violacein inhibition was calculated according to the following equation:

$$[eqn]$$

where “OD” is the optical density, “OD control” is the mean of the control absorbance, and “OD test” is the mean of the absorbance of the samples at different concentrations.

Effect on Swarming Motility

2.6.2

The effect on swarming motility was performed according to the methodology proposed by Packiavathy et al. (2014) with some modifications. Semisolid 0.5% (w/v) LB agar containing different sub‐MIC concentrations of the extracts was poured into Petri dishes and kept at rest for 10 min. Subsequently, 2 µL of A. hydrophila, S. marcescens, and H. alvei were inoculated on the central region of the agar. The plates were then incubated for 24 h. The results of motility reduction were visually evaluated in comparison with the control agar without the addition of extracts.

Effect on Biofilm Formation

2.6.3

The assay was performed according to the methodology proposed by Huber et al. (2003) with some modifications. LB broth (200 µL) containing sub‐MIC concentrations of red (0%, 12.5%, and 25%) and white (0%, 12.5%, 25%, and 50%) pitayas pulp extract and 2 µL of a 10^8^ CFU/mL culture (grown overnight) of A. hydrophila, S. marcescens, and H. alvei were inoculated into sterile 96‐well microplates. Subsequently, the plates were incubated at 30°C (S. marcescens and H. alvei) or 37°C (A. hydrophila) for 72 h. After incubation, the culture medium was removed by inverting the microplate on absorbent paper. The sessile cells were stained with 200 µL of 0.1% (w/v) crystal violet for 30 min and then removed, and the wells were washed three times with 200 µL of sterile distilled water. The plates were then dried in an oven at 40°C for 15 min. The crystal violet retained by the adhered cells was solubilized in 200 µL of ethanol (95%) and the absorbance was read at 630 nm using a microplate spectrophotometer (Epoch, BioTek, USA). Biofilm formation was evaluated relative to the control using the following equation:

$$[eqn]$$

where “OD” is the optical density; “Test” is the LB broth with phenolic extract and bacteria; “Broth extract” is the blank LB broth with phenolic extract; “Control” is the LB broth  with bacteria; and “Broth” is the blank LB broth.

Analysis of Results

2.7

The results of centesimal composition and bioactive compounds were analyzed by analysis of variance (ANOVA) and Tukey's test at 5% significance using the Sisvar software (Ferreira 2011). Analyses of inhibition on solid medium and motility were based on observation and comparison of the control with the tests. The IP and biofilm formation were evaluated by comparing the means of the samples with those of the control.

Results and Discussion

3

Proximal Composition and Physical–Chemical Analysis

3.1

Red pitaya pulp had a higher (p ≤ 0.05) protein content than white pitaya pulp (Table 1). The values obtained were similar to those observed by Abreu et al. (2012), who reported a protein content of 0.78% for WP pitaya, whereas de Mello et al. (2014) and Jeronimo (2016) reported protein contents of 0.84%–2.7% for RP pitaya. Factors, such as the degree of ripeness, soil, and climate, can influence the protein content of fruits, thereby explaining the different values observed (de Mello et al. 2014; Oliveira et al. 2016). Both fruits had high moisture content (Table 1). The moisture contents observed are in agreement with those obtained by Abreu et al. (2012), who found moisture contents of 85.52% and 86.08% for the pulp of red and white pitayas, respectively. In general, fruit pulps have an average moisture content between 65% and 95% (Chitarra and Chitarra 2005). Moisture content can affect the stability and quality of the product and is one of the main intrinsic factors related to microbiological deterioration. It is of fundamental importance to define conservation and storage techniques and strategies.

The ash content, which constitutes the inorganic or mineral fraction of foods, did not differ (p > 0.05) between red (0.78%) and white (0.70%) pulp pitayas. These values differ from those obtained by Abreu et al. (2012), who reported 0.36% for RP pitaya and 0.39% for WP pitaya. This value may be associated with the higher concentration of minerals in the samples (Uchoa et al. 2008). The state of maturity and cultivation can significantly change the ash content. In fresh fruits, the ash content can vary from 0.4% to 2.1%, and large amounts of potassium, sodium, and calcium can be found, as well as small amounts of iron, manganese, and zinc (Correia et al. 2010).

The pH values did not differ (p > 0.05) from each other, and pitaya is considered a low‐acid fruit. The values found corroborate those obtained by Abreu et al. (2012), who reported a pH of 4.88 (RP pitaya) and 5.32 (WP pitaya). The pH values are above 4.5, which delimits the development of various microorganisms and can consider pitaya as a fruit of easy microbial attack (Uchoa et al. 2008).

The acidity is an important factor in the conservation of food products because it influences the ability of microorganisms to grow (Uchoa et al. 2008). The higher the acidity of food, the lower the probability of bacterial and fungal multiplication. Both fruits presented a titratable acidity of 0.26% (Table 1), similar to reported by Abreu et al. (2012), who observed acidities of 0.24% (RP pitaya) and 0.20% (WP pitaya).

Evaluation of Bioactive Compounds and Antioxidant Capacity

3.2

The RP pitaya extract showed higher phenolic compounds content (p ≤ 0.05) than the WP (Table 2). These fruits can be classified as having medium content of phenolic compounds, according to Vasco et al. (2008), who classified fruits as low (<1 mg AGE/g), medium (1–5 mg AGE/g), and high (>5 mg AGE/g) contents of phenolic compounds. Silva e Souza et al. (2023), de Lima et al. (2013), and Abreu et al. (2012) observed lower contents of phenolic compounds for RP pitaya (0.52, 0.21, and 1.18 mg GAE/g, respectively) and WP pitaya (0.52, 0.17, and 1.24 mg GAE/g, respectively). The content of phenolic compounds in fruits and vegetables can be influenced by several factors, such as ripening, species, cultivation practices, geographical origin, growth stage, harvest conditions, and storage process of the fruits, justifying the different results obtained in the present study with literature data (Carvalho et al. 2022; Soares et al. 2008; Silva e Souza et al. (2023).

Although dietary reference intakes exist for macronutrients and selected micronutrients, no official daily intake recommendations are currently established for most secondary bioactive compounds, such as phenolic compounds and betalains. Therefore, the nutritional relevance of these compounds was discussed in the context of comparative literature and functional bioactivity rather than dietary guidelines. In this context, pitaya may be considered a food source that contributes nutrients and bioactive compounds relevant to basic nutrition, as also observed by Silva e Souza et al. (2023).

In this context, there is a positive correlation between the increased consumption of plant‐based foods and the reduction in the occurrence of chronic degenerative diseases, which occurs due to the presence of bioactive compounds in these foods, and we can highlight those with antioxidant capacity, usually attributed to the content of phenolic compounds (Patil et al. 2009; Gordon et al. 2011).

Studies aimed at obtaining phenolic compounds for food preservation should be encouraged, as these compounds are widely distributed in nature (Lourenço et al. 2019). In addition, the use of natural antioxidants has gained increasing attention due to their potential effectiveness and lower toxicity compared to synthetic additives, driving interest in plant‐ and food‐derived compounds as alternative agents for food applications (Tamfu, Ceylan, Fru et al. 2020).

In the antioxidant capacity by ABTS radical, red pitaya showed higher antioxidant capacity than white. The values obtained were higher than those observed by Rocha et al. (2020), but they also observed higher antioxidant capacity for RP pitaya (2.9 µmol Trolox/g) than WP pitaya (1.44 µmol Trolox/g). Fruits grown in different seasons may present changes in their characteristics as a function of photoperiod (time of exposure to light that the plants receive each day), light intensity, and precipitation (Silva et al. 2015), which may explain these differences.

By the DPPH radical scavenging method, the WP pitaya showed higher antioxidant capacity, whereas in ABTS method, the RP pitaya showed the highest capacity. This is because of the specificity of the radicals used in each method, as ABTS can interact more efficiently with hydrophilic, lipophilic, and highly pigmented compounds (Oliveira et al. 2016).

These methods provide a general estimate of antioxidant capacity and are influenced by the chemical nature, polarity, and reaction kinetics of the antioxidant compounds present in the extract. In addition, secondary metabolites other than phenolic compounds, including pigments and non‐phenolic compounds, may contribute to the overall radical scavenging response. Therefore, the combined use of complementary assays is essential to obtain a more comprehensive evaluation of antioxidant capacity, mainly because of the complexity and variety of compounds with antioxidant capacity present in fruits (Sucupira et al. 2012; Bibi Sadeer et al. 2020).

Using the β‐carotene/linoleic acid method, it was verified that RP pitaya showed higher (p ≤ 0.05) antioxidant capacity than the white. Abreu et al. (2012) also observed higher antioxidant capacity in RP pitaya (85%) than in WP pitaya (65%). According to Vaillant et al. (2005), RP pitaya presents higher antioxidant capacity due to the presence of high content of phenolic compounds and, in addition, it has betacyanin, a component that is not present in WP pitaya. The antioxidant capacity of the β‐carotene/linoleic acid method above 70% indicates high capacity, between 50% and 70% indicates moderate capacity, and below 50% indicates low antioxidant capacity (Melo et al. 2008). According to this classification, red and white pitaya pulp are considered to have low antioxidant capacity.

Regarding the betalain content, the RP pitaya contained 3.15 mg/100 g betanine equivalent/g. Among the natural pigments suitable for food coloring, betalains stand out due to their strong dyeing capacity, making them suitable for various food products (Ferreira et al. 2023). Betalains have been identified as natural antioxidants with beneficial effects on human health (Le 2021; Tesoriere et al. 2004), besides anti‐inflammatory, anticancer, and antidiabetic properties (Madadi et al. 2020). Betalains have no acceptable daily intake (ADI), that is, they have no established maximum intake values (Volp et al. 2009).

The chromatograms of betalains responsible for the purple pigmentation of red pitaya are shown in Figure 1 and their respective identification detailed in Table 3. Seven betalains were identified in the extract; two of them belong to betaxanthin group and five to betacyanins group. Betacyanins are often glycosylated at the C5 position and rarely at C6 position (Esatbeyoglu et al. 2015). Peaks 1 and 2 presented molecular ion [M + H]^+^ at m/z 551 releasing MS2 fragment at m/z 389 and were identified as betanidin glucoside. According to the comparison with a betalain extract from beetroot, these compounds were identified as (iso) betanidin 5‐β‐d‐glucoside ((iso)betanin), which agree with betanin found in fresh fruits of red pitaya (Hylocereus spp.) (Barkociová et al. 2021). Otherwise, in two other species of pitaya, Stenocereus Pruinosus and S. stellatus, these compounds were identified as (iso)gomphrenin I (betanidin‐6‐O‐β‐glucoside) once the comparison with a betalain extract from beetroot was discarded (García‐Cruz et al. 2017). The major betalain (Peak 3) was identified as phyllocactin (betanidin 5‐O‐(6′‐O‐malonyl‐β‐d‐glucoside), presenting molecular ion at m/z 637 and releasing MS2 fragment at m/z 389. Two other peaks presented same molecular ion and identified as 4′‐O‐malonyl‐betanin (Peak 4) and isophyllocactin (Peak 5), also described in purple pitaya (García‐Cruz et al. 2017). The two betaxanthins, identified as isocaxanthin and indicaxanthin, were co‐eluted with Peak 1. Total betalain content was 1.569 ± 0.005 mg/mL extract, which phyllocactin was the major betalain compound (0.864 ± 0.006 mg/mL) followed by betanin (0.495 ± 0.03 mg/mL) (Table 3).

HPLC profile of betalains from the RP pitaya extract recorded at 530 nm. Peaks identities are shown in Table 3 under betacyanins. Peak 1: betanin; Peak 2: (iso) betanin; Peak 3: phylocactin; Peak 4: 4′‐O‐malonyl‐betanin; and Peak 5: isophyllocactin.

Four flavonoids were identified in pitaya extract (Table 4), three in purple pitaya, and two of them present in the white one. The major flavonol was identified as quercetin 3‐rutinoside (10.61 µg/mL) presenting a molecular ion at m/z 609 and characteristic MS2 fragments as m/z 301 and 151.

Antimicrobial Activity

3.3

The extracts of both pitaya species showed a mild antimicrobial activity against E. coli, L. monocytogenes, P. aeruginosa, Salmonella spp., and S. aureus at all concentrations tested (Table 5). P. aeruginosa showed a 2.0 log cycle reduction in its population by the RP pitaya extract, and S. aureus showed a 2.4 log cycle reduction by the WP pitaya extract. The phenolic compounds present in the extracts are most likely responsible for the observed antimicrobial action. These compounds can act in three ways: by altering the permeability of the bacterial cell membrane and reducing cellular constituents; by inactivating enzyme systems; or by destroying or functionally inactivating genetic material (Dos Santos Pereira et al. 2018). Except for S. aureus, the RP pitaya extract had greater IP than the white one, possibly by the presence of betalain, which has antimicrobial properties (Zambrano et al. 2019).

Santos et al. (2020) evaluated the IP of jambolan (Syzygium cumini L.) against the same bacteria and obtained values much higher than those found for white and RP pitaya (7.07 for E. coli, 3.83 for L. monocytogenes, 3.50 for P. aeruginosa, 3.82, and 3.89 S. aureus, respectively). Similar to WP pitaya, S. aureus were reduced by 3.89 log cycles.

Determination of the Anti‐QS Activity

3.4

The expression of many virulence genes in bacteria is regulated by QS, icluding biofilm formation, sporulation, expression of virulence genes, bioluminescence, and the production of toxins and pigments (Lima et al. 2023; Bassler and Losick 2006). QS can be inhibited by blocking the synthesis and secretion of AI molecules, by degradation of these signaling molecules, or by competition for binding to receptor proteins, inhibiting target gene expression (Lima et al. 2023; Grandclément et al. 2016; Machado et al. 2020). To assess the anti‐QS effect, all assays must be performed in concentrations that did not interfere with bacterial growth (sub‐MICs), as presented in Table 6.

Violacein production by C. violaceum is a phenotype regulated by QS and has been widely used as a model for evaluating interference with this system. The inhibition of this system can be evidenced by the reduction of pigment production without alteration in the C. violaceum growth (Santos et al. 2020; Tamfu, Ceylan, Fru, et al. 2020; Tamfu, Ceylan, Kucukadyin, et al. 2020).

In the present study, the extracts of RP and WP pitayas inhibited violacein production with inhibition halos (absence of pigment formation) of 27 and 28 mm, respectively. The bacterial growth was observed by the turbidity of the halo, indicating that QS was inhibited and not bacterial growth (Figure 2).

Inhibition of violacein production in Chromobacterium violaceum by WP and RP pitaya extracts. C: control; E: extract.

The results showed that the extracts obtained from the pulp of both fruits had relevant potential for inhibiting violacein production by C. violaceum. Zhu et al. (2011) observed similar results using natural compounds obtained from mushrooms. Oliveira et al. (2016) also observed that wild strawberry extract (at 5902.8 mg GAE/L) was able to inhibit the production of violacein.

In liquid media, the extracts of both pulps presented a relevant potential for inhibition of violacein at all concentrations tested. The highest concentration of red and white pitayas extract inhibited 87% and 89% of violacein, respectively (Figure 3). The other concentrations also showed inhibition without significant variation. Other recent studies have also reported the inhibition of violacein production (in liquid media) by plant extracts, such as those obtained from Annona senegalensis (Tamfu, Ceylan, Fru, et al. 2020), from methanol, ultrasonic, and green extracts of Salvia triloba (Quradha et al. 2024), and for Camellia sinensis and Curcuma longa extracts (Tamfu, Ceylan, Kucukaydin, et al. 2020).

Percentage of inhibition of violacein production by red (A) and white (B) pulp pitaya extract at different concentrations (mg GAE/mL of extract).

The lack of studies evaluating this parameter in pitaya underscores the relevance of the present research, as such data are still unavailable in the literature. In contrast, studies using extracts from other Brazilian fruits, including grumixama (Eugenia brasiliensis), pitanga (Eugenia uniflora L.) (Rodrigues et al. 2016), jambolão (S. cumini L.) (Santos et al. 2020), and pimenta malagueta (Rivera et al. 2019), have reported promising results regarding the inhibition of violacein production. These studies demonstrate how natural compounds and plant extracts can inhibit QS‐regulated phenotypes.

The swarming motility is another phenotype regulated by QS in several bacteria. The cell motility is a mechanism by which bacteria can move to locations favorable for growth when there are changes in the nutritional and physical conditions of the environment. Therefore, they have adaptive advantages (Lima et al. 2025; Quradha et al. 2024). In swarming motility, bacterial cells move in groups parallel to their horizontal axis, leading to an increase in cell contact and colonization of the surface (Lima et al. 2025). The inhibition of this mechanism can affect the pathogenicity of the microorganism because motility is associated with biofilm formation and the action of virulence factors (Quradha et al. 2024; Santos et al. 2021).

In the present study, both pitaya extracts had no significant effects on swarming motility of A. hydrophila, H. alvei, and S. marcescens at the concentrations tested. The motility of the control (without extract) was equal to the motility zone of the treatments added with extracts (data not shown). Although no inhibitory effect was observed, this result is still important, as swarming is a well‐established QS‐regulated phenotype and its evaluation contributes to a comprehensive assessment of anti‐QS activity.

Biofilm formation is an important biological concept in environmental microbiology. Typically, bacteria bind to surfaces and form structured communities inside a self‐produced matrix of extracellular polymeric substances (EPS), which poses a formidable challenge for food industries (Carrascosa et al. 2021; Alain et al. 2022). The establishment and development of biofilm is characterized by five stages: initial attachment of planktonic bacteria; bacterial adhesion and aggregation; microcolony formation; biofilm maturation; and dispersion, when bacteria spread from one part to another to spread infection (Sharma et al. 2023; Quecán et al. 2026).

The biofilm formation is partially regulated by QS in several bacteria. By inhibiting regulatory mechanisms such as QS, reducing bacterial motility, and inhibiting virulence factors, phenolic compounds have the ability to inhibit bacterial biofilm development (Lima et al. 2025; Alain et al. 2022; Quecán et al. 2026).

In the present study, the pitaya extracts inhibited biofilm formation only by S. marcescens. The extracts stimulated biofilm formation by A. hydrophila and H. alvei (Figure 4). It was not possible to perform the analysis for RP pitaya at the highest concentration because there was precipitation of the extract, interfering in the results. The precipitation is attributed to the limited solubility of the extract in the culture medium, a phenomenon commonly reported for plant‐derived extracts, especially at higher concentrations.

Biofilm formation in the presence of white (A) and red (B) pitaya pulp extract. White pitaya extracts at 50%, 25%, and 12.5% correspond to 9.6, 4.8, and 2.4 mg GAE/mL, respectively. Red pitaya extracts at 25% and 12.5% correspond to 5.3 and 2.6 mg GAE/mL, respectively.

Although in the present study, most extracts did not inhibit the biofilm formation at these subinhibitory concentrations, extracts from other plants rich in phenolic compounds showed an anti‐biofilm effect. Oliveira et al. (2016) observed the biofilm inhibition by phenolic extracts of wild strawberry and acerola against A. hydrophila, C. violaceum, and S. marcescens. Zambrano et al. (2019) observed the biofilm inhibition of several foodborne bacteria by yellow pitaya, black grape, and apple extracts. In the study by Quradha et al. (2024), the green extract of S. triloba exhibited high biofilm inhibition against Enterococcus faecalis, whereas the methanolic extract exhibited high inhibition against S. aureus and E. coli at MIC. These studies support the application of plant extracts to control biofilm of food‐related bacteria.

As in our study most pitaya extracts had no inhibitory effect against biofilms, we suggest further studies should be done using higher concentrations or extracts combined with other compounds. The combination of compounds has been shown to be synergistic in inhibiting biofilms, as shown by Vipin et al. (2020) when combining quercetin and antibiotics against biofilm of P. aeruginosa, by Hossain et al. (2020) when combining phenolic compounds and antibiotics against E. coli, and by Lima et al. (2024) when combining four phenolics (baicalein, curcumin, resveratrol, and rosmarinic acid) against biofilm of P. aeruginosa. Thus, the combination of natural and traditional compounds can be a strategy to enhance the effect of both.

It is important to emphasize that the evaluation of QS inhibition, even when using combinations of compounds, must be conducted at sub‐MIC. This approach ensures that the observed effects are specifically related to QS communication rather than to antimicrobial activity. In contrast, when the objective is to achieve direct antimicrobial effects, such as bacterial growth inhibition or cell death, higher concentrations are required (Lima et al. 2023; Tamfu, Ceylan, Fru, et al. 2020).

Finally, although natural compounds have been explored as antimicrobial compounds for food and surface sanitation, their application depends on factors such as concentration, contact time, and food matrix interactions. Despite advantages over synthetic disinfectants, including a lower risk of resistance development, further studies are needed to overcome challenges related to extraction, scalability, and regulatory approval (Machado et al. 2020; Lima et al. 2026).

Conclusion

4

This study provides a comprehensive characterization of RP and WP pitaya extracts, integrating centesimal composition, physicochemical properties, bioactive profile, and biological activities relevant to food systems.

The extracts showed antioxidant capacity, medium content of phenolic compounds, and antimicrobial activity, in addition to being effective in inhibiting violacein production, a phenotype regulated by QS. The RP pitaya stands out due to its betalain content and higher antioxidant performance in selected assays. Although no inhibitory effects were observed on swarming motility or biofilm formation, these findings contribute to a clearer understanding of the specific biological responses modulated by pitaya extracts.

Overall, the results highlight pitaya as a promising source of bioactive compounds with potential technological applications, such as microbial control in the food industry. Further studies exploring formulation strategies, combination of extracts with sanitizers and other antimicrobial compounds, and food‐grade applications, such as edible coatings or films, are encouraged to enhance and expand these effects.

Author Contributions

Raíssa Soares Gomes: writing – original draft, methodology. Alice Mendes de Carvalho: writing – original draft, methodology. Emília Maria França Lima: writing – review and editing, visualization, investigation, writing – original draft. Patrícia Aparecida Pimenta Pereira: writing – review and editing, software. Isabela Pereira Gouveia: methodology. Neuza Mariko Aymoto Hassimotto: writing – review and editing, methodology. Uelinton Manoel Pinto: writing – review and editing, validation and resources. Luciana Rodrigues da Cunha: conceptualization, investigation, funding acquisition, writing – review and editing, methodology, formal analysis, project administration, data curation, supervision, validation, visualization, resources.

Funding

This work was supported by the FAPEMIG (APQ‐02707‐14 e APQ 02053‐22).

Conflicts of Interest

The authors declare no conflicts of interest.