Bioaccessibility of Antioxidants in Prickly Pear Fruits Treated with High Hydrostatic Pressure: An Application for Healthier Foods

Abstract

High hydrostatic pressure (HHP) is a commercial processing technology which can enhance the health potential of foods by improving the bioaccessibility of their bioactive compounds. Our aim was to study the bioaccessibility and digestive stability of phenolic compounds and betalains in prickly pear fruits (Opuntia ficus-indica L. Mill. var. Pelota and Sanguinos) treated with HHP (100, 350, and 600 MPa; come-up time and 5 min). The effects of HHP on pulps (edible fraction) and peels (sources of potential healthy ingredients) were assessed. In pulps, betanin bioaccessibility increased (+47% to +64%) when treated at 350 MPa/5 min. In HHP-treated pulps, increases in the bioaccessibility of piscidic acid (+67% to +176%) and 4-hydroxybenzoic acid glycoside (+126% to 136%) were also observed. Isorhamnetin glycosides in peels treated at 600 MPa/CUT had higher bioaccessibility (+17% to +126%) than their controls. The effects of HHP on the bioaccessibility of health-promoting compounds are not exclusively governed by extractability increases of antioxidants in the food matrix (direct effects). In this work we found evidence that indirect effects (effects on the food matrix) could also play a role in the increased bioaccessibility of antioxidants in fruits treated with HHP.

1. Introduction

Prickly pear (Opuntia ficus-indica L. Mill.) fruits from the Cactaceae family represent nutritious sources of healthy foods adaptable to expanding regions of hot climate. Prickly pears are the most widely consumed fruits from the plants of the Opuntia genus and are cultivated in Latin America, Africa, and the Mediterranean region. They can be found in Mexico and in Spain, where colored varieties such as Pelota and Sanguinos are widely cultivated. Prickly pear fruits may be commercialized as fresh fruits or as derived products such as juices and jams. During the preparation of Opuntia beverages, a large amount of waste and by-products are produced mainly from the peels of the fruits. Prickly pear peels could be sustainable sources of nutrients and bioactive compounds. Moreover, the recovery of high-added value compounds from Opuntia waste and by-products provides dual benefits by addressing both management of bio-waste and societal health [1].

Betalains and phenolic compounds are health-promoting compounds which contribute greatly to the health potential of colored prickly pear varieties. Red-colored betanin and yellow-colored indicaxanthin are the most abundant betalains in these fruits and are responsible for their free radical scavenging and antioxidant activity [2]. Prickly pears are also abundant in phenolic acids such as piscidic acid, which is also found in other members of the Cactaceae family. Piscidic acid, despite being less studied compared to other phenolic compounds, has shown anti-hypercholesterolemia effects by inhibiting cholesterol permeation in vitro as well as anti-inflammatory activity [3,4]. Furthermore, prickly pears are rich sources of flavonoids such as isorhamnetin glycosides, which possess significant antioxidant and anti-inflammatory activities [5].

For betalains and phenolic compounds to be able to exert the mentioned health benefits in vivo, they must be bioaccessible. The bioaccessibility of a bioactive compound refers to the fraction which is released from the food matrix, modified in the gastrointestinal tract, and that is available for potential absorption [6]. This includes the transformations to the food matrix, which occur during digestion, absorption by epithelial cells in the intestine, and finally presistemic metabolism (intestinal and hepatic). It is possible to assess digestive stability and bioaccessibility via standardized static in vitro simulated gastrointestinal digestion methodologies such as the INFOGEST methodology [7] as used in this study.

High hydrostatic pressure (HHP) is a non-thermal technology traditionally used to assure microbiological safety in foods, meanwhile preserving their sensorial characteristics. However, in the last years there has been significant progress made in the use of HHP for promoting healthy attributes in foods. Some potential applications being explored include enhancing or retaining nutritional value, retaining immunoglobulin components in dairy products, increasing resistant starch content in cereals, reducing glycemic index of fruits, and promoting the extraction of bioactive compounds from food waste [8]. This last application has been widely studied in the last years. However, the underlying principles and mechanisms are not yet fully understood and more studies are needed to optimize HHP treatments on different bioactive compounds in food products with important effects on health, opening doors to new HHP applications in the food industry [9].

We recently studied the microstructural changes in prickly pear cells submitted to HHP treatments to identify the mechanisms by which bioactive compounds could be released from their intracellular compartments [10]. This was done to gain more information on the effect of HHP on bioactive extractability and in vitro antioxidant and anti-inflammatory activities in prickly pear fruits [11]. This last study focused on the direct and quantifiable changes of bioactive compound concentration after pressurization. Although insightful, the contribution of high hydrostatic pressure to producing healthier foods should be further substantiated by evidence on the digestive stability and bioaccessibility of their bioactive compounds. Furthermore, confirmation is needed to see if the HHP treatments that result in the highest release of health promoting compounds (direct effect) are, in fact, the most bioaccessible; or if, contrarily, a premature release of bioactive compounds could have a negative effect on their bioaccessibility.

Moreover, studies on the digestive stability of health-promoting compounds during each phase of simulated gastrointestinal digestion can be useful for elucidating other effects of HHP on the bioaccessibility of bioactive compounds (indirect effects). Beneficial indirect effects caused by HHP can include (i) microstructural changes to the food matrix that can promote the release of phytochemicals during digestion because of a higher exposure to enzymes from the digestive tract, (ii) changes in viscosity due to modification of proteins or pectins that can have a protective effect on bioactive compounds from digestive conditions (i.e., pH shifts, enzymes) and even delay their release, and (iii) the activation/inactivation of endogenous enzymes to the food matrix. However, the use of HHP for enhancing the bioaccessibility of bioactive compounds should be considered a double-edge sword since the mentioned changes to the food matrix can also be detrimental to bioactive compound bioaccessibility under certain conditions.

Hence, the aim of this work was to study the changes in digestive stability and bioaccessibility of individual phenolic compounds and betalains in prickly pear fruits (Opuntia ficus-indica L. Mill. Var. Pelota and Sanguinos) treated with HHP (100, 350, and 600 MPa; come-up time and 5 min) and in untreated fruits (control samples). With this study we expect to contribute to the knowledge of the use of HHP treatment for promoting the health potential of foods by enhancing the bioaccessibility of their bioactive compounds, which is a limiting factor for their potential in vivo bioactivities.

2. Results and Discussion

2.1. Main Bioactive Compounds in Pelota and Sanguinos Prickly Pear Fruits

The main bioactive compounds in control and pressurized Pelota and Sanguinos peels and pulps are shown in

Table 1. Betalain and phenolic content (mg/100 g fresh weight) in rehydrated Sanguinos and Pelota prickly pear (Opuntia ficus-indica L. Mill.) peels and pulps.

	Sanguinos		Pelota
Compound	Pulp	Peel	Pulp	Peel
Indicaxanthin	0.90 ± 0.05	0.60 ± 0.01	3.92 ± 0.24 *	1.01 ± 0.01 *
Betanin	2.05 ± 0.02	5.78 ± 0.23	27.91 ± 0.45 *	16.29 ± 0.10 *
Piscidic acid	58.43 ± 3.53	439.45 ± 0.90	195.63 ± 1.48 *	779.17 ± 4.74 *
4-hydroxybenzoic acid glycoside	1.25 ± 0.13	19.12 ± 0.21	4.47 ± 1.17	23.62 ± 0.14 *
Isorhamnetin glucosyl-rhamnosyl-rhamnoside (IG1)	n.d.	0.55 ± 0.03	n.d.	1.44 ± 0.01 *
Isorhamnetin glucosyl-rhamnosyl-pentoside (IG2)	n.d.	1.51 ± 0.13	n.d.	0.35 ± 0.00 *
Isorhamnetin hexosyl-hexosyl-pentoside (IG3)	n.d.	0.32 ± 0.03	n.d.	0.15 ± 0.00 *
Isorhamnetin glucosyl-pentoside (IG4)	n.d.	0.81 ± 0.06	n.d.	0.23 ± 0.00 *
Isorhamnetin glucosyl-rhamnoside (IG5)	n.d.	3.09 ± 0.18	n.d.	2.26 ± 0.01 *

Results are expressed as mean ± standard deviation (n = 3). Lowercase superscript letters indicate statistically significant differences (p ≤ 0.05) between peels and pulps. * Indicate statistically significant differences (p ≤ 0.05) between varieties. n.d. not detected.

. The main betalains (betanin and indicaxanthin), phenolic acids (piscidic acid and 4-hydroxybenzoic acid glycoside), and flavonoids (isorhamnetin glycosides) were quantified. The complete betalain and phenolic profile in Sanguinos and Pelota prickly pear fruit varieties was reported in a previous study [12], where besides the main bioactive compounds shown in the present work, other betalains (portulacaxanthin, vulgaxanthin, betanidin) and phenolic compounds (quercetin glycosides and kaempferol glycosides) were found in minor concentrations.

As shown in Table 1, the red-colored betanin is the most abundant betalain in red-colored Sanguinos and purple-colored Pelota prickly pear tissues. The Pelota variety has 12.6 and 1.8 times more betanin in pulps and peels, respectively, compared to the Sanguinos prickly pear fruit. Yellow colored indicaxanthin was also found in peels and pulps of both prickly pear fruit varieties. The Pelota variety had 3.4 times higher indicaxanthin content in pulps and 0.6 times higher indicaxanthin content in peels than Sanguinos ones.

Besides betalains, prickly pear pulps contained piscidic acid and 4-hydroxybenzoic acid glycoside, being again the Pelota fruits that contain higher content in phenolic acids. In both varieties, peel tissue contained from 3 to 6.5 times higher piscidic acid and from 5.3 to 15.3 times higher 4-hydroxybenzoic acid glycoside than pulp tissue.

Meanwhile, the isorhamnetin glycosides were only detected in prickly pear peels (Table 1). The most abundant flavonoids were, namely, isorhamnetin glucosyl-rhamnosyl-rhamnoside (IG1), isorhamnetin glucosyl-rhamnosyl-pentoside (IG2), isorhamnetin hexosyl-hexoyl-pentoside (IG3), isorhamnetin glucosyl-pentoside (IG4), and isorhamnetin glucosyl-rhanoside (IG5). Purple-colored Pelota prickly pear fruits have a higher IG1 and IG5 content, whereas red-colored prickly pear fruits such as Sanguinos are characterized by a higher IG2 and IG5 profile. Total isorhamnetin glycoside concentration in peels was 6.28 and 4.43 mg/100 g fresh weight in Sanguinos and Pelota prickly pear peels.

2.2. Effect of HPP-Treatment on Individual Phenolic and Betalain Compounds Content

The content of individual betalain and phenolic compounds in control and pressurized (100, 350, and 600 MPa; CUT & 5 min) Sanguinos and Pelota prickly pear tissues (pulp and peel) after HHP-treatment were investigated in a previous study [11].

2.3. Bioaccessibility of Betalains and Phenolic Compounds in HHP-Treated Pulps

Increases in the bioaccessibility of bioactive compounds in prickly pear tissues (peels and pulps) due to HHP treatments may be attributed to direct and/or indirect effects. A direct effect of HHP refers to the increase in the extractability of the bioactive compound observed post-HHP-treatment. An indirect effect of HHP includes any changes to the food matrix and changes in other main components of the fruit tissues, which could promote the bioaccessibility of their bioactive compounds by enhancing their digestive stability.

The bioaccessibility of betalains and phenolic compounds in non-treated prickly pear pulps (control samples), and pressurized ones (100, 350, and 600 MPa; CUT and 5 min) are shown in

Table 2. Initial content (mg/100 g weight) and in vitro bioaccessibility of betalains and phenolic compounds in HHP-treated (100 MPa, 350 MPa, 600 MPa; CUT and 5 min) Sanguinos and Pelota prickly pear pulps and in untreated (control) pulps.

		Prickly Pear (Opuntia ficus-indica) Pulps
		Sanguinos		Pelota
	Treatment	Pulp (mg/100 g Weight) 1	Bioaccessibility (%)	Pulp (mg/100 g Weight) 1	Bioaccessibility (%)
Indicaxanthin	Control	0.90 ± 0.05a	53.05 ± 3.01c	3.92 ± 0.24c	54.87 ± 2.49bc
	100 MPa/CUT	0.73 ± 0.05a	40.93 ± 3.27b	3.62 ± 0.17bc	52.81 ± 5.17bc
	350 MPa/CUT	0.90 ± 0.07a	44.81 ± 2.24bc*	5.38 ± 0.05d	64.83 ± 5.19c
	600 MPa/CUT	0.72 ± 0.06a	25.30 ± 2.02a*	2.85 ± 0.16ab	36.78 ± 2.94a
	100 MPa/5 min	0.86 ± 0.07a	28.84 ± 2.31a*	3.51 ± 0.14bc	46.87 ± 3.28ab
	350 MPa/5 min	0.84 ± 0.06a	46.98 ± 3.29bc	3.21 ± 0.08abc	55.38 ± 2.22bc
	600 MPa/5 min	0.71 ± 0.01a	22.33 ± 1.79a*	2.65 ± 0.50a	43.12 ± 3.45ab
Betanin	Control	2.05 ± 0.02a	42.49 ± 2.69c	27.91 ± 0.45ab	44.92 ± 1.52ab
	100 MPa/CUT	1.80 ± 0.07a	35.10 ± 2.81bc	28.44 ± 0.73ab	48.50 ± 4.74b
	350 MPa/CUT	2.32 ± 0.23a	43.01 ± 3.44c	35.02 ± 0.00b	50.65 ± 4.05b
	600 MPa/CUT	2.14 ± 0.22a	28.67 ± 1.43ab*	27.93 ± 1.64ab	39.23 ± 3.14ab
	100 MPa/5 min	1.98 ± 0.14a	25.98 ± 1.82ab*	26.44 ± 1.79a	35.21 ± 2.46a
	350 MPa/5 min	2.19 ± 0.23a	69.57 ± 5.57d*	25.79 ± 0.27a	65.88 ± 2.64c
	600 MPa/5 min	2.02 ± 0.23a	20.51 ± 1.64a*	24.79 ± 4.91a	46.95 ± 3.76ab
Piscidic acid	Control	58.43 ± 3.53a	53.61 ± 4.29ab*	195.63 ± 1.48a	38.49 ± 3.08a
	100 MPa/CUT	63.32 ± 7.10a	56.66 ± 4.53abc*	255.44 ± 15.40bc	82.25 ± 6.58cd
	350 MPa/CUT	71.46 ± 1.26ab	58.61 ± 3.44abc	223.96 ± 3.82ab	62.71 ± 5.02bc
	600 MPa/CUT	92.66 ± 9.61b	65.68 ± 5.25bc	311.03 ± 17.41d	71.56 ± 3.58bcd
	100 MPa/5 min	62.78 ± 1.89a	73.70 ± 1.05cd*	235.34 ± 15.86abc	54.39 ± 3.81ab
	350 MPa/5 min	73.82 ± 8.70ab	89.36 ± 7.15d	273.68 ± 2.09bc	106.20 ± 8.50e
	600 MPa/5 min	81.97 ± 9.50ab	42.40 ± 3.39a*	287.35 ± 29.07cd	88.43 ± 7.07de
4-hydrozybenzoic acid glycoside	Control	1.25 ± 0.13a	20.00 ± 1.60a	4.47 ± 1.17a	17.00 ± 1.36a
	100 MPa/CUT	1.54 ± 0.40ab	28.53 ± 2.79b	5.76 ± 0.45ab	25.89 ± 2.07b
	350 MPa/CUT	1.87 ± 0.17abc	27.43 ± 2.19ab	4.09 ± 0.03a	21.09 ± 1.24ab
	600 MPa/CUT	2.48 ± 0.28c	20.28 ± 1.62a	9.87 ± 0.47c	16.76 ± 1.34a
	100 MPa/5 min	1.60 ± 0.07ab	22.10 ± 1.55ab	7.69 ± 0.84bc	22.81 ± 1.82ab
	350 MPa/5 min	2.18 ± 0.07bc	47.13 ± 1.89c	6.70 ± 0.14ab	38.44 ± 3.08c
	600 MPa/5 min	2.19 ± 0.27bc	19.18 ± 1.53a*	8.38 ± 1.69bc	37.48 ± 3.00c

Results are expressed as mean ± standard deviation (n = 3). Superscript letters indicate statistically significant differences (p ≤ 0.05) between treatments. * Indicates statistically significant differences (p ≤ 0.05) in bioaccessibility between Pelota and Sanguinos varieties. ¹ Data previously reported [11].

. Graphical representation of this data may be consulted in Supplementary Figure S1.

The bioaccessibility of indicaxanthin (Table 2) in all prickly pear pulps treated with HHP was similar or lower than in their respective control samples (p ≤ 0.05). HPP treatment at 350 MPa/CUT and 350 MPa/5 min did not affect the bioaccessibility of indicaxanthin. However, HPP treatments such as 600 MPa/CUT, 100 MPa/5 min, and 350 MPa/5 min significantly (p ≤ 0.05) reduced the bioaccessibility of indicaxanthin in pulps of both prickly pear varieties. In Sanguinos pulps, bioaccessibility reductions of −53%, −45%, and −58% could be observed after the mentioned HPP treatments. This low bioaccessibility of indicaxanthin in pressurized pulps is most likely attributed to the changes in the components present in the food matrix as enzymes and polysaccharides among others (indirect effects of pressure).

On the other hand, betanin was more bioaccessible in Sanguinos and Pelota pulps treated at 350 MPa/5 min than in their respective controls (Table 2). After a HPP treatment at 350 MPa/5 min, the bioaccessibility of betanin in Pelota and Sanguinos pulps was 70% and 66%, compared to 42% and 45% in untreated pulps, respectively. Despite conserving betanin content post-pressurization, other HHP treatments caused negative effects (decreases) in the betanin bioaccessibility. Similar to what was observed for indicaxanthin, the bioaccessibility of betanin in pressurized pulps was mostly affected by changes to the food matrix caused by pressurization (indirect effects), rather than by degradation of the mentioned betalains during treatment (direct effects).

Contrarily, the bioaccessibility of phenolic acids in prickly pear pulps treated with high hydrostatic pressure was enhanced by most treatments (Table 2). Sanguinos and Pelota pressurized pulps processed at 350 MPa/5 min showed +68% and +179% higher bioaccessibility of piscidic acid, respectively, compared to their controls. Pulps processed at 100 MPa/5 min also showed a higher bioaccessibility of piscidic acid than their respective controls. In this case, the enhanced extractability of piscidic acid (direct effect) observed after treatment in pressurized prickly pear pulps was a main factor that strongly contributed to its higher bioaccessibility. Similarly, researchers [13] studied the effect of HHP on phenolic content in fruit juices and suggested that the phenols linked to the food matrix are released during pressurization, improving their extractability and therefore their content. Furthermore, these authors found 38% higher bioaccessibility of phenolic acids (caffeic, p-coumaric, chlorogenic, and ferulic) in pressurized juice-based beverages.

Meanwhile, 4-hydroxybenzoic acid glycoside showed lower bioaccessibility (Table 2) compared to other bioactive phenolic compounds. In untreated Sanguinos and Pelota pulps (control samples) its bioaccessibility was 20% and 17%, respectively, which is in agreement to what has been reported in orange-, red-, and white-colored prickly pear pulps from the Canary Islands [14]. The best treatments were at 100 MPa/CUT (+45% to +53% higher bioaccessibility) and 350/5 min (+135% to +124% higher bioaccessibility). These treatments have previously shown to enhance phenolic acid extractability in prickly pear pulps [11].

Processing at 350 MPa/5 min was the best treatment for increasing the extractability of betanin, piscidic acid, and 4-hydroxybenzoic acid glycoside in the pulps of both prickly pear varieties. Hence, the digestive stability of these samples during each stage of digestion was further studied and discussed in Section 2.5.1.

2.4. Bioaccessibility of Betalains and Phenolic Compounds in HHP-Treated Peels

The bioaccessibility of betalains and phenolic compounds in prickly pear peels is shown in

Table 3. Initial content (mg/100 g weight) and in vitro bioaccessibility of betalains and phenolic compounds in HHP-treated (100 MPa, 350 MPa, 600 MPa; CUT and 5 min) Sanguinos and Pelota prickly pear peels and in untreated (control) peels.

		Prickly Pear (Opuntia ficus-indica) Peels
		Sanguinos		Pelota
Compound	Treatment	Peel (mg/100 g Fresh Weight) 1	Bioaccessibility (%)	Peel (mg/100 g Fresh Weight) 1	Bioaccessibility (%)
Indicaxanthin	Control	0.60 ± 0.01b	61.98 ± 3.70d	1.01 ± 0.01c	55.39 ± 2.90d
	100 MPa/CUT	0.53 ± 0.01ab	23.64 ± 1.89ab*	0.85 ± 0.06bc	49.35 ± 3.95cd
	350 MPa/CUT	0.58 ± 0.03ab	41.54 ± 3.32c	0.80 ± 0.01b	48.92 ± 3.91cd
	600 MPa/CUT	0.53 ± 0.03ab	33.50 ± 4.55bc	0.57 ± 0.06a	38.58 ± 3.09abc
	100 MPa/5 min	0.55 ± 0.03ab	19.93 ± 1.59a*	0.74 ± 0.01ab	45.76 ± 3.66bcd
	350 MPa/5 min	0.62 ± 0.04b	37.82 ± 3.03c	0.92 ± 0.12bc	35.23 ± 2.82ab
	600 MPa/5 min	0.49 ± 0.00a	33.50 ± 4.21bc	0.55 ± 0.02a	30.24 ± 2.42a
Betanin	Control	5.78 ± 0.23d	28.88 ± 2.31d*	16.29 ± 0.10d	46.11 ± 3.69b
	100 MPa/CUT	3.77 ± 0.23b	3.58 ± 0.29a*	15.39 ± 0.89cd	29.54 ± 2.36a
	350 MPa/CUT	4.48 ± 0.38bc	11.98 ± 0.96b*	12.30 ± 0.00b	32.13 ± 2.57a
	600 MPa/CUT	4.52 ± 0.28bc	22.84 ± 1.83c*	13.92 ± 1.06bc	31.36 ± 2.51a
	100 MPa/5 min	3.76 ± 0.44b	1.17 ± 0.09a*	12.66 ± 0.24b	31.60 ± 2.53a
	350 MPa/5 min	5.26 ± 0.53cd	20.16 ± 1.61c*	15.39 ± 0.45cd	30.36 ± 2.43a
	600 MPa/5 min	2.02 ± 0.23a	36.80 ± 2.94e	10.30 ± 058a	32.36 ± 2.59a
Piscidic acid	Control	439.45 ± 0.90c	52.36 ± 4.19a	779.17 ± 4.74ab	52.71 ± 4.22ab
	100 MPa/CUT	358.85 ± 5.05ab	55.90 ± 4.47a	821.22 ± 60.40ab	53.79 ± 4.30ab
	350 MPa/CUT	398.36 ± 14.95bc	45.09 ± 3.61a	721.33 ± 0.18ab	59.40 ± 4.75ab
	600 MPa/CUT	346.51 ± 18.65ab	54.78 ± 4.38a	713.46 ± 59.11ab	46.83 ± 3.75a
	100 MPa/5 min	393.96 ± 13.37bc	48.68 ± 3.89a	689.55 ± 0.20a	55.06 ± 4.40ab
	350 MPa/5 min	385.99 ± 35.08bc	45.88 ± 3.67a	847.09 ± 31.12b	62.40 ± 4.99ab
	600 MPa/5 min	311.32 ± 0.51a	47.26 ± 3.78a*	750.60 ± 33.40ab	69.96 ± 5.60b
4-hydroxybenzoic acid glycoside	Control	19.12 ± 0.21d	60.67 ± 4.85ab	23.62 ± 0.14a	65.55 ± 5.24a
	100 MPa/CUT	16.37 ± 0.28bc	65.38 ± 5.23abc	24.12 ± 2.67a	63.29 ± 5.06a
	350 MPa/CUT	17.47 ± 1.61cd	50.81 ± 4.06a	21.04 ± 0.09a	67.86 ± 5.43ab
	600 MPa/CUT	14.38 ± 0.73b	80.54 ± 6.44c	25.67 ± 2.20a	89.34 ± 7.15bc
	100 MPa/5 min	19.54 ± 0.18d	74.89 ± 5.99bc	36.83 ± 1.04b	102.28 ± 8.18c
	350 MPa/5 min	19.08 ± 0.18d	61.62 ± 4.93ab	23.02 ± 0.90a	71.36 ± 5.71ab
	600 MPa/5 min	12.07 ± 0.02a	60.19 ± 4.82ab	25.92 ± 0.59a	80.91 ± 6.47abc
IG1 2	Control	0.54 ± 0.02ab	56.20 ± 4.50ab	1.44 ± 0.01b	53.30 ± 4.26bc
	100 MPa/CUT	0.61 ± 0.03bc	82.13 ± 6.57c*	0.86 ± 0.08a	25.64 ± 2.05a
	350 MPa/CUT	0.63 ± 0.01bc	72.46 ± 5.80bc	2.39 ± 0.04c	76.75 ± 6.30d
	600 MPa/CUT	0.66 ± 0.06c	84.06 ± 6.72c*	2.79 ± 0.17d	120.15 ± 9.61e
	100 MPa/5 min	0.67 ± 0.04c	67.63 ± 5.41abc*	1.44 ± 0.05b	40.29 ± 3.22ab
	350 MPa/5 min	0.67 ± 0.01c	77.29 ± 6.18c*	3.21 ± 0.04e	108.06 ± 8.64e
	600 MPa/5 min	0.50 ± 0.00a	48.31 ± 3.86a	3.18 ± 0.01e	65.93 ± 5.27cd
IG2 3	Control	1.50 ± 0.13a	41.80 ± 3.34a*	0.35 ± 0.00b	72.56 ± 5.80bc
	100 MPa/CUT	1.37 ± 0.24a	54.32 ± 4.35bc*	0.20 ± 0.01a	24.27 ± 1.94a
	350 MPa/CUT	1.44 ± 0.11a	40.56 ± 3.24a*	0.56 ± 0.03c	85.27 ± 6.82cd
	600 MPa/CUT	1.50 ± 0.06a	59.10 ± 4.73c*	0.64 ± 0.05c	99.02 ± 7.92de
	100 MPa/5 min	1.71 ± 0.12a	38.03 ± 3.04a	0.34 ± 0.01b	34.93 ± 2.79a
	350 MPa/5 min	1.70 ± 0.01a	43.88 ± 3.51ab*	0.73 ± 0.00d	110.44 ± 8.84e
	600 MPa/5 min	1.30 ± 0.00a	36.55 ± 2.92a*	0.74 ± 0.03d	63.44 ± 5.08b
IG3 4	Control	0.32 ± 0.03ab	48.05 ± 3.84c	0.15 ± 0.00c	56.89 ± 4.55bc
	100 MPa/CUT	0.29 ± 0.03ab	42.56 ± 3.40bc*	0.11 ± 0.00b	23.52 ± 1.88a
	350 MPa/CUT	0.31 ± 0.02ab	33.20 ± 2.66b*	0.20 ± 0.01d	59.04 ± 4.72bc
	600 MPa/CUT	0.32 ± 0.01ab	62.51 ± 5.00d*	0.23 ± 0.01e	93.69 ± 7.50d
	100 MPa/5 min	0.36 ± 0.00b	31.78 ± 2.54b*	0.15 ± 0.00c	15.68 ± 1.25a
	350 MPa/5 min	0.34 ± 0.01ab	37.74 ± 3.02bc*	0.08 ± 0.00a	61.50 ± 4.92c
	600 MPa/5 min	0.28 ± 0.00a	16.63 ± 1.33a*	0.08 ± 0.02a	43.97 ± 3.52b
IG4 5	Control	0.82 ± 0.06ab	59.49 ± 4.76cd	0.23 ± 0.00b	58.74 ± 4.70bc
	100 MPa/CUT	0.52 ± 0.20a	52.26 ± 4.18c*	0.12 ± 0.00a	22.68 ± 1.81a
	350 MPa/CUT	0.55 ± 0.09ab	61.83 ± 4.95cd	0.42 ± 0.00c	85.85 ± 6.87de
	600 MPa/CUT	0.71 ± 0.03ab	68.59 ± 5.49d	0.44 ± 0.04cd	75.20 ± 6.02cd
	100 MPa/5 min	0.87 ± 0.05b	21.56 ± 1.72a	0.23 ± 0.01b	24.88 ± 1.99a
	350 MPa/5 min	0.86 ± 0.01b	39.20 ± 3.14b*	0.49 ± 0.00d	99.19 ± 7.93e
	600 MPa/5 min	0.56 ± 0.01ab	35.00 ± 2.80b*	0.51 ± 0.04d	53.90 ± 4.31b
IG5 6	Control	3.10 ± 0.18b	29.77 ± 2.38b*	2.27 ± 0.01b	55.45 ± 4.44b
	100 MPa/CUT	2.44 ± 0.18ab	43.05 ± 3.44c*	1.22 ± 0.06a	24.45 ± 1.96a
	350 MPa/CUT	2.12 ± 0.15a	26.81 ± 2.14b*	4.31 ± 0.45c	99.96 ± 8.00c
	600 MPa/CUT	2.82 ± 0.10ab	42.10 ± 3.37c*	5.10 ± 0.49c	97.67 ± 7.81c
	100 MPa/5 min	3.11 ± 0.35b	11.94 ± 0.96a*	2.20 ± 0.03b	39.91 ± 3.19ab
	350 MPa/5 min	3.14 ± 0.39b	28.05 ± 2.24b*	5.21 ± 0.05c	137.52 ± 11.00d
	600 MPa/5 min	2.16 ± 0.00a	25.38 ± 2.03b*	5.12 ± 0.45c	59.11 ± 4.73b

Results are expressed as mean ± standard deviation (n = 3). Superscript letters indicate statistically significant differences (p ≤ 0.05) between treatments. * Indicates statistically significant differences (p ≤ 0.05) in bioaccessibility between Pelota and Sanguinos varieties. ¹ Data previously reported [11], ² isorhamnetin glucosyl-rhamnosyl-rhamnoside (IG1), ³ isorhamnetin glucosyl-rhamnosyl-pentoside (IG2), ⁴ isorhamnetin hexosyl-hexosyl-pentoside (IG3), ⁵ isorhamnetin glucosyl-pentoside (IG4), ⁶ isorhamnetin glucosyl-rhamnoside (IG5).

. Graphical representation of data may be consulted in Supplementary Figure S2.

The bioaccessibility of indicaxanthin in untreated Sanguinos and Pelota peels was 62% and 55%, respectively. In Sanguinos peels, all pressurized samples had lower indicaxanthin bioaccessibility (−11% to −58%) than the untreated control. Meanwhile, Pelota peels treated at lower pressure-time combinations (100 MPa/CUT, 100 MPa/5 min and 350 MPa/CUT) showed no statistical differences (p ≤ 0.05) in indicaxanthin bioaccessibility compared to controls. Similarly, lower bioaccessibility values for betanin could be observed in peels treated with high hydrostatic pressure. The only increase (+27%) in betanin bioaccessibility was observed in Sanguinos peels treated at 600 MPa/5 min. In this study, HHP promoted the degradation of betalains, which had a negative effect on their bioaccessibility. It is likely that HHP enhanced the rupture of cell walls and favored enzymatic activity in the fruit tissues, which contributed to the loss of these compounds. Similarly, a previous study showed that betalain content decreased during HHP processing in beetroot juice [15]. It has been suggested that betalains are pressure-liable due to their high sensitivity to oxygen, pH, and enzymatic activity [16].

Submitting prickly pear peels to HHP caused little changes in the bioaccessibility of piscidic acid. The only significant increase (+32%) in the bioaccessibility of this phenolic acid was observed in Pelota peels treated at 600 MPa/5 min.

The bioaccessibility of 4-hydroxybenzoic acid glycoside was 61% and 66% in untreated Sanguinos and Pelota peels. Although 4-hydroxybenzoic acid in control samples had a high bioaccessibility, treating the samples with HHP resulted in an even higher bioaccessibility in the peels. The treatments with the highest bioaccessibility increase in 4-hydroxybenzoic acid glycoside were 600 MPa/CUT (+33% in Sanguinos and 35% in Pelota) and 100 MPa/5 min (+55% in Pelota). The rest of the assayed HPP conditions showed no statistically significant differences. Bioaccessibility increases of 4-hydroxybenzoic acid glycoside in pressurized peels are attributed to favorable changes in the food matrix by the HHP treatments (indirect effects) since little or no changes in phenolic acid content were observed post treatment.

Isorhamnetin glycoside bioaccessibility in prickly pear peels significantly improved with pressurization (Table 3). At the lowest and highest pressure-time combinations (100 MPa/CUT, 100 MPa/5 min and 600 MPa/5 min), the bioaccessibility of isorhamnetin glycosides was lower than in the correspondent controls. However, at intermediate-high pressure-time combinations, their bioaccessibility was similar (350 MPa/CUT) and even higher (350 MPa/5 min and 600 MPa/CUT). The bioaccessibility of isorhamnetin glucosyl-rhamnosyl-rhamnoside (IG1) in Pelota and Sanguinos prickly pears treated at 600 MPa/CUT was 120% and 84%, compared to 53% and 56% in untreated peels, respectively. Similarly, the bioaccessibility of isorhamnetin glucosyl-rhamnosyl-pentoside (IG2) was higher in Pelota (+36%) and Sanguinos (+40%) prickly pear peels processed at 600 MPa/CUT, compared to their controls. At this same condition, the bioaccessibility of isorhamnetin hexosyl-hexosyl-pentoside (IG3) was 63% and 94%, compared to 48% and 57% in untreated Pelota and Sanguinos peels, respectively.

Furthermore, the best treatments to improve the bioaccessibility of isorhamnetin glucosyl-pentoside (IG4) were at 350 MPa/CUT (+5 to 46% increase in bioaccessibility) and 600 MPa/CUT (+17 to 27% increases in bioaccessibility). Similarly, the best treatment to increase the bioaccessibility of isorhamnetin glucosyl-rhamnoside (IG5) in both Sanguinos and Pelota prickly pear peels was also at 600 MPa/CUT. Isorhamnetin glycoside extractability has been shown to be considerably enhanced in prickly pear peels submitted to high hydrostatic pressure, particularly at 350 MPa/CUT, 350 MPa/5 min, and 600 MPa/CUT [11]. This suggests that the high bioaccessibility of isorhamnetin glycosides in prickly pear peels could be driven by enhanced extractability due to HHP processing (direct effect). Previous studies have shown that high pressure increases the content of phenolic substances due to the breakdown of the cell wall structure and hydrolysis of polysaccharides [17]. This disruption of the plant cell walls induced by high hydrostatic pressure results in the release of bioactive compounds and mineral and starch content into the extracellular environment [18]. A previous study also showed the release of phenolic compounds from the cell walls and the modification and rearrangement of microfibrilated cellulose in prickly pear chlorenchyma cells treated with HHP [10].

The best HHP treatment to improve the bioaccessibility of phenolic bioactive compounds in prickly pear peels was 350 MPa/5 min because it increased the extractability of 4-hydroxybenzoic acid derivative and isorhamnetin glycosides. Hence, the digestive stability of bioactive compounds during each stage of digestion in prickly pear peels treated at 600 MPa/CUT is reported and discussed in Section 2.5.2.

2.5. Digestive Stability of Betalains and Phenolic Compounds

The best HHP treatments to achieve a higher bioaccessibility of bioactive compounds in prickly pear fruits were selected. For pulps, the highest bioaccessibility was obtained at 350 MPa/5 min. For peels, the highest bioaccessibility was obtained at 600 MPa/CUT. In this section, the concentration and the recovery of each bioactive compound in each phase of the simulated gastrointestinal digestion (oral, gastric, and intestinal phases) was studied. This information allowed us to elucidate potential mechanisms that could contribute to the effects of HHP treatments, and how they affect the bioaccessibility of betalains and phenolic compounds in pressurized prickly pear fruits.

2.5.1. Stability and Recovery of Bioactives in Prickly Pear Pulps Treated with HHP at 350 MPa/5 min

The best treatment for increasing the bioaccessibility of most bioactive compounds in prickly pear pulps of both studied varieties was at 350 MPa/5 min. The content and recovery (%) of each bioactive compound in the pulp (control) and in oral, gastric, and intestinal phases of the simulated gastrointestinal digestion is shown in

Table 4. In vitro digestive stability (mg/100 g fresh weight) of betalains and phenolic compounds in HHP-treated (350 MPa/5 min) Sanguinos and Pelota prickly pear pulps.

	Treatment	Pulp	Oral Phase		Gastric Phase		Intestinal Phase
		Content 1	Content 1	Recovery (%)	Content 1	Recovery (%)	Content 1	Recovery (%)
Indicaxanthin	Sanguinos Control	0.90 ± 0.05c	0.82 ± 0.02bc	91 ± 3b	0.75 ± 0.02b	83 ± 3b	0.48 ± 0.03a	53 ± 3a
	Sanguinos 350 MPa/5 min	0.84 ± 0.06a	1.10 ± 0.06a	131 ± 16b	1.01 ± 0.34a	120 ± 11b	0.42 ± 0.03a	51 ± 4a
	Pelota Control	3.92 ± 0.24b	4.09 ± 0.32b	104 ± 2c	3.57 ± 0.09b	91 ± 3b	2.15 ± 0.17a	54 ± 2a
	Pelota 350 MPa/5 min	3.21 ± 0.08b	3.60 ± 0.43b	112 ± 11b	3.46 ± 0.17b*	108 ± 3b*	2.17 ± 0.17a	68 ± 5a
Betanin	Sanguinos Control	2.05 ± 0.02d	1.31 ± 0.07c	64 ± 3c	1.04 ± 0.05b	51 ± 2b	0.87 ± 0.06a	42 ± 3a
	Sanguinos 350 MPa/5 min	2.19 ± 0.23ab	3.67 ± 0.08b*	168 ± 14b*	3.24 ± 0.79b	148 ± 21b*	1.42 ± 0.11a*	65 ± 5a*
	Pelota Control	27.91 ± 0.45c	29.68 ± 1.31c	106 ± 3c	25.76 ± 0.82b	92 ± 1b	12.54 ± 0.50a	45 ± 3a
	Pelota 350 MPa/5 min	25.79 ± 0.27b*	23.54 ± 2.01b	91 ± 7a	22.78 ± 1.47b	88 ± 5a	18.39 ± 1.47a*	71 ± 6a*
Piscidic acid	Sanguinos Control	58.43 ± 3.53b	50.40 ± 2.52b	86 ± 1b	54.87 ± 2.74b	94 ± 1c	31.32 ± 2.51a	54 ± 4a
	Sanguinos 350 MPa/5 min	73.82 ± 8.70ab	78.30 ± 12.98ab	106 ± 5b*	107.36 ± 17.19b*	145 ± 6c*	52.21 ± 4.18a*	71 ± 6a*
	Pelota Control	195.63 ± 1.48d	163.24 ± 1.52c	83 ± 0c	106.36 ± 3.62b	54 ± 1b	75.30 ± 6.02a	38 ± 3a
	Pelota 350 MPa/5 min	273.68 ± 2.09b*	267.30 ± 25.06b*	98 ± 8a	238.01 ± 19.18ab*	87 ± 6a	207.76 ± 16.62a*	76 ± 6a*
4-hydroxy benzoic acid derivative	Sanguinos Control	1.25 ± 0.13b	1.05 ± 0.05b	84 ± 5b	2.05 ± 0.10c	164 ± 9c	0.25 ± 0.02a	20 ± 2a
	Sanguinos 350 MPa/5 min	2.18 ± 0.07b*	3.06 ± 0.61b*	140 ± 23b	3.49 ± 0.50b	160 ± 18b	0.59 ± 0.05a*	27 ± 2a*
	Pelota Control	4.47 ± 1.17b	6.39 ± 0.12b	143 ± 35b	6.02 ± 0.24b	135 ± 31b	0.76 ± 0.06a	17 ± 1a
	Pelota 350 MPa/5 min	6.70 ± 0.14b	7.40 ± 0.57b	110 ± 6b	8.49 ± 0.57c*	127 ± 1b	1.72 ± 0.14a*	26 ± 2a

¹ mg/100 g fresh weight. Results are expressed as mean ± standard deviation (n = 3). Superscript lowercase letters indicate statistically significant differences (p ≤ 0.05) between digestive phases.* Indicate statistically significant differences (p ≤ 0.05) between treatments for the same variety.

. The graphical representation of this data is available in the Supplementary Figure S3.

In pulps treated at 350 MPa/5 min, indicaxanthin showed a similar digestive stability to its respective controls (Table 4).

Pelota pulps treated at 350 MPa/5 min had a similar initial betanin content (25.8 mg/100 g fresh weight) to unpressurized pulps (27.9 mg/100 g fresh weight) [11]. However, this betacyanin showed a better digestive stability than indicaxanthin (Table 4). During gastro-intestinal digestion, pressurized pulps from both prickly pear varieties had statistically significant (p ≤ 0.05) higher betanin content in the intestinal phase, than their respective controls. The recoveries of Sanguinos and Pelota in the intestinal phase were 65% and 71%, respectively. These results support the theory that increases in betanin bioaccessibility are mainly driven by changes in the food matrix as an indirect effect of HHP. It has been suggested that processing technologies (such as HHP) influence enzymatic pectin conversion reactions [19,20]. This could have significant effects on pectin by denaturing of pectinases, enhancing the catalytic activity of pectinases and enhancing nonenzymatic (chemical) pectin conversions [21]. In the present study, gelatinization was observed visually after processing prickly pear pulps at 350 MPa/5 min. In more viscous pulps, betanin could be less exposed to enzymes and pH shifts in the gastrointestinal tract, which could have contributed to its higher digestive stability in pressurized samples than in unpressurized samples.

It has been shown that dominant factors involved in the influence of dietary fiber on digestion are (i) physical trapping of bioactive compounds within structured assemblies such as fruit tissue, and (ii) enhanced viscosity of gastric fluids restricting the peristaltic mixing process that promotes transport of enzymes to their substrates, bile salts to un-micellized fat, and soluble antioxidants to the gut wall [22]. Secondary factors may include binding of bile salts (and perhaps enzymes) to specific fiber components and inhibition of diffusion [23]. These effects of dietary fiber on digestion can be modified when applying HHP. In this study it is likely that treatments such as 350 MPa/5 min promoted changes in soluble fiber (i.e., pectins) from high methoxyl to low methoxyl. Low methoxyl pectins can increase the viscosity of the digesta and limit the interactions of betalains and digestive enzymes among other digestive components, which may degrade these antioxidants.

In the case of phenolic acids, piscidic acid in prickly pear pulps treated at 350 MPa/5 min (Table 4) showed both higher extractability and better digestive stability than unpressurized pulps. Higher digestive stability of piscidic acid in HHP-treated Pelota pulps is evidenced by the increasing content observed in each phase of the simulated gastrointestinal digestion; however, the pressurized Sanguinos pulp showed only a statistically significant (p ≤ 05) higher content in the gastric phase compared to its control. It is possible that HHP treatments could promote interactions between the prickly pear phenolic compounds and dietary fiber, due to the modifications in the tissue microstructure [10] producing the liberation of linked phenolics to the cell walls, which include the formation of junctions stabilized by an array of noncovalent bonds between hydroxide groups from phenolic compounds and polar groups from polysaccharide molecules (hydrogen bonds, electrostatic and dipolar interactions, and van der Waals attractions) [24]. Because these bonds are individually weak, their formation and disruption often occur as sharp, cooperative processes in response to comparatively small changes (pH or solvent quality in the gastrointestinal tract) [23].

In pulps processed at 350 MPa/5 min, differences in 4-hydroxybenzoic derivative content in the intestinal phase correlate with its initial content in the pressurized material. The stability of 4-hydroxybenzoic acid glycoside during the gastro-intestinal digestion was similar in treated and untreated prickly pear pulps. High recoveries in the gastric phase of all samples were observed (127% to 164%). However, the recovery of 4-hydroxybenzoic acid glycoside in the intestinal phases of Pelota and Sanguinos pulps treated with HHP was higher than in their untreated pulps.

2.5.2. Stability and Recovery of Bioactives in Prickly Pear Peels Treated with HHP at 600 MPa/CUT

As shown previously, the pressurization of prickly pear peels at 600 MPa/CUT (come-up time) enhanced the bioaccessibility of 4-hydroxybenzoic acid glycoside (+33 to +37%) and isorhamnetin glycosides (+15 to +125%) in Sanguinos and Pelota varieties. This HHP treatment was chosen to study the digestive stability and recovery of its bioactive compounds because it was considered the best treatment to obtain the higher values of bioaccessibility for phenolic compounds (even when the betalain bioaccessibility decreased at this HHP treatment). Hence, the digestive stability and recovery of betalains, phenolic acids, and flavonoids in pulps treated at 600 MPa/CUT was studied in each stage of in vitro simulated gastrointestinal digestion to identify the factors that could influence their bioaccessibility (

Table 5. In vitro digestive stability (mg/100 g fresh weight) of betalains and phenolic compounds in HHP-treated (600 MPa/CUT) Sanguinos and Pelota prickly pear peels.

	Treatment	Peel	Oral Phase		Gastric Phase		Intestinal Phase
		Content 1	Content 1	Recovery (%)	Content 1	Recovery (%)	Content 1	Recovery (%)
Indicaxanthin	Sanguinos Control	0.60 ± 0.01b	0.63 ± 0.03b	105 ± 3b	0.40 ± 0.01a	66 ± 1a	0.37 ± 0.02a	62 ± 4a
	Sanguinos 600 MPa/CUT	0.53 ± 0.03a	0.55 ± 0.08a	104 ± 8a	0.50 ± 0.16a	94 ± 25a	0.20 ± 0.03a*	39 ± 5a*
	Pelota Control	1.01 ± 0.01c	0.98 ± 0.06c	97 ± 5c	0.78 ± 0.07b	77 ± 6b	0.56 ± 0.03a	55 ± 3a
	Pelota 600 MPa/CUT	0.57 ± 0.06b*	0.65 ± 0.04b*	113 ± 5b	0.64 ± 0.07b	112 ± 0b*	0.39 ± 0.03a*	68 ± 5a
Betanin	Sanguinos Control	5.78 ± 0.23c	5.40 ± 0.50c	93 ± 5c	4.06 ± 0.46b	70 ± 5b	1.67 ± 0.13a	29 ± 2a
	Sanguinos 600 MPa/CUT	4.52 ± 0.28b*	5.18 ± 0.06b	115 ± 6b	5.00 ± 0.53b	111 ± 5b*	1.32 ± 0.11a*	29 ± 2a
	Pelota Control	16.29 ± 0.10b	17.73 ± 1.47b	109 ± 8b	15.90 ± 2.13b	98 ± 12b	7.51 ± 0.60a	46 ± 4a
	Pelota 600 MPa/CUT	13.92 ± 1.06b	13.20 ± 0.59b	95 ± 3b	12.50 ± 1.06b	90 ± 1b	5.11 ± 0.75a*	37 ± 3a
Piscidic acid	Sanguinos Control	439.45 ± 0.90b	463.24 ± 5.65b	105 ± 1b	401.20 ± 33.23b	91 ± 7b	230.10 ± 18.41a	52 ± 4a
	Sanguinos 600 MPa/CUT	346.50 ± 18.65b*	373.12 ± 8.66b*	108 ± 3b	454.47 ± 34.38c	131 ± 3c*	208.83 ± 16.70a	60 ± 5a
	Pelota Control	779.17 ± 4.74b	785.43 ± 40.00b	101 ± 4b	772.12 ± 36.45b	99 ± 4b	410.70 ± 32.86a	53 ± 4a
	Pelota 600 MPa/CUT	713.46 ± 59.11b	720.35 ± 39.82b	101 ± 3b	839.20 ± 49.96b	118 ± 3c*	364.88 ± 29.19a	51 ± 4a
4-hydroxy benzoic acid derivative	Sanguinos Control	19.12 ± 0.21b	20.57 ± 1.90b	108 ± 9b	17.45 ± 2.31b	91 ± 11ab	11.60 ± 0.93a	61 ± 5a
	Sanguinos 600 MPa/CUT	14.38 ± 0.73a*	16.47 ± 1.46a	114 ± 4b	17.61 ± 0.46a	122 ± 3ab	15.40 ± 1.23a	107 ± 9a*
	Pelota Control	23.62 ± 0.14ab	27.44 ± 3.71b	116 ± 15a	25.33 ± 3.75ab	107 ± 15a	15.48 ± 1.24a	66 ± 5a
	Pelota 600 MPa/CUT	25.67 ± 4.28a	32.74 ± 1.64ab	128 ± 15b	41.83 ± 5.00b	163 ± 8b*	21.10 ± 2.20a	82 ± 9a
IG1 2	Sanguinos Control	0.55 ± 0.03ab	0.63 ± 0.09b	115 ± 11b	0.85 ± 0.13b	156 ± 17b	0.31 ± 0.03a	56 ± 5a
	Sanguinos 600 MPa/CUT	0.67 ± 0.06a	0.74 ± 0.26a	111 ± 29a	0.81 ± 0.24a	122 ± 25a	0.46 ± 0.04a*	69 ± 6a*
	Pelota Control	1.44 ± 0.01b	1.55 ± 0.15b	108 ± 10b	1.45 ± 0.23b	101 ± 15b	0.77 ± 0.06a	53 ± 4a
	Pelota 600 MPa/CUT	2.79 ± 0.17b*	2.74 ± 0.09b*	98 ± 3b*	2.72 ± 0.14b*	97 ± 1b*	1.73 ± 0.14a*	62 ± 5a*
IG2 3	Sanguinos Control	1.51 ± 0.13b	1.47 ± 0.14b	98 ± 1c	0.87 ± 0.10a	58 ± 2b	0.63 ± 0.05a	42 ± 3a
	Sanguinos 600 MPa/CUT	1.50 ± 0.06b	1.61 ± 0.18b*	107 ± 8b	1.60 ± 0.13b*	107 ± 5b*	0.89 ± 0.07a	59 ± 5a*
	Pelota Control	0.35 ± 0.00a	0.45 ± 0.05a	130 ± 14a	0.42 ± 0.09a	121 ± 26a	0.25 ± 0.02a	73 ± 6a
	Pelota 600 MPa/CUT	0.64 ± 0.05b*	0.56 ± 0.01b	89 ± 6b	0.55 ± 0.01b	87 ± 5b	0.35 ± 0.03a	55 ± 4a
IG3 4	Sanguinos Control	0.32 ± 0.03b	0.33 ± 0.04b	103 ± 2c	0.26 ± 0.03b	83 ± 2b	0.15 ± 0.01a	48 ± 3a
	Sanguinos 600 MPa/CUT	0.32 ± 0.01b	0.33 ± 0.03b	105 ± 6b	0.30 ± 0.02b	96 ± 3b*	0.20 ± 0.02a*	63 ± 5a*
	Pelota Control	0.15 ± 0.00ab	0.18 ± 0.00b	125 ± 5b	0.18 ± 0.03b	120 ± 26b	0.08 ± 0.01a	57 ± 4a
	Pelota 600 MPa/CUT	0.23 ± 0.01c	0.20 ± 0.01b	85 ± 1b*	0.19 ± 0.00b	80 ± 1b	0.14 ± 0.1a*	59 ± 5a
IG4 5	Sanguinos Control	0.81 ± 0.06b	0.73 ± 0.04b	89 ± 2b	0.53 ± 0.06a	65 ± 3a	0.49 ± 0.04a	60 ± 5a
	Sanguinos 600 MPa/CUT	0.71 ± 0.03a	0.83 ± 0.05a	116 ± 1a*	0.85 ± 0.19a	120 ± 21a	0.56 ± 0.04a	79 ± 6a*
	Pelota Control	0.23 ± 0.00a	0.24 ± 0.03a	104 ± 14a	0.23 ± 0.10a	97 ± 41a	0.14 ± 0.01a	59 ± 4a
	Pelota 600 MPa/CUT	0.44 ± 0.04c*	0.38 ± 0.00bc*	85 ± 6b	0.32 ± 0.01b	72 ± 5b	0.18 ± 0.01a	40 ± 3a*
IG5 6	Sanguinos Control	3.09 ± 0.18c	2.89 ± 0.07bc	93 ± 3c	2.56 ± 0.08b	83 ± 2b	0.92 ± 0.07a	30 ± 2a
	Sanguinos 600 MPa/CUT	2.82 ± 0.11b	2.57 ± 0.11b	91 ± 2b	2.49 ± 0.42b	88 ± 11b	1.30 ± 0.11a*	46 ± 4a*
	Pelota Control	2.26 ± 0.01b	2.29 ± 0.21b	101 ± 9b	2.26 ± 0.19b	100 ± 8b	1.26 ± 0.10a	56 ± 4a
	Pelota 600 MPa/CUT	5.10 ± 0.49c*	4.78 ± 0.07bc*	94 ± 8b	3.91 ± 0.09b*	77 ± 5b	2.21 ± 0.18a*	43 ± 4a*

Results are expressed as mean ± standard deviation (n = 3). Superscript lowercase letters indicate statistically significant differences (p ≤ 0.05) between digestive phases. * Indicate statistically significant differences (p ≤ 0.05) between treatments for the same variety. ¹ mg/100 g fresh weight, ² isorhamnetin glucosyl-rhamnosyl-rhamnoside (IG1), ³ isorhamnetin glucosyl-rhamnosyl-pentoside (IG2), ⁴ isorhamnetin hexosyl-hexosyl-pentoside (IG3), ⁵ isorhamnetin glucosyl-pentoside (IG4), and ⁶ isorhamnetin glucosyl-rhamnoside (IG5).

). The graphical representation of this data may be consulted in Supplementary Figure S4.

Betalain compounds, betanin and indicaxanthin, in prickly pear peels processed at 600 MPa/CUT suffered a considerable degradation due to the HPP treatment (−12 to −77%). Mentioned degradation of these pigments due to pressurization contributed to their lower content in the intestinal phase and bioaccessibility. A group of researchers applied HHP treatments of 650 MPa at different processing times (3, 7, 15, and 30) on beetroot slices (var. Red cloud) as an alternative to blanching pretreatment (90 °C for 7 min) and observed higher betalain degradation at higher time-exposure to high pressure [18].

The bioaccessibility (Table 3) and digestive stability (Table 5) of piscidic acid in prickly pear peels treated at 600 MPa/CUT showed no statistically significant differences compared to the control. On the other hand, 4-hydroxybenzoic acid glycoside showed noticeable increases in the gastric phase of HHP-treated Pelota prickly pear peels (Table 5). A previous study showed that treating prickly pear peels at 600 MPa/10 min at 22 °C and 55 °C increased the content of low molecular weight fractions of soluble dietary fiber [25]. An increase in low molecular weight soluble dietary fiber could have a positive effect on the digestive stability of phenolic acids in the gastric phase of digestion.

Regarding flavonoids, most isorhamnetin glycoside (IG) were more abundant in peels treated at 600 MPa/CUT than in their respective controls (Table 5). This contributed greatly to their high bioaccessibility. Pelota peels treated with HHP showed higher IG1 content throughout the complete digestive process. This included the intestinal phase where the recovery of HHP-treated Pelota peels was 62% compared to 53% in untreated ones. However, IG1 content in Sanguinos peels was not statistically different between pressurized and control samples. In other terms, the digestive stability of IG2 in HHP-treated prickly pear peels showed different behavior (Table 5). IG2 content in Pelota peels treated at 600 MPa/CUT was higher in the starting material (0.64 mg/100 g fresh weight), which contributed to its higher content in the intestinal phase (direct effect). However, in pressurized Sanguinos peels, IG2 content was similar in the starting material, but reached a higher concentration in the intestinal phase due to a higher digestive stability in the gastric phase (+107% higher than the control) (indirect effect).

HHP treatments did not enhance the release of this IG3 at 600 MPa/CUT (Table 5). Contrarily, in Sanguinos peels treated with HHP, IG4 showed no statistically significant differences in the intestinal phase, despite showing a high recovery of 120% in the gastric phase. Pressurized and control Pelota peels did not show any differences in IG4 content in the intestinal phase, either. Finally, a higher IG5 content was observed throughout the whole digestive process in Pelota peels. Despite higher content in the intestinal phase, Pelota showed lower (−23%) overall recovery compared to untreated peels.

3. Materials and Methods

3.1. Solvents, Reagents, and Standards

Ultra-pure water was obtained from a Milipak^® Express 40 system (Merck-Milipore, Dormstadt, Germany). Methanol (99.8% LC-MS) was purchased from VWR International (Barcelona, Spain). Pepsin (P6887; 791 U mg/L), α-amylase (10080; 79 U mg/L), pancreatin (P7545; 17 U/mg), bile (B8381), and other reagents used for the in vitro digestion assay were purchased from Sigma-Aldrich (St. Louis, MO, USA).

Piscidic acid was purified from prickly pear peels by semi-preparative high-performance liquid chromatography (HPLC) [12]. Betanin was purified from a betalain-rich concentrate extracted from commercial beetroot and indicaxanthin was semi-synthesized using purified betanin [12]. Commercial standards isorhamnetin and 4-hydroxybenzoic acid were purchased from Sigma-Aldrich (St. Louis, MO, USA). All isolated and semi-synthetized standards were analyzed for authenticity and purity by HPLC-ESI-MS-QTof.

3.2. Prickly Pear Fruits

Pelota prickly pears were provided by Agroproductores La Flor de Villanueva in San Sebastián Villanueva Acatzingo (Puebla, Mexico; 19°1’ N, 97°4′ W; 121 m. a. s. l.). Sanguinos prickly pears were purchased from Bioarchen in Archen (Murcia, Spain; 38°7′ N, 1°180′ W; 121 m. a. s. l.). Fruits were selected according to size, peel coloration, and ripeness, and were prepared for HHP treatments according to Figure 1. For each treatment, 16 fruits were sliced into quarters and one quarter of each fruit was placed in a bag (four bags of identical composition). Then, samples were vacuum sealed and treated with high hydrostatic pressure. Afterwards, samples were separated into pulps (mesocarp) and peels (endocarp and exocarp) and were frozen with liquid nitrogen. Samples were freeze dried at −45 °C and 1.3 × 10⁻³ MPa for 5 days (LyoBeta 15, Azbil Telstar, S.L., Terrasa, Spain). Freeze-dried prickly pear tissues were pulverized in a knife mill (Grindomix GM200, Retsch, Germany) to a small particle size (< 2 mm) and sieved to remove seeds in pulps. Samples were vacuum-sealed and stored at −20 °C until analysis.

3.3. High Hydrostatic Pressure Treatments

Prickly pears fruits were processed as described previously [11]. The purple Pelota variety was processed in Mexico in a pilot-scale high pressure equipment (Model 2 L, Flow Autoclave Systems, Columbus, OH, USA) and the red Sanguinos variety was processed in Spain in an equipment of comparable characteristics (Model 2 L, Stansted SFP 7100:9/2C, Harlow, Essex UK). Pressure conditions were based on commercially used intensities of 100 MPa (low), 350 MPa (intermediate), and 600 MPa (high) and time (5 min). The effect of heir come-up times (CUTs) was evaluated as well. Compression rates were 7 MPa/s and decompression occurred in under 1 s. The come-up times (CUTs) at 100, 350, and 600 MPa were 14.3 ± 1, 50.0 ± 4, and 85.7 ± 6 s, respectively. Pressure, time, and temperature were controlled by a computer program. Average maximum temperatures reached in the Flow Autoclave Systems (Pelota variety) were 17, 25, and 32 °C for CUT and 19, 27, and 34 °C for holding times at 100, 350, and 600 MPa, respectively. In both varieties, the average maximum temperature reached was 20 °C since it was constantly cooled by means of a thermostat jacket. The processing of each treatment was performed three times.

3.4. In Vitro Simulated Gastrointestinal Digestion

The in vitro simulated gastrointestinal digestion was performed according to the standardized INFOGEST protocol [7,26] using prickly pear rehydrated puree (control and HPP treated samples). Prior to digestion, the rehydrated puree was extracted according to [12] to analyze the concentration of bioactive compounds in the starting material. The simulated saliva fluid, simulated gastric fluid, and simulated duodenal fluid were prepared according to [7]. The oral phase was made up of 5 g rehydrated fruit puree, 4 mL of electrolyte stock solution (SSF), 0.025 mL of 0.3 M CaCl₂(H₂O)₂, 0.75 mL salivary amylase (75 U/mL activity; 10 mg/mL concentration), and 0.225 mL of water. The gastric phase was made up of 10 mL from the oral phase, 8 mL of electrolyte stock solution (SGF), 0.005 mL of 0.3 M CaCl₂(H₂O)₂, 0.667 mL of pepsin (2000 U/mL activity, 20 mg/mL concentration), 0.928 mL of water, and approximately 0.4 mL of HCL (5 M) to adjust the pH to 3. The intestinal phase was composed of 20 mL from the gastric phase, 8 mL of electrolyte stock solution, 0.04 mL of CaCl₂(H₂O)₂, 5 mL of trypsin in pancreatin (100 u/mL activity, 133.3 mg/mL concentration), 3 mL of bile acid mixture from bovine and ovine (10 mM), 3.16 mL of water, and approximately 0.8 mL of NaOH (5 M) to adjust the pH to 7. Enzymes were prepared and added to the simulated fluids prior to the digestive assay.

To study bioaccessibility, all untreated and HHP-treated prickly pears were submitted to the complete digestive simulation and their intestinal phases were frozen with liquid nitrogen and stored at −20 °C. Samples were extracted from thawed intestinal phases and analyzed by HPLC-DAD-MS.

The bioaccessibility of betalains and phenolic compounds was calculated according to Equation (1). This quantification differs from the traditional computation which, by definition, divides the bioactive content in HHP-treated fruit by the bioactive content in the intestinal phase of HHP-treated fruit. However, when talking about processed foods, any positive or negative effects on bioactive compounds due to processing would directly influence the bioaccessibility parameter, providing false positive or false negative effects. Therefore, to assess the bioaccessibility of all HHP-treated samples in a first instance, the bioactive content in the untreated fruit was divided by the bioactive content in the intestinal phase of the HHP-treated fruit tissue.

$$Bioaccessibility \left(\%\right)=\frac{Bioactive conten{t}_{untreated fruit}}{Bioactive conten{t}_{intestinal phase of HHP treated fruit}}$$

(1)

After analyzing this bioaccessibility data of Opuntia samples, we conducted the study of the digestive stability of selected samples chosen on the basis on the HHP treatments, which resulted in the highest bioaccessibility. These selected HPP-treated samples were digested again, and a collection of each digestive phase (oral, gastric and intestinal) fractions were made in order to analyze the bioactive compounds by HPLC-DAD-MS. These digesta were frozen immediately with liquid nitrogen and stored at −20 °C. Samples were extracted from thawed digestive phases and analyzed by HPLC-DAD-MS.

The stability of betalains and phenolic compounds in each digestive phase (oral, gastric and intestinal) of simulated gastrointestinal digestion is shown in Equation (2). This stability could be expressed by the recovery of bioactive compounds during simulated gastrointestinal digestion and provides relevant information related to digestive stability and was calculated by dividing the bioactive content in samples treated with HHP by the bioactive content in the digestive phase of HHP-treated samples.

$$Recovery \left(\%\right)=\frac{Bioactive conten{t}_{HHP treated fruit}}{Bioactive conten{t}_{digestive phase of HHP treated fruit}}$$

(2)

3.5. Extraction of Betalains and Phenolic Compounds for HPLC Analysis

Betalains and phenolic compounds were extracted simultaneously from fruit tissues [12]. One gram of freeze-dried sample was extracted with 5 mL of methanol:water in a 1:1 (v:v) proportion. Samples were vortexed and sonicated for 4 min. Then they were centrifuged at 4 °C at 10,000× g for 10 min. The supernatant was recovered and the pellet was re-extracted two more times with methanol:water and one last time with pure methanol, while recovering the supernatant after each extraction. The supernatants were combined and concentrated in a rotavapor by evaporating methanol. The aqueous extracts were then made up to 5 mL with water, filtered, and analyzed by HPLC.

Betalains and phenolic compounds were extracted simultaneously from thawed digestive phases according to the procedure described by [27]. An aliquot of the digestive phase was weighed (10g oral phase or 20 g gastric and intestinal phase) and placed in an assay tube. The pH was adjusted to 4 and pure methanol was added in a 1:1 (v:v) proportion. The sample was homogenized for 2 min at 700× g using a ultrahomogenizer (Omnimixer ES-207, Omni International Inc, Gainsville, FL, USA) in an ice bath and then centrifuged at 4 °C for 15 min at 9000 rpm. The supernatant was concentrated using a rotavapor and the sample was made up to 5 mL (oral phase) or 20 mL (gastric and intestinal phase), then filtered for HPLC analysis.

3.6. Quantification of Betalains and Phenolic Compounds by HPLC

Betalains and phenolic compounds were quantified simultaneously by high-performance liquid chromatography using a 1200 Series Agilent HPLC System (Agilent Technologies, Santa Clara, CA, USA) with a reverse-phase C18 column (Zorbax SB-C18, 250 × 4.6 mm i.d., S-5 µM; Aglient) at 25 °C [10,12]. Mobile phase A was 1% formic acid (v/v) in ultrapure water and mobile phase B was 1% formic acid (v/v) in methanol. Separation was achieved using an initial composition of 15% B during 15 min, increased to 25% within 10 min, subsequentially ramped to 50% B within 10 min, increased to 75% B in 15 min, and finally followed by a decrease period of 15% B in 5 min prior to isocratic re-equilibration for 10 min. The flow rate was 0.8 mL/min and the injection volume was 20 μL. The UV-vis photodiode array detector was set at four wavelengths to detect phenolic acids (280 nm), flavonoids (370 nm), betaxanthins (480 nm), and betacyanins (535 nm). UV/Vis spectra were also recorded between 200 and 700 nm. The HPLC-DAD was coupled to a mass spectrometry detector (LCMS SQ 6120, Agilent, Agilent Technologies, Santa Clara, CA, USA) with an electrospray ionization (ESI) source operating in positive ion mode. The drying gas was nitrogen at 3 L/ min at 137.9 KPa. The nebulizer temperature was 300 °C and the capillary had 3500 V potential. The coliseum gas was helium, and the fragmentation amplitude were 70 V. Spectra were recorded m/z from 100 to 1000.

Further mass spectrometry analyses were performed in a maXis II LC-QTOF equipment (Bruker Daltonics, Bremen, Germany) with an ESI source and the same chromatographic conditions. The ESI-QTOF detector worked in positive ion mode and recorded spectra m/z from 50 to 3000. Operation gas at 6 L/min. MS/MS analysis used the bbCID (Broad Band Collision Induces Dissociation) method at 30 eV.

Compounds were identified and quantified according to their retention times, UV/Vis and mass spectral data compared to their respective standards and calibration curves. These are in agreement with what was reported previously by various authors [12,28,29,30]. For indicaxanthin standard, the retention time was 10.5 min; UV λ_max at 478 nm; [M + H]⁺ ion at 309.11 m/s; MS/MS fragments at m/s 263.10, 217.10, 70.06; and calibration curve was y = 0.0105x (R² = 0.9974) where x = peak area and y = concentration (ug/mL). For betanin standard, the retention time was 15.7 min; UV λ_max at 534 nm; [M + H]⁺ ion at 551.15 m/s; MS/MS fragments at m/s 390.10, 389.10; and calibration curve was y = 0.0175x (R² = 0.9944), where x = peak area and y = concentration (ug/mL). For hydroxybenzoic acid standard, the retention time was 23.5 min; UV λ_max at 274 nm; [M+H]⁺ ion at 205.05; and calibration curve was y = 0.0409x (R² = 0.9999) where x = peak area and y = concentration (ug/mL). For piscidic acid standard, the retention time was 14.0 min; UV λ_max at 232 and 275 nm; [M + H]⁺ ion at 257.07 m/s; MS/MS fragments at m/s 191.07, 147.04, and 119.05; 107.05; and calibration curve was y = 0.4341x (R² = 0.9967) where x = peak area and y= concentration (ug/mL). Isorhamnetin glycosides (IG1, Rt = 40.0 min; IG2, Rt = 40.4 min; IG3, Rt = 40.9 min; IG4, Rt = 41.2 min; and IG5, Rt = 44.5 min) all showed the MS/MS fragment at m/s 317.07 for isorhamnetin. Hence, they were quantified with the isorhamnetin standard. For isorhamnetin standard, the retention time was 49.9 min; UV λ_max at 370 nm; [M + H]⁺ ion at 317.07 m/s; and calibration curve was y = 0.0324x (R² = 0.9820) where x = peak area and y= concentration (ug/mL). Details on the elucidation of the isorhamnetin glycosides were reported in previous research articles [5,12,31].

3.7. Statistical Analysis

Data were expressed as mean ± standard deviation of three determinations. Significant differences were calculated by one-way analysis of variance (ANOVA) and a post hoc Tukey’s test (p ≤ 0.05). Pearson’s correlation was calculated at p ≤ 0.05 and p ≤ 0.01. Statistical analyses were performed with IBM SPPS Statistics 23.0 (IBM Corp, Armonk, NY, USA).

4. Conclusions

Sanguinos and Pelota prickly pear fruits were treated with high hydrostatic pressure (100, 350, and 600 MPa; CUT and 5 min), which had different effects on the bioaccessibility of their betalains, phenolic acids, and flavonoids in their peel and pulp tissue-sections. The bioaccessibility of betalains in pressurized prickly pear tissue-sections was mostly negatively affected by their degradation during processing. However, the bioaccessibility of betalains in pulps could be enhanced by HHP at specific conditions such as 350 MPa/5min. This effect was a result of a higher digestive stability in the gastric and intestinal phase of simulated digestion, which is likely influenced by HPP-induced changes to soluble dietary fiber. Phenolic acids were more pressure-resistant than betalains and their bioaccessibility was mostly favored by changes to the food matrix, which enhanced their extractability in the gastric phase of digestion. Curiously, the effect of HHP on isorhamnetin glycosides (flavonoids) depended greatly on the type of glycoside and pressurization conditions. The bioaccessibility of isorhamnetin glycosides in peels was greatly favored at 600 MPa/CUT by both effects of (i) enhanced extractability and (i) higher digestive stability.

In general terms, the best treatments for enhancing the bioaccessibility of bioactive compounds in prickly pear pulps (edible fraction) was at 350 MPa/5 min, and in peels (potential healthy ingredient) was at 600 MPa/CUT. Future studies require the assessment of changes in polysaccharides such as dietary fiber and, more specifically, pectic substances to study the interactions between bioactive compounds and the food matrix. In this study, we expect to contribute to the use of high hydrostatic pressure to promote the healthy attributes of foods by increasing the bioaccessibility of their bioactive compounds. To take advantage of the nutritional aspects presented in this work, HPP-treated prickly pear fruits can be further processed into juices, purees, and jams. Further research regarding the shelf life stability of HHP-treated prickly pear fruit products to promote their commercialization is needed.