Letter pubs.acs.org/JAFC
Volunteer Stratification Is More Relevant than Technological Treatment in Orange Juice Flavanone Bioavailability ABSTRACT: The effect of two technological treatments on orange juice flavanone bioavailability in humans was assessed. Processing affected flavanone solubility and particle size of the cloud. Volunteers were stratified in high, medium, and low urinary excretion capabilities. Flavanones from high-pressure homogenized juice showed better absorption than those of conventional pasteurized juice in high excretors. These differences were not observed in medium and low excretors. High flavanone excretors took advantage of the high-pressure homogenization juice attributes (smaller cloud particle size) and showed an improved absorption/excretion. Stratification of the individuals by their excretion capability is more relevant than technological treatments in terms of flavanone bioavailability. This stratification should be considered in clinical studies with citrus juices and extracts as it could explain the large interindividual variability that is often observed. KEYWORDS: bioavailability, flavanones, orange juice, technological treatments, volunteer stratification
■
tempered at 31 °C in a steam-jacketed kettle and homogenized at 150 MPa in a high-pressure homogenizer (NS3015H model, GEA Niro Soavi S.p.A., Parma, Italy). During homogenization, the juice was heated at 68 °C for 10 s, and when it reached the outlet section of the system, it was immediately cooled at 7 °C in a plate heat-exchanger (model junior, APV Ibérica S. A., Madrid, Spain). Samples were bottled also in 1 L glass jars with twist-off lids (both previously sterilized with fluent steam). Conventional pasteurization (CP) of samples was carried out using a plate heat-exchanger (model Junior, APV Ibérica S.A) fed at 1 L/min, where the juice was heated at 92 °C for 40 s and finally cooled at 7 °C, and aseptically bottled in 1 L glass jars with twist-off lids (both were previously sterilized with fluent steam). Qualitative and Quantitative Analyses of Juice Flavanones. These were carried out as reported previously by HPLC-DAD-MS.4 The different flavanones were quantified as hesperidin. Volunteer Stratification and Intervention Study. Volunteers’ (n = 18) flavanone absorption/excretion capability after the intake of orange juice had been characterized in previous studies.4 All volunteers had a consistent behavior over the past two years regarding their individual capability for flavanone excretion after orange juice consumption. High, medium, and low excretors were identified. High (flavanone excretion values >10% of the ingested flavanones), medium (excretion values between 5 and 10%), and low excretors (excretion values < 5%) were characterized. A crossover study (10 males and 8 females; 20−50 years old) was carried out as reported previously.4 The volunteers ingested 400 mL of the juices. Flavanone intake was quoted as hesperetin and naringenin equivalents. Analysis of Flavanone Metabolites in Urine. Volunteers collected 24 h urine, and samples were stored and processed as reported previously.4 Phase II metabolites of flavanones were identified and quantified by a UPLC 1290 Infinity series (Agilent Technologies) equipped with a triple-quadrupole mass spectrometer (6460 Jet Stream series, Agilent Technologies). A Poroshell 120 C18 column (100 × 3.0 mm i.d., 2.7 μm) (Agilent, Palo Alto, CA, USA) was used at room temperature, and the injected volume was 3 μL. Gradient elution was carried out using water/formic acid (99:1, v/v) and acetonitrile at a constant flow rate of 0.5 mL/min. The gradient used was [t (min), % acetonitrile v/v] (0, 1), (10, 40), (12, 90), and (15, 1). The optimum mass spectrometer parameters for detection of hesperetin and naringenin aglycones, hesperetin 7- and 3′-glucuronide,
INTRODUCTION Hydrostatic high pressure and pulsed electric fields have consolidated as alternatives to conventional citrus processing, as these mild treatments produce juices with quality characteristics similar to those of fresh juice.1 Although the impact of these novel technologies on orange juice bioactives has been studied,2 their effect on flavanone bioavailability in humans has not been studied to the same extent. Conventional processing leads to flavanone precipitation from the soluble fraction to the cloud.3 As flavanone solubility is a key factor for their bioavailability,4 there is an interest in developing new technologies that increase orange juice flavanone solubility. High-pressure homogenization with moderate heat treatment is able to reduce orange juice particle size (micrometer scale), leading to microsuspensions of the cloud that might increase flavanone bioavailability.5 The systemic health effects of citrus flavanones depend on their absorption. Hesperidin is not absorbed in the small intestine, and it reaches the colon, where it is hydrolyzed by gut microbiota to release hesperetin that is then absorbed.4,6 Depending on the gut bacterial composition of the different volunteers, they can have different levels of rhamnosidase activity and therefore different capabilities to absorb and excrete hesperetin.7,8 Due to the large interindividual variability in flavanone absorption and excretion, a previous stratification of volunteers by their absorption/excretion capability was suggested to be desirable in nutritional studies.4 The aim of this study was to evaluate the effect of the highpressure homogenization versus conventional pasteurization on citrus juice flavanone bioavailability in humans. This will be assessed by taking into account the stratification of volunteers in different flavanone absorption/excretion levels.
■
MATERIALS AND METHODS
Orange Juices. Fruits from a Citrus hybrid between mandarin and sweet orange (Citrus sinensis L. var. ‘Ortanique’) were harvested at optimal maturity index during the season 2011−2012 from an orchard located in Lliria (Valencia, Spain) and kept at 8 °C before being processed. Fresh hand-squeezed juice was prepared using a handsqueezer (Braun MPZ22, Madrid, Spain) and then was filtered using a 2 mm steel sieve. The juice was immediately consumed after preparation. High-pressure homogenized (HPH) juice was previously © 2013 American Chemical Society
Received: Revised: Accepted: Published: 24
November 3, 2013 December 15, 2013 December 17, 2013 December 17, 2013 dx.doi.org/10.1021/jf4048989 | J. Agric. Food Chem. 2014, 62, 24−27
Journal of Agricultural and Food Chemistry
Letter
Table 1. Flavanone Content of the Orange Juicesa fresh flavanone naringenin-7rutinoside-4′glucoside hesperetin-7rutinoside-3′glucoside naringenin-7rutinoside hesperetin-7rutinoside naringenin-4′methyl-7rutinoside total a
soluble
cloud
homogenized (HPH) total
soluble
cloud
pasteurized (CP) total
soluble
cloud
0.31 (0.01)b
nd
0.62 (0.02)
nd
0.51 (0.02)
0.11 (0.01)
0.51 (0.02)
nd
1.43 (0.04)
0.11 (0.01)
1.43 (0.04)
0.12 (0.01)
3.32 (0.04)
0.11 (0.01)
9.04 (0.12)
1.93 (0.02)
8.92 (0.06)
2.33 (0.04)
8.63 (0.11)
0.32 (0.01)
10.72 (0.13)
13.61 (0.14)
10.64 (0.09)
16.01 (0.06)
0.92 (0.01)
nd
1.84 (0.04)
1.21 (0.02)
1.61 (0.03)
1.54 (0.02)
13.69 (0.05)C
0.43 (0.01)C
23.54 (0.07)A
16.86 (0.05)B
23.09 (0.05)A
20.08 (0.05)A
14.12 (0.05)C
40.40 (0.05)B
total
43.17 (0.05)A
b
Values are expressed as mg/100 mL. Standard deviation (n = 3). Means followed by the same letter are not significantly different according to Duncan’s test (P < 0.05). naringenin 7- and 4′-glucuronide, and hesperetin 7- and 3′-sulfate (50 μM) were optimized, connecting directly the column to the Jet Stream. Pure standards were synthesized by Villapharma (Murcia, Spain). Source parameters were as follows: capillary voltage, −3500 V; charging potential, −500 V; nebulizer pressure, 40 (psi); auxiliary gas, heated to 275 °C and introduced at a flow rate of pure nitrogen, 9 L/min. The multiple reaction monitoring (MRM) method monitored five transitions for each analysis: hesperetin, m/z 301→ 164; hesperetin glucuronide, m/z 477→301; hesperetin diglucuronide, m/z 653→(477)→301; hesperetin sulfoglucuronide, m/z 557→ (477)→301; hesperetin sulfate, m/z 381→301, with a dwell time for each transition of 8 ms. Concentrations of hesperetin diglucuronide and hesperetin sulfoglucuronide metabolites were estimated by synthesized hesperetin glucuronide calibration curves (20, 10, 5, 2.5, 1 μM). For naringenin, metabolites were as follows: naringenin, m/z 271→113; naringenin glucuronide, m/z 447→271; naringenin diglucuronide, m/z 623→(447)→271; naringenin sulfoglucuronide, m/z 527→(447)→271; naringenin sulfate, m/z 351→271, with a dwell time for each transition of 8 ms. Concentrations of naringenin diglucuronide and naringenin sulfoglucuronide metabolites were estimated by synthesized naringenin glucuronide calibration curves (20, 10, 5, 2.5, 1 μM). Limits of quantification (LOQs) were 80, 50/80, and 40/50 nM for hesperetin, hesperetin 7- and 3′-glucuronides, and hesperetin 7- and 3′-sulfates, respectively. Limits of detection (LODs) were 30, 20/30, and 15/20 nM, respectively. In the case of naringenin metabolites, LOQs were 30 and 70/500 nM for naringenin and naringenin 7- and 4′-glucuronides, and LODs were 10 and 20/200 nM, respectively. The intraday repeatability of the UPLC-QqQ method was assessed from 10 consecutive chromatographic runs using a standard solution with 2.5 μM of every standard in MeOH/0.1% (v/v) formic acid. The interday repeatability of the method was assessed by analyzing the same standard solution for two consecutive days. The relative standard deviation (RSD) for peak area was in the range of 0.5−4.7% in the intraday test and 1.3−3.5% in the case of the interday test. Finally, an analysis of variance (ANOVA) was carried out, and means were separated by Duncan’s test (P < 0.05%).
Figure 1. Representative particle size distribution of citrus juices produced by conventional pasteurization (CP) and high-pressure homogenization (HPH).
and the lack of a cloud fraction in the fresh juice. However, flavanones were mainly in the soluble phase. After juice intake, either hesperetin 7-sulfate or 7-glucuronide was the most abundant urinary metabolites. Their isomers in the 3′-position were found in lower concentrations (between 2 and 5 times). Hesperetin sulfoglucuronide and hesperetin diglucuronide were minor metabolites. The same trend was found for naringenin metabolites. Volunteers were stratified after the intake of fresh orange juice, and high excretors showed mean excretion values of 15% of the flavanone intake; medium excretors, 9%; and low excretors, 3%, after fresh juice intake (Figure 2). Homogenization and pasteurization led to a decrease in the percentage of ingested flavanones that were excreted with regard to hand-squeezed juice (Table 2; Figure 2A). This decrease was not observed when only the soluble flavanones were considered. The total excretion percentage of fresh hand-squeezed orange juice reached mean values of 8.1 and 11.0% for hesperetin and naringenin, respectively (Table 2). Those values were in the range of previous papers.9,10 High flavanone excretors showed no significant differences in flavanone excretion (% ingested dose) after the intake of fresh
■
RESULTS AND DISCUSSION Table 1 shows that both pasteurized (CP) and homogenized (HPH) juices had similar flavanone contents with slight differences in the soluble and cloud fractions. The particle size, however, was much smaller in the homogenized juice (Figure 1). The flavanone content of fresh hand-squeezed orange juice was considerably lower than those of thermally processed juices, consistent with the milder extraction process 25
dx.doi.org/10.1021/jf4048989 | J. Agric. Food Chem. 2014, 62, 24−27
Journal of Agricultural and Food Chemistry
Letter
Table 2. Flavanone (Hesperetin and Naringenin) Excretion after the Intake of Fresh, Homogenized, and Pasteurized Citrus Juices and Stratification of Volunteers by Their Excretion Capabilities (High, Medium, and Low Excretors)a fresh
homogenized (HPH)
pasteurized (CP)
ingested dose (mg equivalents hesperetin)
18.8
51.6
56.2
ingested soluble dose (mg hesperetin)
18.2
24.2
24.0
all excretors
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
1.5 (±0.3)A 8.1 (±1.4)A 8.4 (±1.5)A
2.5 (±0.6)A 4.8 (±1.1)B 10.1 (±2.4)A
1.9 (±0.3)A 3.3 (±0.5)B 7.7 (±1.1)A
high excretorsb
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
2.8 (±0.4)Ba 14.8 (±2.0)Aa 15.3 (±2.1)Ba
6.3 (±0.8)A 12.2 (±1.6)Aa 26.0 (±3.4)Aa
3.0 (±0.5)B 5.4 (±0.8)Ba 12.6 (±1.9)Ba
medium excretorsc
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
1.7 (±0.2)Ab 9.0 (±1.3)Ab 9.3 (±1.4)Ab
2.0 (±0.6)Ab 3.9 (±1.1)Bb 8.4 (±2.3)Ab
2.1 (±0.3)Ab 3.7 (±0.6)Bb 8.8 (±1.3)Ab
low excretorsd
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
0.5 (±0.1)Ac 2.9 (±0.5)Ac 3.0 (±0.5)Ac fresh
0.7 (±0.2)Ac 1.3 (±0.4)Bc 2.9 (±0.9)Ac homogenized (HPH)
1.0 (±0.2)Ac 1.7 (±0.3)Bc 4.0 (±0.8)Ac pasteurized (CP)
7.0
21.9
22.5
ingested dose (mg equivalents naringenin)
6.8
18.3
17.9
all excretors
ingested soluble dose (mg naringenin) excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
0.8 (±0.1)B 11.0 (±1.8)A 11.4 (±1.9)A
1.7 (±0.4)A 7.7 (±1.9)B 9.3 (±2.3)A
1.5 (±0.3)A 6.7 (±1.2)B 8.5 (±1.6)A
high excretorsb
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
1.4 (±0.1)Ba 19.9 (±1.6)Aa 20.5 (±1.7)Aa
4.6 (±0.9)Aa 20.8 (±4.0)Aa 24.9 (±4.8)Aa
3.1 (±0.8)Aa 14.7 (±2.8)Aa 18.4 (±3.5)Aa
medium excretorsc
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
1.0 (±0.1)Ab 14.2 (±2.1)Ab 14.6 (±2.1)Ab
1.1 (±0.1)Ab 5.1 (±0.6)Bb 8.6 (±2.5)Bb
1.5 (±0.2)Ab 6.6 (±0.9)Bb 10.2 (±2.4)Bb
low excretorsd
excreted equivalents (mg) total excreted equivalents 24 h (%) relative % of the soluble intake
0.3 (±0.2)Bc 5.0 (±2.5)Ac 5.1 (±2.6)Ac
0.7 (±0.2)Ac 3.2 (±1.0)Ac 3.8 (±1.2)Ac
0.7 (±0.1)Ac 2.9 (±0.5)Ac 3.6 (±0.6)Ac
a Values are mean (n = 18) ± standard errors. Means followed by the same capital letter within a row are not significantly different according to Duncan’s test (P < 0.05). Means followed by the same lower case letter within a column are not significantly different according to Duncan’s test (P < 0.05). bn = 4. cn = 7. dn = 7.
hand-squeezed orange juice and that of HPH juices, whereas differences with fresh juice persisted for both medium and low excretors (Figure 2B). High excretors must have a gut microbiota very active in releasing hesperetin from hesperidin, and therefore they take advantage of the smaller cloud particle size in the HPH juice with more hesperidin available to interact with the gut bacteria than in pasteurized juice. Meanwhile, the lower levels of the appropriate microbiota to hydrolyze rutinosides in the medium and low excretors did not allow enhanced flavanone bioavailability when the flavanone accessibility was improved, and no significant differences between HPH and pasteurized juices were found (Figure 2). Although pasteurized juice showed the lowest absorption and excretion rates, high excretors were able to maximize flavanone absorption up to 49 and 57% higher than those of the low excretors for both hesperetin and naringenin, respectively (Table 2). The results show that high flavanone excretors have the appropriate microbiota and the intestinal transporters necessary to efficiently absorb and excrete flavanones after orange juice intake and can better benefit from improvements in flavanone
solubility and accessibility than low excretors who do not respond in the same way. The amounts of hesperetin glycosides present in the soluble fraction of the three juices obtained were quite similar, ranging between 9.1 mg/100 mL for fresh hand-squeezed and 12.1 for the HPH juice (Table 1). Thus, the relative urinary excretion expressed as percentage of the soluble intake was calculated. This ranged for all excretors from 7.7% for the pasteurized juice to 10.1% for the homogenized juice with no statistically significant differences among treatments (Table 2). The same trend was found for naringenin equivalents present in the soluble fraction. Therefore, the intake of similar amounts of soluble flavanones leads to similar relative urinary excretion of metabolites. However, significant differences in excretion were found when volunteers were stratified for their excretion capabilities. Thus, the differences observed for flavanone excretion after the intake of HPH and CP juices by high excretors clearly indicated that other juice characteristics, such as particle size, can also be relevant for absorption in addition 26
dx.doi.org/10.1021/jf4048989 | J. Agric. Food Chem. 2014, 62, 24−27
Journal of Agricultural and Food Chemistry
Letter
Funding
This work has been supported by Projects CSD2007-00063 (Fun-C-Food; Consolider Ingenio 2010) and Fundación Séneca (grupo de excelencia GERM 06 04486). Notes
The authors declare no competing financial interest.
■
REFERENCES
(1) Sánchez-Moreno, C.; Plaza, L.; Elez-Martínez, P.; De Ancos, B.; Martín-Belloso, O.; Cano, M. P. Impact of high pressure and pulsed electric fields on bioactive compounds and antioxidant activity of orange juice in comparison with traditional thermal processing. J. Agric. Food Chem. 2005, 53, 4403−4409. (2) Sanchez-Moreno, C.; Plaza, L.; De Ancos, B.; Cano, M. P. Effect of high-pressure processing on health-promoting attributes of freshly squeezed orange juice (Citrus sinensis L.) during chilled storage. Eur. Food Res. Technol. 2003, 216, 18−22. (3) Baker, R. A.; Cameron, R. G. Clouds of Citrus juices and juice drinks. Food Technol. 1999, 53, 64−69. (4) Vallejo, F.; Larrosa, M.; Escudero, E.; Zafrilla, M. P.; Cerdá, B.; Boza, J.; García-Conesa, M. T.; Espín, J. C.; Tomás-Barberán, F. A. The concentration and solubility of flavanones in orange beverages affect their bioavailability in humans. J. Agric. Food Chem. 2010, 58, 6516−6524. (5) Sentandreu, E.; Gurrea, M. D.; Betoret, N.; Navarro, J. L. Changes in orange juice characteristics due to homogenization and centrifugation. J. Food Eng. 2011, 105, 241−245. (6) Mullen, W.; Archeveque, M. A.; Edwards, C. A.; Matsumoto, H.; Crozier, A. Bioavailability and metabolism of orange juice flavanones in humans: impact of a full-fat yogurt. J. Agric. Food Chem. 2008, 56, 11157−11164. (7) Gonzalez-Barrio, R.; Trindade, L. M.; Manzanares, P.; De Graaff, L. H.; Tomas-Barberan, F. A.; Espin, J. C. Production of bioavailable flavonoid glucosides in fruit juices and green tea by use of fungal α-Lrhamnosidases. J. Agric. Food Chem. 2004, 52, 6136−6142. (8) Scalbert, A.; Williamson, G. Dietary intake and bioavailability of polyphenols. J. Nutr. 2000, 130, 2073S−2085S. (9) Nielsen, I. L. F.; Chee, W. S. S.; Poulsen, L.; Offord-Cavin, E.; Rasmussen, S. E.; Frederiksen, H.; Enslen, M.; Barron, D.; Horcajada, M. N.; Williamson, G. Bioavailability is improved by enzymatic modification of the citrus flavonoid hesperidin in humans: a randomized, double-blind, crossover trial. J. Nutr. 2006, 136, 404−408. (10) Borges, G.; Mullen, W.; Mullen, A.; Lean, M.; Roberts, S.; Crozier, A. Bioavailability of multiple components following acute ingestion of a polyphenol-rich juice drink. Mol. Nutr. Food Res. 2010, 54, S268−S277. (11) Stinco, C. M.; Fernández-Vázquez, R.; Escudero-Gilete, M. L.; Heredia, F. J.; Meléndez-Martínez, A. J.; Vicario, I. M. Effect of orange juice’s processing on the color, particle size, and bioaccessibility of carotenoids. J. Agric. Food Chem. 2012, 60, 1447−1455.
Figure 2. Urinary excretion of hesperetin after the intake of juices produced by different technological treatments in volunteers stratified as high, medium, and low excretors: (A) mean values for all volunteers (n = 18); (B) stratification in high (n = 4), medium (n = 7), and low (n = 7) excretors. Error bars are standard errors. Values are percentage of the total hesperetin intake excreted in urine. ∗, p < 0.05, significantly different according to Duncan’s test.
to flavanone solubility, in agreement with previous results on carotenoid bioavailability.11 These results suggest that volunteer stratification for flavanone excretion should be considered in future studies to evaluate the bioavailability and health effects of citrus flavanones.
María Tomás-Navarro† Fernando Vallejo*,† Enrique Sentandreu§ Jose L. Navarro§ Francisco A. Tomás-Barberán† †
■
Quality, Safety and Bioactivity of Plant Foods, Food Science and Technology Department, CEBAS-CSIC, Campus de Espinardo, 30100 Murcia, Spain § IATA-CSIC, Agustı ́n Escardino 7, 46980 Paterna, Valencia, Spain
AUTHOR INFORMATION
Corresponding Author
*(F.V.) E-mail:
[email protected]. Phone: +34 968 396 374. 27
dx.doi.org/10.1021/jf4048989 | J. Agric. Food Chem. 2014, 62, 24−27
