vitro bioaccessibility in cloudy pomegranate juice

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Characterization of the �avor, sensory quality and invitro bioaccessibility in cloudy pomegranate juicetreated by high pressure and thermal processingJun Tian 

Kunming University of Science and TechnologyFengyun Cheng 

Kunming University of Science and TechnologyYurou Yun 

Kunming University of Science and TechnologyJunjie Yi 

Kunming University of Science and TechnologyShengbao Cai 

Kunming University of Science and TechnologyLinyan Zhou  ( [email protected] )

Kunming University of Science and Technology

Research Article

Keywords: Cloudy pomegranate juice, High hydrostatic pressure processing, Thermal sterilization,Phenolics, Flavor change, In vitro bioaccessibility

Posted Date: May 27th, 2022

DOI: https://doi.org/10.21203/rs.3.rs-1684319/v1

License: This work is licensed under a Creative Commons Attribution 4.0 International License.   ReadFull License

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AbstractRecently, cloudy pomegranate juice (PJ) becomes popular due to its rich in phenolics and health-promotingeffects. The present work aimed to evaluate the application of high hydrostatic pressure processing (HPP),pasteurization (PT) and high-temperature short-time sterilization (HTST) on physicochemical properties(color, �ow behaviour, turbidity, sugars, organic acids, aroma and sensory evaluation) and in vitrobioaccessibility of total phenols content (TPC), entire �avonoids content (TFC) and phenolics of cloudy PJ.Compared to HPP, thermal sterilization signi�cantly increased the L*, a*, ΔE and turbidity and decreased theTPC, and TFC. HPP better maintained the volatile pro�le of cloudy PJ, while thermal sterilizationsigni�cantly changed it by reducing alcohols by 23.8%-32.7% and increasing acids by 33.6%-182.8%. Thebioaccessibility of �avonoids, phenolic acids, and tannins in control cloudy PJ after in vitro oral-gastric-intestinal digestion was 1.5%, 4.9%, and 9.0%, respectively, which were not signi�cantly changed bydifferent treatments. These results contributed to promoting the color quality and health bene�ts of cloudyPJ rich in phenolics by optimizing the processing conditions in the food industry.

IntroductionPomegranate (Punica granatum L.), one of the most ancient fruits, is native to the Middle East and widelycultivated in tropical and subtropical countries (Li et al., 2015). Signi�cant phytochemicals have been foundin pomegranate, including phenolics and �avonoids (Türkyılmaz et al., 2022). The interest in pomegranatehas recently increased mainly because of its health-promoting effects. Previous studies have reported thatthe intake of pomegranate and its functional compounds could reduce blood pressure and low-densitylipoprotein oxidation, and exhibit potential health bene�ts in antioxidant, antiatherogenic, antihypertensive,and anticancer (Asgary et al., 2014; Bassiri-Jahromi, 2018).

Recently, cloudy pomegranate juice (PJ) has become popular due to its convenient use, pleasant �avor, andhigh nutritional value. Thermal sterilization is the traditional technology used to extend the shelf life ofcloudy PJ. However, thermal sterilization inevitably results in the irreversible loss of sensory characteristicsand quality, as well as degradation of functional compounds, particularly phenolics. Phenolics in PJ mainlyinclude anthocyanins, tannins, ellagic acid, and �avonoids (Kalaycıoğlu and Erim, 2017). As one of themost essential phenolics in PJ, anthocyanins exhibit excellent performance in enhancing antioxidativecapacity and improving glucose and lipid metabolism, but anthocyanins are very heat-sensitive (Wu et al.,2021a). Thus, non-thermal technologies, such as High hydrostatic pressure processing (HHP), are usuallyapplied to processing juice rich in phenolics to preserve the nutrients better. In our previous study,pasteurization (PT, 85 ℃/30 s) and high-temperature short-time sterilization (HTST, 110 ℃/8.6 s)treatments signi�cantly decreased total monomeric anthocyanins content in PJ by 27.5% and 29.3%,respectively, while its content was not signi�cantly changed under HPP treatment (at 350/450/550MPa for3/5 min) (Yuan et al., 2022). Varela-Santos et al. (2012) con�rmed the positive in�uences of HPP onpolyphenols stability in PJ, and results showed that the polyphenol contents of PJ were increased by 3.38%and 11.99% after HPP at 350 MPa and 550 MPa, respectively. It might be due to an improved extractione�ciency of polyphenolic compounds from plant matrices caused by HPP, which increased the permeabilityof pressurized cells (Putnik et al., 2019). Moreover, after ingested, they are susceptible and signi�cantly

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degraded by internal environmental factors such as pH, digestive enzymes and temperature duringdigestion, and only small amounts of phenolics are absorbed (Spínola et al., 2018). For example, thebioaccessibility of polyphenols, anthocyanins and �avonoids of blueberries was signi�cantly reduced afterintestinal digestion, with bioaccessibility of only 13.93%, 1.95% and 15.68%, respectively (Jiao et al., 2018).Furthermore, processing methods may have different impacts on the release, transformation andabsorption of polyphenols (Parada and Aguilera, 2007). For example, the phenolics bioaccessibility of HPP(600 MPa/6 min) treated cloudy hawthorn berry juice was 35.12%, which was signi�cantly higher than thatvalue (30.54%) of the untreated sample (Lou et al., 2022a). Thus, an in vitro study was necessary toevaluate the effects of different processing conditions on digestion and the bioaccessibility of thephenolics in food.

In addition, processing can change the composition of the food matrix and signi�cantly alters the �avorcomponents, which then affects consumer acceptance. Previous studies have proved that thermalsterilization usually induced the decrease of essential aroma compounds and gave rise to the distinctcooked off-�avor of fruits and vegetables (Cheng et al., 2020; Li et al., 2009). It has been pointed out thatlychee juice after thermal sterilization at 100 ℃ for 5 min presented cooked cabbage/potato, garlic/onionand sulfurous impression, due to the formation of sulfur volatiles such as dimethyl sul�de, methional,dimethyl disul�de, dimethyl trisul�de and 2,4-dithiapentane (An et al., 2019).

With consumers’ growing awareness of healthy, green, minimally processed, and clean labelling foods, HPPtreated juices would exhibit an advantage in the fruit juice market. Therefore, the objective of this study wasto 1) investigate the change of physicochemical characteristics, including color, TSS, pH, turbidity,rheological properties, and the chemical compounds, including sugars, organic acids, as well as untargetedvolatile pro�les of cloudy PJ after the treatments of PT/HTST/HPP; 2) evaluate the change of the totalphenolics, total �avonoids and individual phenolics bioaccessibility of cloudy PJ after the treatments ofPT/HTST/HPP, to provide a comprehensive multi-quality analysis for the novel application of HPP infunctional cloudy PJ.

Materials And Methods2.1. Chemicals

Fructose and glucose were obtained from Aladdin Chemical Reagent Co., Ltd. (Shanghai, China). Methanol(HPLC grade reagents), α-Amylase (porcine pancreas, ten units/mg), pepsin (porcine gastric mucosa, 595units/mg protein), pancreatin (porcine pancreas, eight × USP speci�cations), bile salts were purchased fromSigma-Aldrich Chemical Co. (St. Louis, MO, USA). n-alkanes (C3–C9, C10–C25) were supplied by O2si(North Charleston, SC). All other chemicals were of analytical grade.

2.2. Pomegranate juice preparation and processing

2.2.1. Preparation of cloudy pomegranate juice (PJ)

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Freshly harvested pomegranate fruit was purchased from local markets in Jianshui County, YunnanProvince, China, and stored at 4 ℃ until processing. Arils were manually separated from the pomegranatefruits. Pomegranate juice was collected by pressing the arils with a domestic juicer (JYZ-E21C, Joyoung,China) and then �ltrated through gauze, and centrifuged at 6000 g and 4 ℃ for 5 min. The obtained juicewas stored at 4 ℃ before further processing and analysis.

2.2.2. High hydrostatic pressure processing (HPP) treatment

In the food industry, HPP treatments of 500–600 MPa have yielded food products with good quality andsafety. Thus, 450 MPa and 550 MPa for 5 min were selected for low intensity (HPP1) and high intensity(HPP2) treatments. HPP was carried out in a high hydrostatic pressure machine (HHP-600, Baotou Kefa CO,Ltd, China). The cloudy PJ was �lled in 200 mL polyethene terephthalate (PET) bottles, placed in a 10 Lpressure container, and pressurized at ambient temperature (25 ℃) for 5 min at 450 MPa and 550 MPa.The duration of treatment did not include the boosting time and releasing time.

2.2.3. Pasteurization (PT) treatment

For PT treatment, cloudy PJ was treated in a multipurpose UHT sterilization unit (ST-20, Shanghai SunyiTech. Co, Ltd, China). The procedure of cloudy PJ processing was to preheat the cloudy PJ to 65 ℃, thensterilize it at 85 ℃ for 30 s (Wibowo et al., 2019). After PT treatment, the cloudy PJ was immediately cooledby an ice bath and packed into aseptic PET bottles.

2.2.4. High-temperature short-time sterilization (HTST) treatment

HTST treatment was also conducted in the multipurpose UHT sterilization unit (ST-20, Shanghai SunyiTech. Co, Ltd, China). Cloudy PJ was preheated to 95 ℃ and pasteurized at 110 ℃ for 8.6 s (You et al.,2018). After HTST treatment, the cloudy PJ was immediately cooled by an ice bath and packed into asepticPET bottles.

2.3. Color measurement

The CIE color of cloudy PJ was determined by colorimeter (Agera, Hunter Associate Laboratory, Inc., Fairfax,USA). For each measure, 50 mL of the sample was added to the quartz cup and covered with a black lid.The color parameters, including brightness (L*), redness (a*), and yellowness (b*), were obtained by settingthe light source to D65 and observation angle of 10°. The hue angle (H*), saturation chroma (C*), and totalcolor difference (ΔE) were calculated by equations (1), (2) and (3), respectively.

H* = arctan (b*/ a*) (1)

C* = [(a*)2+(b*)2]1/2 (2)

    (3)

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where subscript ‘0’ indicates the values of control cloudy PJ color. L*, a*, and b* are values of cloudy PJafter different treatments.

2.4. Total soluble solid and sugars pro�le measurement

Total soluble solids (TSS) were measured at ambient temperature using a refractometer (TD-45, Jinkelida,China) at 20 ℃ and expressed as °Brix (%).

According to the method of Yi et al. (2018), the sugars pro�le was analyzed by high-performance liquidchromatography (HPLC) system and an evaporative light scattering detector (1260 Series, AgilentTechnologies, USA) equipped with a column (Asahipak NH2P-50 4E, Shodex, Japan). Firstly, 10 mL cloudyPJ was mixed with 1 mL extraction solution (150 g/L k4[Fe (CN)6] and 300 g/L ZnSO4), and then the mixturewas centrifuged at 22000 g for 20 min at 4 ℃. The obtained supernatant was diluted 25 times using HPLC-grade water and �ltered through a 0.45 µm syringe �lter. The injection volume was 5 µL, and the mobilephase (75% v/v, acetonitrile/water) was used at a �ow rate of 1 mL/min at 30 ℃. Identi�cation andquanti�cation were performed based on retention times and a calibration curve of standard solutions.

2.5. pH and organic acid pro�le measurement

The pH of the cloudy PJ was determined at ambient temperature with a pH meter (FE28-Standard, MettlerToledo, Zurich).

The procedure for extracting organic acids was the same with that of sugars described in Section 2.4. Theextract was diluted 4 times with HPLC-grade water and analyzed by a reversed phase HPLC system (Agilent1200, Agilent Technologies, USA) with a DAD detector set at 210 nm. The stationary phase used was aprevail 5 µm organic aid column (250 ×4.6 mm, Avantor, USA) coupled to a Prevail C18 guard cartridge (7.5mm×4.6 mm, 5 mm particle size, Alltech Grace, Deer�eld, IL). The injection volume was 30 µL, and isocraticelution was performed using a potassium dihydrogen phosphate buffer (25 mmol/L, pH 2.5) at a �ow rateof 0.8 mL/min (25 ℃).

2.6. Identi�cation and quanti�cation of aroma compounds by HS-SPME-GC-MS

The analysis of the volatile fraction was performed using the method of Colantuonode et al. (2018) withsome modi�cations. Volatile compounds were extracted from cloudy PJ using headspace solid phasemicro-extraction (HS-SPME). A total of 5 mL sample and 1.8 g NaCl were mixed in a 10 mL screw-cappedamber glass vial with a PTFE/silicone septum seal, and 10 µL of 2-octanol was added as internal standard.The vials were incubated at 40 ℃ for 5 min under agitation at 500 rpm to equilibrate the solution andheadspace. Headspace volatiles were extracted by an SPME �ber composed of 50/30 µmdivinylbenzene/carboxen/polydimethylsiloxane (Zhenzheng, Qingdao, China). The �ber was exposed to theheadspace of the capped vial to adsorb volatile substances under agitation at 500 rpm and 40 ℃ for 40min and then inserted into the GC injection port immediately after extraction and desorbed at 250 ℃ for 5min. The GC-MS system (QP2010, Shimadzu, Japan) was equipped with an HP-5 column (30 m×0.32mm×0.25 µm, Agilent Technologies). Analyses were carried out using helium as carrier gas at a �ow rate of

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0.8 mL/min and the following program: (a) 40 ℃ for 2 min; (b) rate of 5 ℃/min from 40 to 110 ℃; (c) rateof 8 ℃/min from 110 to 160 ℃ and hold for 2 min; (d) rate of 15 ℃/min from 160 to 250 ℃ and hold for2 min. In full scan acquisition mode, the mass spectrometer was operated with a scan range of m/z 30–300. The MS ion source temperatures were 200 ℃. The volatile content of each sample was determined intriplicate. Retention Index (RI) was calculated using n-alkanes (C5-C25) as the external reference under thesame operating conditions for further con�rmation. Volatile compounds were tentatively identi�edaccording to the database of the NIST 2014 library, the NIST 2014s library and the RI.

2.7. Rheological characteristics and turbidity measurements

Rheological measurements were carried out using a Modular Compact Rheometer (MCR 102, Anton Paar,Germany) �tted with a Couette-geometry sensor (concentric cylinder, Anton Paar CC27) and a four-bladedvane geometry (ST22-4V-40). Twenty milliliters of cloudy PJ were poured slowly into the cylinder, then thespindle was submerged in the sample and the apparent viscosity was measured along with a shear rateranging between 0.1 and 100 s− 1 at 25 ℃.

Turbidity, expressed in nephelometric turbidity units (NTU), served as a measure of juice clarity andconcentration of the suspended particles and colloids presented in the juice. A Hach Model TL2300turbidimeter (Hach, Loveland, USA) was used and calibrated with formazin standards (0-4500 NTU). Fiftymilliliters sample was taken in a clean vial and placed into the measurement cell in the turbidimeter.

2.8. In vitro-simulated gastrointestinal digestion

The in vitro-simulated gastrointestinal digestion process, including three main consecutive steps - oral,gastric, and intestinal digestion, was performed according to the procedure described by Minekus et al.(2014), with a slight modi�cation.

For the oral digestion, each sample (20 mL, in triplicate) was mixed with 14 mL of simulated salivary �uidelectrolyte stock solution (SSFESS), then 3.9 mL of deionized water, 0.1 mL of 0.3 M CaCl2, and 2 mL of α-

amylase (1500 U∙mL− 1) made up in SSFESS were added. The samples were incubated in the dark withcontinuous shaking for 2 min at 37 ℃. Afterwards, 20 mL of the oral bolus was mixed with 12.8 mL ofsimulated gastric �uid electrolyte stock solution (SGFESS), 10 µL of 0.3 M CaCl2, 3.2 mL of porcine pepsin

(25000 U∙mL− 1), and pH was adjusted to 3.0 using 0.5 mL of 1 M HCl and 1.29 mL in SGFESS for gastricdigestion. The samples were incubated in the dark with continuous shaking for 120 min at 37 ℃. Forsimulation of intestinal digestion, 20 mL gastric chyme was mixed with 11 mL of simulated intestinal �uidelectrolyte stock solution (SIFESS), 0.04 mL of 0.3 M CaCl2, 2.5 mL of freshly prepared bile (160 mM), 5 mL

of pancreatin (800 U∙mL− 1) in SIFESS. Then, 0.15 mL of 1 M NaOH and 1.31 mL of deionized water wereadded to reach pH 7.0. The samples were incubated in the dark with continuous shaking for 120 min at 37℃. After the digestion process, the samples were centrifuged at 10000 g and 4 ℃ for 20 min, and thesupernatants were collected and frozen at -80 ℃ for further analyses.

The supernatant of each sample with or without digestion was recovered and puri�ed by solid-phaseextraction (SPE) column using HF Bond Elut C18 cartridges (Agilent Technologies, Santa Clara, CA).

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Cartridges were pre-conditioned with 18 mL of MeOH, MeOH-H2O (1:1 v/v), and distilled water. To removesalts or sugar contamination in the samples, all columns loaded with samples were �rstly washed with 1%(v/v) formic acid in water. The sample was subsequently eluted with 1% (v/v) formic acid in 80% (v/v)acetonitrile. Finally, the eluent was collected and stored at − 80 ℃ for further analysis.

The bioaccessibility was calculated using Eq. (4) and explained as percentage.

Bioaccessibility (%) = (BCdigested/BCnon−digested) *100 (4)where BCdigested was the amount of bioactive compounds (TPC, TFC, �avonoids, phenolic acids andtannins) obtained in the supernatants of the �nal digestion and BCnon−digested was the amount of bioactivecompounds in non-digested cloudy PJ.

2.8.1. Total phenols content (TPC) and total �avonoids content (TFC) measurements

According to method of Wang et al., (2019) with modi�cations, 5 mL eluent was dissolved in 20 mLmethanol, and treated with ultrasonic for 20 min, then centrifuged at 6000 g for 5 min at 4 ℃. The �nalmethanolic extract was collected for TPC and TFC analysis.

TPC was determined by the Folin-Ciocalteu method (Tezcan et al., 2009) with some modi�cations. Tenmicroliters of the methanolic extract was mixed with 500 µL of Folin-Ciocalteu reagent (previously diluted10-fold with distilled water), and 450 µL of 7.5% of sodium carbonate was added 5 min later. The mixturewas allowed to set for 1 h in the dark at room temperature, then measured by a microplate reader (EPOCH2,BioTek 199 Instrument Co., Ltd, Vinooski, USA) at 760 nm. Results were expressed as mg of gallic acidequivalents (GAE) / L cloudy PJ.

TFC was measured using a colorimetric assay (Elena et al., 2007). Brie�y, at time zero, 100 µL ofmethanolic extract were added with 15 µL of 5% NaNO2. Then, 15 µL of 5% AlCl3 was added 5 min later. At11 min, 100 µL of 1 M NaOH was added and mixed. The absorbance of each reaction mixture wasmeasured at 510 nm by a microplate reader (EPOCH2, BioTek Instrument 208 Co., Ltd, Vinooski, USA). Theresults are expressed as rutin equivalents (RE) / L cloudy PJ.

2.8.2. Identi�cation and quanti�cation of phenolic acids, �avonoids, and tannins by UHPLC-ESI-HRMS/MS

The methanolic extract were analyzed by a Thermo Fisher Ultimate 3000 UHPLC System equipped with a Q-Exactive Orbitrap mass spectrometer (Thermo Fisher Scienti�c, Bremen, Germany). A rapid resolution HDC18 column (2.1 mm ×100 mm ×1.8 µm, Agilent, USA) was used for the separation of phenolics with thebinary mobile phase. The mobile phase consisted of 0.1% (v/v) formic acid in water (eluent A) and 0.1%(v/v) formic acid in acetonitrile (eluent B). The �ow rate was 0.2 mL/min, and the injection volume for allsamples was 1 µL. The gradient elution was applied as: 0–3 min, 95% A isocratic; 3–15 min, linear gradientfrom 95–80% A; 15–25 min, linear gradient from 80–70% A; 25–45 min, linear gradient from 70–35% A;45–48 min, linear gradient from 35–95% A, then the column was re-equilibrated for 5 min at 95% A. The MSconditions are listed as follows: positive and negative ion scanning mode, scanning range: m/z 100–1500;spray voltage, 3.3 kV; capillary temperature, 320 ℃; heater temperature, 350 ℃; auxiliary gas �ow, 8.0

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L/min; sheath gas �ow rate, 32.0 L/min; sweep gas, 4.0 L/min; and S-lens RF level, 50%. Compounds wereidenti�ed by comparing their MS data with those of the corresponding standards, database, or thosereported in the references. The identi�ed phenolics were quanti�ed or semi-quanti�ed based on theircorresponding standard (or at least with similar aglycone) calibration.

2.9. Sensory evaluation

The sensory pro�les of cloudy PJ were evaluated by thirty trained panelists. According to the methodreported by Laboissière et al. (2007) with some modi�cations, standard QDA (quantitative descriptiveanalysis) procedure was used to investigate the sensory characteristics of �ve kinds of cloudy PJ. Thesensory evaluation was conducted in a room at constant temperature and panel members were seated inindividually partitioned booths. After reaching consensus on the standard sample (untreated cloudy PJ), aset of coded capped glass bottles containing 5 mL juices was presented to the panelists for sni�ng andsipping. During the evaluation process, the assessors were asked to rate each descriptor the intensity from1 (no attribute) to 9 (very intense). The standard score sheet for the sensory evaluation was presented inSupplementary Table S1. The average score for each descriptive attribute was plotted in a web diagram.

2.10. Statistical analysis

All the data were collected in triplicates and presented as means value ± SD (standard deviation). Theresults in each experimental group were compared by one-way analysis of variance (ANOVA), and meandifference was determined by Tukey’s multiple comparison test at p < 0.05 using SPSS 26 software. UHPLC-ESI-HR-MSn data were analyzed by the Xcalibur 3.0 software (Thermo Fisher Scienti�c, USA). Variableimportance in projection (VIP) coe�cients were calculated to select discriminant compounds, and thosevalues with the largest |VIP| > 1 were selected for principal component analysis (PCA).

Results And Discussion3.1. Color

Color is one of the main quality attributes of fruit-based products that strongly affects the choice andacceptability of consumers on food, and it is also an important indicator of showing the changes in colorrelated phytochemicals during food processing (Lou, et al., 2022b). Effects of HHP, PT and HTSTprocessing on the color were shown in Table 1. PT and HTST treated juices showed signi�cant differencesfrom the untreated juice with higher lightness (L*) values and redness (a*) values, indicating that the PTand HTST treated samples became darker. Compared to untreated cloudy PJ, HPP showed no in�uence onL*, a*, and b* values of cloudy PJ. The chroma value (C*) indicates the degree of color saturation. ΔE valuesigni�es the color alteration of samples is signi�cant or not (Chen et al., 2013), and it could be a noticeabledifference in numerous situations when its value was greater than or equal to 3.0 (Wu et al., 2021b). Therewere no signi�cant changes obtained in ΔE value after both HPP1 and HPP2 (ΔE = 0.58, 0.54), while thosevalues of the PT and HTST treated cloudy PJ signi�cantly increased to 5.19 and 6.62, respectively,indicating that signi�cant visual differences were observed between thermal treated and untreated cloudyPJ. Similar changes were observed for the C* values. The result indicated that HPP could better retain

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cloudy PJ’s original color, which was also found in pineapple fruit juice, cloudy hawthorn berry juice, andcloudy apple juice (Lou et al., 2022b; Wibowo et al., 2019; Wu et al., 2021b). Oey et al., (2008) reported thatHPP could preserve color due to its minimal effect on the covalent bonds of low molar-mass compoundssuch as color compounds. Anthocyanins were mainly responsible for the bright red color of PJ (Chen et al.,2013). Generally, total monomeric anthocyanins in PJ during processing and storage were very unstable.Chen et al., (2013) reported that HTST (110 ℃/8.6 s) resulted in a 13.2% reduction in total monomericanthocyanins content in PJ, while HPP (400 MPa/5 min) signi�cantly reduced the its degradation.

3.2. TSS, pH, sugars, and organic acids

The overall taste of cloudy PJ is provided by a good balance of sugar and organic acids. As shown inTable 2, all processing technologies including HHP1, HPP2, PT, and HTST, did not cause signi�cantdifferences in pH and TSS. Only fructose and glucose were detected in cloudy PJ, with initial contents of85.9 and 79.7 g/L, respectively. Mphahlele et al., (2016) also reported that there were only fructose andglucose were found in PJ with contents of 2.52 ± 0.02 ~ 17.6 ± 0.07 g/100 mL and 2.42 ± 0.02 ~ 13.8 ± 0.01g/100 mL, respectively. Consistent with the most studies, little effects of the processing technologies onindividual sugars could be observed (Zhang et al., 2016). Acidity in cloudy PJ is mainly attributed to malicacid, lactic acid and citric acid, accounting for 29.7%, 20.4% and 14.3% in untreated cloudy PJ, respectively.Overall, all processing technologies had no signi�cant effect on the organic acids pro�le as compared tountreated cloudy PJ, which was similar with previous studies (Wibowo et al., 2019). In general, treatmentsdid not change the sugars and organic acids contents of cloudy PJ, and therefore they could preserve thetaste of cloudy PJ regarding to sweet and sour taste.

3.3. Rheological characteristics and Turbidity

The rheological behavior and turbidity of juice impact much of consumer experience in relation to particlesize and mouthfeel (Salehi, 2020). As shown in Fig. 1a, the viscosities of all cloudy PJ samples weresigni�cantly increased with increasing shear rate, which exhibited the non-Newtonian characteristics of apseudoplastic nature �uid. Similar �ow behavior was observed in carrot juice and PJ in previous studies(Bodbodak et al.,2013; Zhang et al., 2016). Bodbodak et al., (2013) found that PJ samples under differenttemperatures (10–70℃) and concentrations (12–52 °Brix) all showed the shear thickening nature, and theHerschel-Bulkley model was selected to describe the rheological behavior with �ow behavior index (n)ranged from 0.97 to 1.45. The �ow characteristics of cloudy PJ were not changed by all processingtechnologies, indicating that the rheological behavior of cloudy PJ was stable towards both thermal andnon-thermal treatments. Zhang et al., (2016) reported that HPP had no signi�cant effects on the viscosity ofcarrot juice compared to the control, while HTST (110 ℃/8.6 s) signi�cantly decreased the viscosity by21.1% possibly attributed to the degradation of pectin in the cell walls. However, Lou et al., (2022b) reportedthat signi�cant increases of 70.2%-926.9% in viscosity of cloudy hawthorn berry juice were achieved byHPP (300/600 MPa, for 2/6 min) in comparison with the control sample with the initial viscosity of 53.33 ± 3.89 mPa·s. The different changes in rheological behavior found for different juice were possibly dependenton their varieties, stage of ripening, concentration and temperature variation (Salehi, 2020).

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Figure 1b showed that HPP did not change the turbidity signi�cantly. However, the value was signi�cantlyincreased by 79.4% and 110.4% after PT and HTST, respectively. Similar results were reported that thermalsterilization signi�cantly increased the turbidity of grapefruit juice (80 ℃/11 s) and apple juice (98 ℃/30 s)(Igual et al., 2014; Tian et al., 2018). It could be due to the increasing concentration of colloidal pectin injuice because thermal sterilization greatly ruptured cell structure and allowed pectin to leak out (Tian et al.,2018). Igual et al., (2014) also reported the increase of turbidity in grapefruit juice after heat treatment dueto the combination of pectin and protein.

3.4. Volatile compounds

Aroma is one of the sensory properties affecting quality perception and consumer acceptance of fruit juiceto a large extent. Pomegranate cultivars are considered to have a slight aromatic intensity, and their volatilecompounds are easy to lose during juice processing (Beaulieu et al., 2015). A total of 26 volatilecompounds were identi�ed in control cloudy PJ as listed in Table 3, including 12 alcohols ((Z)-3-Hexen-1-ol,(E)-3-Hexen-1-ol, 1-Hexanol, 2-Ethyl-1-hexanol, Linalool, 1-Octanol, 1-Nonanol, 4-Terpineol, a-Terpineol, 1-Dodecanol, 1-Tetradecanol, Farnesol), 5 aldehydes (Nonanal, Decanal, Undecanal, Dodecanal,Tetradecanal), 3 Ketones (4-methyl-2-Heptanone, 6-methyl-5-Hepten-2-one, 6,10-dimethyl-5,9-Undecadien-2-one), 1 terpenes (D-Limonene), and 5 acids (Nonanoic acid, Decanoic acid, Dodecanoic acid, Tetradecanoicacid, Pentadecanoic acid). Most of these compounds mainly imparted green, citrus, orange, lemon, �oral,and fruity notes to fresh cloudy PJ. The top three compounds in cloudy PJ were 1-Nonanol, (E)-3-Hexen-1-oland a-Terpineol with the concentrations of 121.8 µg/L, 37 µg/L and 6 µg/L, respectively. The study onSpanish pomegranates showed that hexanal, limonene, (E)-3-hexenal, and (E)-3-Hexen-1-ol were the mostabundant compounds in pomegranate (Melgarejo et al., 2011). Moreover, the volatile compounds ofpomegranates from two different regions of Mexico were analyzed and the most abundant compoundswere hexanol, (Z)-3-exen-1-ol, hexanal, 4-Terpineol, α-Terpineol, and 2-Ethyl-1-hexanol (Hernández Escarcegaet al., 2020). Most of the volatile compounds identi�ed in this study have been reported in PJ in previousstudies (Andreu-Sevilla et al., 2013; Beaulieu et al., 2015). But to our knowledge, there were 8 compoundsdetected in this study that were not reported in previous studies yet, namely, Dodecanal, Tetradecanal, 1-Tetradecanol, 4-methyl-2-Heptanone, Decanoic acid, Dodecanoic acid, Tetradecanoic acid, Pentadecanoicacid. Several factors might affect the characteristics of volatile compounds of cloudy PJ, such as genotypeof pomegranate, planting environment, maturity, and aroma extraction (Mayuoni-kirshinbaum et al., 2012;Mphahlele et al., 2016; Tapia-Campos et al., 2016).

To evaluate the homogeneous clusters of samples, the original concentrations of volatile compounds were“Log transformation” and “Pareto scaling”. As shown in Fig. 2, cloudy PJ samples after different processingtechnologies were clearly clustered into two groups. Cluster A consisted of 16 chemical markers, whichincluded alcohols, aldehydes, ketones, and acids. Cluster B consisted of 10 chemical markers, and 6 ofwhich were alcohols. It was obvious that thermal sterilization signi�cantly decreased the concentrations ofvolatile compounds in cluster B as compared with the control and HPP treated sample. The juices treatedby HHP at 450 MPa and 550 MPa were closer to fresh cloudy PJ according to the relationship in clusteranalysis. It revealed that HPP better preserved the volatile compounds of cloudy PJ than HTST and PT, dueto that the structures of these small-molecule �avor compounds in the fresh fruits and vegetables were not

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directly affected by HHP processing. Similar result was found by Zhang et al., (2019) that the major aroma-active compounds detected in Keitt mango juices after HHP at 400 MPa and 200 MPa were closer to freshsample according to Hierarchical cluster analysis. Bi et al., (2020) also found that the effects of HHP at 500MPa for 10 min on the contents of esters in orange juice, such as Ethyl butanoate, Ethyl hexanoate, wereless than those of pasteurization at 86 ℃ for 10 min. Chen et al., (2016) reported that the monoterpenoidcontent, which was the main volatile aroma compound in ginger juice, was not signi�cantly in�uenced byHHP treatment (500 MPa/10 min), but its content decreased signi�cantly by 2.27% after HTST (110°C/8.6s). Noteworthily, Linalool with a woody aroma was only identi�ed in both PT and HTST treated cloudy PJ,suggested that the compound was speci�c to thermally treated cloudy PJ and was possibly formed byreactions during thermal sterilization. It was consistent with a previous study that thermally treated orangejuice showed a signi�cantly greater content of linalool than the fresh sample (Bazemore et al., 2003).

It was seen from Fig. 3 that �ve kinds of volatiles compounds were presented in cloudy PJ, among whichalcohols and acids were the main two kinds of compounds, accounted for 92.1% and 4.8% in control cloudyPJ, respectively. It was found that the proportion of alcohols in total volatiles compounds was signi�cantlydecreased after thermal sterilization, especially a decrease of 32.7% detected in the HTST treated sample.For example, 3-Hexen-1-ol, 1-Hexanol, and 2-Ethyl-1-hexanol in cloudy PJ were decreased about 3.8 ug/L,43.8 ug/L and 0.4 ug/L after HTST, respectively. Alcohols showed more sensitivity to thermal sterilizationthan other kinds of volatiles compounds. Previous studies also showed that the proportion of (Z)-nonel-3-olin Hami lemon juice was deceased from 27.51–7.44% after thermal sterilization (90 ℃, 1 min) (Chen et al.,2009). In addition, both 1-Hexanol and (E)-2-Hexenol in cloudy kiwifruit juice were decreased sharply afterthermal sterilization at 80 ℃ for 20 min (Zhao et al., 2021). Moreover, the proportion of acids in cloudy PJwas signi�cantly increased by 33.6% and 182.8% after PT and HTST except for Nonanoic acid, respectively.The pentadecanoic acid showed the largest increase in thermally treated cloudy PJ, which was increased by131.1% and 228.8% after PT and HTST, respectively. This compound was recognized as the aroma of waxy,possibly contributing to the off-odour of cloudy PJ induced by thermal sterilization. Thus, these alcoholsand acids were potential to be strong markers that distinguished HPP and thermal treated cloudy PJ.

3.5. Sensory pro�le

Sensory pro�ling of cloudy PJ showed that there were differences among PT, HTST, HPP and the control(Fig. 4). For all samples, similar scores were obtained for overall taste, sweet, fermented and cooked aroma,while PT and HTST treated juice had lower brightness, redness and clarity than the other juices. As foroverall aroma pro�le, HPP treated cloudy PJ had higher scores in natural cloudy PJ aroma than thosevalues of thermal treated samples, con�rming that HPP better preserved the �avor of cloudy PJ. Especially,an acid �avor was perceived for the PT and HTST treated cloudy PJ, which was in consistent with theprevious result of acids increases. Increasing sourness and cooked �avor for thermally processed (72 ℃ for15 s) apple juice was also reported by Lee et al., (2017). In contrast, HPP treated juices were perceived asfresh, natural, sweet and balanced �avor. Results of sensory comparisons indicated that HPP retainedbetter sensory properties of cloudy PJ, which were also supported by the results on physicochemical andsensory-related chemical indicators in this study.

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3.6. Bioaccessibility of total phenols content (TPC), total �avonoids content (TFC), and �avonoids, phenolicacids and tannins

The changes of TPC and TFC, �avonoids, phenolic acids and tannins in control, HHP1, HPP2, PT, and HTSTtreated cloudy PJ before (undigested) and after simulated oral, gastric, and intestinal conditions wereshown in Fig. 5. The TPC and TFC in control cloudy PJ were 684.2 ± 3.1 mg/L and 440.6 ± 5.2 mg/L,respectively. Compared with the undigested control group, the TPC and TFC in PT treated sample weredecreased by 7.5% and 13.9%, respectively, and those values in HTST treated sample were furtherdecreased by 10.4% and 20.5%, respectively. The decrease of total phenolics by thermal sterilization wasdue to the oxidative degradation of thermally unstable phenolics (Nayak et al.,2015). As expected, bothHPP1 and HPP2 showed no signi�cant in�uence on TPC and TFC of cloudy PJ.

TPC in the control cloudy PJ showed sequential decreases of 28.0%, 19.3% and 37.7% after oral, gastric andintestinal digestion, respectively, and corresponding decreases of 35.6%, 27.1% and 11.4% were found inTFC, respectively. The result indicated that the maximum degradation of TPC and TFC occurred in theintestinal and oral digestion, respectively. Phenolics were easily degraded by the presence of α-amylase andother digestive enzymes during oral digestion, and highly sensitive to gastrointestinal pH variations,resulting in a considerable decrease in their quantity throughout the prolonged digestion process (Spínola etal., 2018). After gastric digestion, the TPC of HPP treated samples was consistent or slightly increasedcompared to the control, while the thermal treated samples had lower levels. The TFC of HPP1 and HPP2treated samples were 11.4% and 20.2% higher than control group during the gastric digestion phase,respectively. Both TPC and TFC of HPP treated samples after intestinal digestion were higher than thosevalues of PT and HTST treated samples. Generally, a slight but signi�cant advantage for bioaccessibility ofTPC and TFC in cloudy PJ was found for HPP treated samples as compared to thermal treated samples.Food processing had varied impact on the bioaccessibility of phenolic substances in samples, mainlydepending on the food matric and the type and intensity of the treatment. Ozkan et al., (2022) reported thatincreasing trends were also obtained in TPC and TFC bioaccessibility in HPP (600 MPa/5 min) treatedcranberrybush puree in comparison to the untreated sample, since HPP might accelerate the liberation ofbioactive from the food matrix under the effect of the digestive enzymes action, temperature and pHconditions. Rodríguez-Roque et al., (2015) demonstrated that thermal sterilization (90 ℃/60 s) signi�cantlydecreased the bioaccessibility of TFC in mixed fruit juice beverage (orange, kiwi, pineapple and mango) by2.7% as compared with untreated sample, which could be explained by that high temperature resulted in theloss of thermo-labile phenols (Wang et al., 2014).

Twenty-two phenolics were identi�ed in cloudy PJ (Table 4), including �fteen �avonoids (Cyanidin-3-O-glucoside, Cyanidin-3,5-O-diglucoside, Delphinidin-3-O-diglucoside, Delphinidin-3,5-O-diglucoside,Afzelechin-delphinidin-3-O-hexosid, Pelargonidin-3-O-glucoside, Pelargonidin-3,5-O-diglucoside, Rutin, (+)-Catechin, Epicatechin, Phlorizin, Quercetin, Quercetin-3-O-rhamnoside, Quercetin-3-O-glucoside, Kaempferol,Kaempferol hexoside), �ve phenolic acids (Caffeic acid, Caffeic acid hexose, Gallic acid, Ferulic acidhexose, Protocatechuic acid 4-O-β-hexoside), and 2 tannins (Ellagic acid and Ellagic acid hexoside). Thehighest content of phenolics in cloudy PJ was �avonoids, accounting for 66.5%, followed by phenolic acid(32.8%) and tannin (0.7%).

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In comparison with the initial content of �avonoids in control cloudy PJ, its content sequentially decreasedby 37%, 24%, and 38% in the oral, gastric, and intestinal phase, respectively. Anthocyanins were the maincomponent of �avonoids in cloudy PJ, accounting for 91.7% of them. During gastric digestion process, thedecrease of �avonoids was less since they could maintain the natural structural form under a lower pHvalue around 1.5–3.5 (Castañeda-Ovando et al., 2009). Signi�cantly higher decreases of �avonoids in oraland intestinal digestion mainly due to its extremely instability in neutral and weakly alkaline environments(Pinto et al., 2017). It was reported that �avonoids were completely converted to lower molecular weightmolecules such as phenolic acids and catechol in the intestinal phase (pH 6.7–7.4) (Braga et al., 2018). Thesame phenomenon was found for the in vitro simulated digestion of apple juice, which the phloridzin andhesperidin content remained unchanged after gastric digestion but decreased by 30.1% and 26.3% afterintestinal digestion, respectively. (He et al., 2016). As compare to undigested control sample, the content of�avonoids was signi�cantly reduced by 16.2% and 50.2% after PT and HTST, respectively, while HPP2slightly increased its content by 3.7%. However, there was no signi�cant different in �avonoids contentsafter intestinal digestion among different treatments (HHP, PT and HTST), and their bioaccessibility were inthe range of 1.5%-3.5%. That was to say, HPP could retain more �avonoids content prior to digestion, butthe �nal bioaccessibility of �avonoids did not differ between treatments.

The phenolic acids content in cloudy PJ was signi�cantly reduced by 33.4% and 70.2% after oral andintestinal digestion, respectively. Overall, different technologies showed no signi�cant difference on theinitial content and bioaccessibility of phenolic acids during entire digest process. Tannins showed a similarchange during digestion process, but signi�cantly higher contents were found for untreated sample afterintestinal digestion with the �nal bioaccessibility of 9.0%. In addition, the caffeic acid and protocatechuicacid 4-O-β -hexose were not detected at the end of the intestinal stage. It was possibly attributed to that thelight alkaline condition could activate the hydrophobic interactions between tannins and protein, andresulted in precipitation (Wojtunik-Kulesza et al., 2020).

Among the three kinds of phenolics evaluated, after oral-gastric-intestinal digestion, the �nalbioaccessibility of �avonoids, phenolic acids and tannins was 1.50%-2.03%, 4.43%-6.34% and 4.07%-8.98%,respectively. Additionally, HPP resulted in the largest preservation of phenolics before digestion, however,the �nal bioaccessibility after the entire oral-gastro-intestinal in-vitro digestion was not signi�cantlyaffected by different treatments.

3.7. Principal Component Analysis (PCA)

As shown in Fig. 6, all samples were basically clustered according to the processing method. PC1 explained62.1% variation, whereas PC2 accommodated 18.0% variability. The results showed that PC1 allowed todifferentiate cloudy PJ subjected to thermal sterilization from control and HPP treated sample. Ascompared to control and HPP treated sample, the thermal sterilization treated cloudy PJ was characterizedby the highest values of turbidity, color (ΔE and H*), TSS and the lowest values of TPC, TFC, alpha-Terpineol, (E)-hex-3-en-1-ol and 1-Hexanol. This result suggested that HPP could maintain the stability ofalcohols and phenolics. On the other hand, PC2 allowed the discrimination of HTST treated sample from PT

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treated one. The markers that differentiated PT and HTST treated samples were that the HTST resultedhigher values of turbidity, TSS, H*, ΔE and the lower values of glucose and fructose.

ConclusionIn this study, detailed analysis of HPP, PT, and HTST treated cloudy PJ was compared. HPP had nosigni�cant in�uence on physicochemical properties of cloudy PJ, while apparent increases of color andturbidity were found in PT and HTST treated samples. Twenty-six volatile compounds and twenty-twophenolics were identi�ed in control cloudy PJ. The volatile compounds of HPP treated cloudy PJ was closerto fresh cloudy PJ, while PT and HTST signi�cantly changed it by decreasing the alcohols and increasingthe acids. HPP better maintained the contents of TPC and TFC in cloudy PJ before and after digestion, whilethe bioaccessibility of �avonoids, tannins and phenolic acids in cloudy PJ after different treatments werenot altered by different treatments after digestion. This was possibly due to colorimetric assays of TPC andTFC were not selective to polyphenols and could react with metabolites of TPC and TFC during digestion,which was not unidenti�ed by HPLC. The study provided a scienti�c basis for further study of the effect ofthese technologies on metabolic pathways of phenolics in juices rich in phenolics.

DeclarationsAuthor Statement

J. Tian: Conceptualization, Formal Analysis, Investigation, Data curation, Visualization, Writing–Originaldraft preparation.

F. Y. Cheng: Methodology, Resources, Data curation.

Y. R. Yun: Methodology and editing.

J. J. Yi: Methodology, Supervision.

S. B. Cai: Methodology, Supervision.

L. Y. Zhou: Conceptualization, Resources, Supervision, Writing - review and editing.

Funding Declaration

This work was supported by the Natural Science Foundation of Yunnan Province, China (No.202001AU070029), Special Foundation for Excellent Youth Scholars of Yunnan Province, China (No. YNQR-QNRC-2018-102), the Undergraduate Research Program (No. 202010674111), and the Science andTechnology Project of Yunnan Province (No. 202102AE090050, 202202AG050009).

Data Availability

The data and material presented in this study will be made available on reasonable request.

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Con�ict of Interest Statement

The authors have no competing interests to declare that are relevant to the content of this article.

Statements and Declarations

The authors have no competing interests to declare that are relevant to the content of this article.

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TablesTable 1 Color characteristics (L*, a*, b*, total color difference (ΔE), chroma (C*) and hue angle (H*)) ofcontrol, PT (85 ℃/30 s), HTST (110 ℃/6 s), HPP1 (450 MPa/5 min), HPP2 (550 MPa/5 min) treatedcloudy pomegranate juice

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Treatment L* a* b* ΔE C* H*

Control 19.36±0.07a 10.65±0.02a 2.63±0.02a 0.00±0.00a 10.97±0.02a 13.86±0.09b

PT (85℃/30 s)

23.62±1.03b 13.49±0.52b 2.84±0.02a 5.19±0.45b 13.78±0.51b 10.90±0.50a

HTST(110℃/8.6 s)

24.43±1.10b 14.11±0.67b 4.85±0.40b 6.62±0.34c 14.92±0.76c 18.95±0.62c

HPP1(450MPa/5min)

19.94±0.22a 10.65±0.09a 2.61±0.05a 0.58±0.30a 10.97±0.10a 13.80±0.15b

HPP2(550MPa/5min)

19.74±0.41a 10.78±0.17a 2.68±0.07a 0.54±0.24a 11.10±0.15a 13.95±0.54b

Values are means and standard errors of three determinations. Values with the different letters within onecolumn are signi�cantly different (p < 0.05).

Table 2 Sugars and organic acids content of control, PT (85 ℃/30 s), HTST (110 ℃/6 s), HPP1 (450MPa/5 min), HPP2 (550 MPa/5 min) treated cloudy pomegranate juice

Values are means and standard errors of three determinations. Values with the different letters within onecolumn are signi�cantly different (p < 0.05).

Table 3 Aroma compounds identi�ed in control, PT (85 ℃/30 s), HTST (110 ℃/6 s), HHP1 (450 MPa/5min), HPP2 (550 MPa/5 min) treated cloudy pomegranate juice using headspace solid phasemicroextraction coupled with gas chromatography‐mass spectrometry (HS‐SPME‐GC‐MS)

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No. Compounds RIa Odor descriptionb Identi�cationc

  Alcohols      

1 (E)-hex-3-en-1-ol 890 green  MS, RI

2 (Z)-hex-3-en-1-ol 892 Green, grassy MS, RI

3 1-Hexanol 895 fruity, green MS, RI

4 2-Ethyl-1-hexanol 1035 citrus, fresh  MS, RI

5 1-Octanol 1076 orange, rose  MS, RI

6 Linalool 1099  rose, woody, green  MS, RI

7 1-Nonanol 1175 fresh, rose, orange, dusty  MS, RI

8 4-Terpineol 1178 musty, sweet MS, RI

9 α-Terpineol 1190 woody, �oral MS, RI

10 1-Dodecanol 1475  waxy, fatty  MS, RI

11 1-Tetradecanol 1679 fruity, waxy  MS, RI

12 Farnesol 1844 fresh, sweet  MS, RI

  Terpenes      

13 D-Limonene 1029 lemon, fruity MS, RI

  Aldehydes      

14 Nonanal 1105 waxy, orange, peel, fatty MS, RI

15 Decanal 1207 orange, peel  MS, RI

16 Undecanal 1308  fatty, fresh  MS, RI

17 Dodecanal 1409 soapy, waxy, green  MS, RI

18 Tetradecanal 1615 fatty, waxy  MS, RI

  Acids      

19 Nonanoic acid 1274 waxy, dirty, cheese MS, RI

20 Decanoic acid 1369  sour, fatty, citrus MS, RI

21 Dodecanoic acid 1562 mild, fatty MS, RI

22 Tetradecanoic acid 1764 waxy fatty soapy coconut MS, RI

23 Pentadecanoic acid 1863 waxy MS, RI

  Ketones      

24 4-methyl-2-heptanone 905 NF MS, RI

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25 6-methyl-5-hepten-2-one 1008 apple MS, RI

26 6,10-Dimethyl-5,9-undecadien-2-one 1455 fresh, rose, green, fruity MS, RI

a Calculated retention index (RI) on HP-5 column.

b Odor description were obtained from literature data (http://www.thegoodscentscompany.com). 

c Identi�cation methods: MS, mass spectrometry; RI, retention indices

“NF” = Not found.

Table 4 Phenolic metabolites detected in cloudy pomegranate juice

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Compounds MolecularFormula

RetentionTime (min)

MS(m/z)

MS/MS (m/z) IonizationModel

Gallic acid C7H6O5 2.630 169.0135 125.0234 [M-H]-

ProtocatechuicAcid

C7H6O4 5.25 153.0186 109.0283, 108.0206 [M-H]-

Caffeic acidhexose

C15H18O9 8.14, 10.15 341.0884 135.0443, 161.0238,179.0344

[M-H]-

Caffeic acid C9H8O4 11.70 179.0344 135.0442, 134.0363 [M-H]-

Ferulic acid hexose C16H20O9 12.23,13.03

355.1042 175.0396, 193.0504160.0160, 134.0365,217.0509

[M-H]-

Ferulic acid C10H10O4 16.89 193.0499 134.0361, 133.0283 [M-H]-

Rutin C27H30O1 17.26 609.1451 447.0919, 284.0399 [M-H]-

(+)-Catechin C15H14O6 10.52 289.0722 109.0283, 123.0441,25.0234

[M-H]-

Epicatechin C15H14O6 13.32 289.0722 109.0283, 123.0441 [M-H]-

Phlorizin C21H24O10 21.43 435.1289 167.0343, 273.0772 [M-H]-

Quercetin C15H10O7 25.46 301.0347 151.0029, 178.9981,107.0127, 121.0285

[M-H]-

Quercetin-3-O-rhamnoside

C20H16O12 16.52 447.0561 229.9907, 300.9981 [M-H]-

Quercetin-3-O-glucoside

C21H20O12 11.05 463.0877 300.0269, 271.0251,301.0337, 255.0301

[M-H]-

Kaempferol C15H10O6 29.90 285.0414 285.0413, 185.0606 [M-H]-

Kaempferolhexosylhexoside

C27H30O16 9.67 609.1453 285.0414, 447.0942,489.1039

[M-H]-

Ellagic acid C14H6O8 16.97 300.9990 300.9999 283.9970299.9921 302.0032

[M-H]-

Ellagic acidhexoside

C20H16O13 13.70 463.0518 300.9994 299.9918 [M-H]-

Cyanidin-3-O-glucoside

C21H21O11 12.41 449.1076 449.1090, 287.0552 [M]+

Cyanidin-3,5-O-diglucoside

C27H31O16 9.80 611.1606 449.1079, 287.0551 [M]+

Delphinidin-3-O-glucoside

C21H21O12 11.21 465.1026 303.0502 [M]+

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Delphinidin-3,5-O-diglucoside

C27H31O17 8.28 627.1557 465.1031, 303.0501,304.0534

[M]+

Afzelechin-delphinidin-3-O-hexosid

C36H33O17 9.68 737.1711 575.1188, 557.1089,439.0632, 287.0551

[M]+

Pelargonidin-3-O-glucoside

C21H21O10 13.68 433.1127 271.0602 [M]+

Pelargonidin-3,5-O-diglucoside

C27H31O15 10.94 595.1663 595.1663, 287.0551 [M]+

Figures

Figure 1

The rheological properties (a) and turbidity (b) in control, PT (85 ℃/30 s), HTST (110 ℃/6 s), HPP1 (450MPa/5 min), HPP2 (550 MPa/5 min) treated cloudy pomegranate juice

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Figure 2

Hierarchical clustering of the 26 quantitated volatile compounds in control, PT (85 ℃/30 s), HTST (110℃/6 s), HPP1 (450 MPa/5 min), HPP2 (550 MPa/5 min) treated cloudy pomegranate juice

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Figure 3

The relative composition of the major classes of volatile compounds of control, PT (85 ℃/30 s), HTST(110 ℃/6 s), HPP1 (450 MPa/5 min), HPP2 (550 MPa/5 min) treated cloudy pomegranate juice

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Figure 4

Spider plot of sensory evaluation of control, PT (85 ℃/30 s), HTST (110 ℃/6 s), HPP1 (450 MPa/5 min),HPP2 (550 MPa/5 min) treated cloudy pomegranate juice

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Figure 5

Total phenols content (TPC/ (mg GAE/L)), total �avonoids content (TFC/ (mg GAE/L)), �avonoids (mg/L),phenolic acids (mg/L), and tannins (mg/L) of cloudy pomegranate juice treated with PT (85 ℃/30 s), HTST(110 ℃/8.6 s), HPP1 (450 MPa/5 min) and HPP2 (550 MPa/5 min) before (undigested) and after to in vitrosimulated oral, gastric, and intestinal conditions

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Figure 6

Principal component analysis (PCA) plot of control, PT (85 ℃/30 s), HTST (110 ℃/6 s), HPP1 (450 MPa/5min), HPP2 (550 MPa/5 min) treated cloudy pomegranate juice

Supplementary Files

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SupplementalFiles.docx