INFECCION POR Salmonella spp y Campylobacter spp EN EL CERDO

Further analysis was carried out to show a range of compounds were produced during berry development. Berry samples were initially analysed by HPLC and the total UV-absorbing compounds were separated by their retention times (RT). The main peaks were collected and identified using LC- MS (Table 4.1). These results showed most of these UV-absorbing compounds were flavonoids (Total UV-absorbing compounds will be mentioned as total flavonoids in the following contents).

Table 4.1 The identification of major flavonols in Sauvignon blanc berries using LC-MS analysis

a, compounds confirmed from reference spectral library and or chromatography.

b, high [Y0-H]- radical suggests rutinoside or mono glycoside position 7 substitution on tandem quadrupole instruments but have not confirmed for this instrument.

The major compounds were tentatively identified based on known structural fragmentation characteristics and or reference standards and literature. Major components responding well in the UV at 352 nm are highlighted in bold. Substitution has been indicated on previously published data (Downey & Rochfort 2008) for grapes and some spectral interpretation (Cuyckens & Claeys 2004; Hvattum & Ekeberg 2003). The identification of flavonols was carried out and analysed by Plant and Food Research, Canterbury, New Zealand.

HPLC RT, min UV nm m/z [M- H]-

ms2_(ms3_(ms4_{))(relative abindance) Tentative ID}

9.2 - 625 -162463(100)(-162301, 300, -120343), -324301(20)(179, 151), 505(5) Quercetin--diglucoside 13.2 - 625 -162463(100)(-162301, 300, -120343), -324301(10)(179, 151),505(0.1) Quercetin-diglucoside 13.8 236 - 257sh, ₃₅₄ 609 -308301, (179, 151, 257), 300 Quercetin-3-O-rutinoside (Rutin)a 14.6 - 593 -308285(257, 229, 267, 241, 163) Kaempferol-3-O-rutinoside a 14.8 234-254sh, ₃₅₄ 463 -162301(179, 151, 257, 273) Quercetin-3-O-glucoside a 15.7 255-234sh, ₃₅₄ 477 -176301(179, 151, 257, 273) Quercetin-3-O-glucuronide 16.6 234, 265, 348 447 284(255(227, 211, 237, 167), 256, 227), -162285, 327, 255 Kaempferol-3-O-glucoside b 16.8 - 477 314(285(270), 271, 286, 300, 243), 315, 301, _{357, 271} Isorhamnetin-3-O-glucoside 17.2 234, 265, 348 461 -176285(257, 229, 267, 241, 163) Kaempferol-3-O-glucuronide 18.4 - 489 -204285(257, 229, 267, 241, 163) Kaempferol-acetyl-glucoside 19.2 - 447 -132315, 161 Methylquercetin-pentoside 67

Flavonoid composition in whole berry during berry development

Figure 4.6 shows theseparation of major flavonols and their concentrations in whole grape berries during berry development, from -4 weeks post-veraison to harvest (6 weeks post-veraison). Flavonoid composition showed a developmental regulation in whole grape berries. At all of four developmental stages measured, quercetin-3-O-glucuronide (RT 15.7) accounted for the majority of the flavonols detected, followed by quercetin-3-O-glucoside (RT 14.8) and kaempferol-3-O-glucoside (RT 16.6). The concentration of quercetin-3-O-glucuronide peaked at -1 week post-veraison and subsequently declined to harvest, while quercetin-3-O-glucoside and kaempferol-3-O-glucoside increased throughout berry development and reached the greatest levels at harvest. For example, the relative ratio of quercetin-3-O-glucuronide to quercetin-3-O-glucoside in the Control treatment was 20:1 at -4 weeks post-veraison, decreased to be 16:1 at -1 week post-veraison and thereafter decreased to be 0.56:1 at harvest.

A significant UV-B response was also observed in flavonol concentrations during berry development. When compared with the Control treatment, leaf removal and UV exposure in the LR and ACRYLIC treatments significantly increased concentrations of flavonols at all stages of development measured. In contrast, the UV/UV-B exclusion in the POLYCARB and PETG treatments significantly reduced flavonol concentrations down to, or below (some flavonols) the control levels. The compounds produced in response to UV exposure were mainly flavonols, specifically quercetin and kaempferol glycosides. Levels of the major flavonol through berry development, quercetin-3-0-glucuronide, were significantly increased in the LR and ACRYLIC treatments. While UV/UV-B exclusion in the POLYCARB and PETG treatments inhibited accumulation of this flavonol with concentrations similar to controls. Post-veraison, a second flavonol, quercetin-3-0-glucoside, became prominent and was also significantly increased in the LR and ACRYLIC treatments, when compared to the Control and UV/UV- B exclusion treatments (POLYCARB and PETG). Other flavonols were present at much lower levels but nonetheless, were significantly induced in UV exposure treatments (LR and ACRYLIC).

Figure 4.6 The effects of leaf removal and UV radiation on flavonoid composition in Sauvignon blanc berries during berry development in the 2010 UV exclusion trial

Data shown are the average mean ± standard error of three replicates (n=3). Different letters indicate statistical significance (P<0.05) among different treatments according to One-way ANOVA and a Fisher’s LSD test at the 5% level. The treatments are: vines with leaves maintained and no screen applied (Control); leaves removed and maintained to harvest (LR); leaves removed and bunches covered with an acrylic screen (ACRYLIC); leaves removed and bunches covered with a polycarbonate screen (POLYCARB); and leaves removed and bunches covered with a PETG screen (PETG). The leaf removal and screens were applied to the vines at -5 weeks (pre- veraison). Samples were collected at (a) -4 weeks, pre-veraison, (b) -1 week, pre-veraison, (c) 3 weeks, post- veraison and (d) harvest (6 weeks post-veraison). Flavonols are shown in retentive time as separated in HPLC analysis and identified by LC-MS are quercetin-3-O-rutinoside (RT 13.8), kaempferol-3-O-rutinoside (RT 14.6), quercetin-3-O-glucoside (RT 14.8), quercetin-3-O-glucuronide (RT 15.7), kaempferol-3-O-glucoside (RT 16.6), isorhamnetin-3-O-glucoside (RT 16.8), and kaempferol-3-O-glucuronide (RT 17.2).

These results were supported by the UV exclusion trial in 2011, in which flavonol concentrations were analysed at six stages of berry development (Figure 4.7). Consistently, the flavonol with the highest level detected in grape berries at all six developmental stages was quercetin-3-O-glucuronide (RT 15.7), followed by quercetin-3-O-glucoside (RT 14.8) and kaempferol-3-O-glucoside (RT 16.6). In the Control treatment, the concentration of quercetin-3-O-glucuronide (RT 15.7) peaked around veraison (1 week post-veraison) at the level of 36 µg/g in the whole fresh berries, and then subsequently declined to 22 µg/g at harvest. While quercetin-3-O-glucoside (RT 14.8) and kaempferol-3-O-glucoside (RT 16.6) increased gradually during berry development, from very low concentrations (0.97 and 0.57 µg/g, respectively) at -3 weeks post-veraison increased to the greatest levels (30.6 and 8 µg/g, respectively) at harvest. Another compound demonstrated a similar increasing trend during berry development was kaempferol-3-O-rutinoside (RT 14.6), which was hardly to be detected in berries at a very early stage (-3 weeks post-veraison), but significantly increased and reached the level of 4.3 µg/g in the harvested berries. Additionally, when relative to the Control treatment, these flavonol concentrations were significantly higher in the leaf removal and UV-B-transmitting treatments LR and ACRYLIC at all stages of development measured. The concentration of quercetin-3-O-glucuronide at -3 weeks post-veraison were similar between the LR and ACRYLIC treatments (58 and 51 µg/g), then gradually increased to 84 and 80 µg/g at 1 week post-verasion, which were 236% and 226%, respectively, higher than that in the Control treatment. These increases became even more significant at 2 weeks post-veraison when quercetin-3-O-glucuronide were 71 and 76 µg/g in the LR and ACRYLIC treatments, which were 339% and 362%, respectively, higher than the Control treatment. Similar increases were found in quercetin- 3-O-glucoside and kaempferol-3-O-glucoside during berry development. The LR and ACRYLIC treatments had significantly higher concentrations of these flavonols when compared with the Control treatment, while the UV exclusion treatment PETG had even lower flavonol concentrations than the control levels at all stage of development measured.

Figure 4.7 The effects of leaf removal and UV radiation on flavonoid composition in Sauvignon blanc berries during berry development in the 2011 UV exclusion trial

Data shown are the average mean ± standard error of three replicates (n=3). Different letters indicate statistical significance (P<0.05) among different treatments according to One-way ANOVA and a Fisher’s LSD test at the 5% level. The treatments are: vines with leaves maintained and no screen applied (Control); leaves removed and maintained to harvest (LR); leaves removed and bunches covered with an acrylic screen (ACRYLIC); leaves removed and bunches covered with a PETG screen (PETG). The leaf removal and screens were applied to the vines at -4 weeks (pre-veraison). Samples were collected at (a) -3 weeks, pre-veraison, (b) -1 week, pre-veraison, (c) 1 week, post-veraison, (d) 2 weeks, post-veraison, (e) 4 weeks, post-veraison, (f) and harvest (6 weeks post- veraison). Flavonols are shown in retentive time as separated in HPLC analysis and identified by LC-MS are quercetin-3-O-rutinoside (RT 13.8), kaempferol-3-O-rutinoside (RT 14.6), quercetin-3-O-glucoside (RT 14.8), quercetin-3-O-glucuronide (RT 15.7), kaempferol-3-O-glucoside (RT 16.6), isorhamnetin-3-O-glucoside (RT 16.8), kaempferol-3-O-glucuronide (RT 17.2), and kaempferol-acetyl-glucoside (RT 18.4).

More supporting results came from two additional treatments in 2011. Figure 4.8 shows the changes of flavonol levels in the PETG-BV and PETG-AV treatments during grape berry development. The treatments PETG and PETG-BV had similar levels of flavonols from screen establishment to veraison. Once the PETG screen was removed at veraison and berries were exposed to UV-B radiation in the PETG-BV treatment, the concentration of flavonols showed a significant increase and this increase lasted throughout the rest of developmental stages measured (Figure 4.8a). In contrast, flavonol levels in the PETG treatment completely removed this increase and remained at relatively static levels until harvest. Additionally, flavonols remained at similar levels in the LR and PETG-AV treatments from -4 to 2 weeks post-veraison (Figure 4.8b). After 2 weeks post-veraison, the UV-B exposure was continued in the LR treatment but berries in the PETG-AV treatment were covered by a PETG screen. While the berries were continued to UV-B exposure in the LR treatment, the flavonol concentrations increased slowly from 2 weeks post-veraison to harvest. However, once the berries were covered by a PETG screen in the PETG-AV treatment, the levels of flavonols stopped this increasing pattern and significantly declined to harvest. These changes in flavonol levels were mainly reflected by changes in several major flavonols, leaded by quercetin-3-O-glucuronide (RT 15.7), quercetin-3-O-glucoside (RT 14.8) and kaempferol-3-O-glucoside (RT 16.6). Other flavonols were present at much lower levels but nonetheless, were significantly increased in the PETG-BV treatments or decreased in the PETG-AV treatments.

Figure 4.8 The effects of leaf removal and UV radiation on flavonoid composition in Sauvignon blanc berries during development in the 2011 UV exclusion trial

Data shown are the average mean ± standard error of three replicates (n=3). *Significant difference comparing the PETG treatment according to One-way ANOVA test (*P<0.05, **P<0.01). Ɨ Significant difference comparing the LR treatment according to One-way ANOVA test (Ɨ P<0.05, ƗƗ P<0.01). The treatments are: (a) leaves removed and bunches covered with a PETG screen until harvest (PETG); (b) leaves removed and bunches covered with a PETG screen from -4 weeks (pre-veraison) to veraison, then removed screen and bunches exposed until harvest (PETG-BV); (c) leaves removed and maintained to harvest (6 weeks post-veraison) (LR); and (d) leaves removed and bunches fully exposed from -4 weeks (pre-veraison) to 2 weeks post-veraison, then covered with a PETG screen until harvest (PETG-AV). The leaf removal and screens were applied to the vines at -4 weeks (pre- veraison). Flavonols are shown in retentive time as separated in HPLC analysis and identified by LC-MS are quercetin-3-O-rutinoside (RT 13.8), kaempferol-3-O-rutinoside (RT 14.6), quercetin-3-O-glucoside (RT 14.8), quercetin-3-O-glucuronide (RT 15.7), kaempferol-3-O-glucoside (RT 16.6), and kaempferol-3-O-glucuronide (RT 17.2).

Flavonoid composition in berry tissue at harvest

As shown previously, the spatial separation determined that the major significant increase of total flavonoids induced by UV-B radiation was located in the skin of berries (Figure 4.5). Further analysis of the skin flavonols by HPLC and LC-MS showed the same flavonol composition as whole berries and a response to the light environment in a similar manner. Table 4.2 and Figure 4.9 show the HPLC profile of skin flavonols at harvest. The leaf removal treatments LR and ACRYLIC had higher concentrations of flavonols in the skin, while UV/UV-B exclusion (POLYCARB and PETG treatments) significantly reduced flavonol concentrations down to, or below control levels. For example, the main flavonols present specifically in the control skins were quercetin-3-O-glucuronide (143.6 µg/g, 47.7% of total extracted flavonoids), followed by quercetin-3-O-glucoside (88.6 µg/g, 29.4%) and kaempferol-3-O-glucuronide (18.8 µg/g, 6%). After UV-B exposure, the quercetin-3-O-glucuronide increased to be 391.6 and 395.4 µg/g (273% and 275%, respectively) in the LR and ACRYLIC treatments, while quercetin-3-O-glucoside increased to be 334.7 and 356.5 µg/g (378% and 402%), respectively. A consistent UV-B response was found in the skin samples at harvest in 2010 (Appendix H).

According to the total UV absorbance, the seeds contained similar levels of total flavonoids to the skin of grape berries. However, HPLC analysis showed significantly lower flavonol concentrations were detected in the seeds compared with the berry skins (Appendix F). An unknown peak (RT 17.8) was determined to be the major flavonol (22.5% of total extracted flavonoids) in grape seeds, followed by quercetin-3-O-glucoside (RT 14.8). This unknown peak was significantly increased after leaf removal and UV-B exposure in the LR and ACRYLIC treatments (169% and 137%, respectively). There were very low levels of flavonols detected in grape pulp when compared with the skin and seeds, and the flavonoid composition of the pulp was similar to that in the skin (Appendix G). Although the concentrations of flavonols were very low in the pulp, significant increases in these major flavonols still could be detected after UV-B exposure in the LR and ACRYLIC treatments.

Table 4.2 The effects of leaf removal and UV radiation on flavonoid composition in the fresh skin, seeds and pulp of Sauvignon blanc grape berries at harvest in 2011

Data shown are the average mean ± standard error from three replicates (n=3). P-values for statistical significance comparing the different treatments according to One-way ANOVA and a Fisher’s LSD test. Different lower case superscript letters within rows indicate grouping information using a Fisher’s LSD test at the 5% level. NS, no significant difference. The treatments are: vines with canopy leaves maintained and no screen applied (Control); leaves removed and bunches exposed to sun until harvest, 6 weeks post-veraison (LR); leaves removed and bunches covered by an acrylic screen (ACRYLIC); leaves removed and bunches covered by a PETG screen (PETG). The leaf removal and screens were applied to vines at 4 weeks pre-veraison (˚Brix 4.5).

Tissue HPLC RT Flavonol

Concentration (µg/g fresh tissue)

P-value

Control LR ACRYLIC PETG

Skin 13.8 Quercetin-3-O-rutinoside 9.12b _{± 0.91 34.94}a _{± 5.78 34.4}a _{± 1.06 5.54}b _{± 0.95 ˂0.001} 14.6 Kaempferol-3-O-rutinoside 11.42b _{± 1.18 39.21}a _{± 4.53 44.48}a _{± 4.21 4.76}b _{± 1.01 ˂0.001} 14.8 Quercetin-3-O-glucoside 88.62b _{± 13.90 334.73}a _{± 16.52 356.49}a _{± 44.33 46.41}b _{± 6.75 ˂0.001} 15.7 Quercetin-3-O-glucuronide 143.56b _{± 14.23 391.57}a _{± 52.99 395.74}a _{± 13.15 85.95}b _{± 11.13 ˂0.001} 16.6 Kaempferol-3-O-glucoside 18.79b _{± 3.04 162.95}a _{± 11.74 147.74}b _{± 14.17 3.22}a _{± 1.68 ˂0.001} 16.8 Isorhamnetin-3-O-glucoside 4.13b _{± 0.78 21.21}a _{± 2.22 17.90}a _{± 2.88 3.56}b _{± 0.34 ˂0.001} 17.2 Kaempferol-3-O-glucuronide 6.84b _{± 0.73 43.84}a _{± 4.52 39.66}a _{± 0.89 1.03}b _{± 0.34 ˂0.001} Seed 13.8 Quercetin-3-O-rutinoside 1.42 ± 0.34 1.57 ± 0.34 1.23 ± 0.27 1.03 ± 0.15 NS 14.8 Quercetin-3-O-glucoside 3.37 ± 0.35 3.26 ± 0.68 3.64 ± 0.38 3.44 ± 0.23 NS 16.6 Kaempferol-3-O-glucoside 1.21 ± 0.14 0.75 ± 0.21 1.24 ± 0.32 0.91 ± 0.09 NS 17.2 Kaempferol-3-O-glucuronide 0.37 ± 0.16 0.27 ± 0.05 0.31 ± 0.05 0.29 ± 0.01 NS 17.8 Unknown peak 7.96 ± 0.70 13.41 ± 2.79 10.87 ± 1.47 6.67 ± 0.56 NS Pulp 13.8 Quercetin-3-O-rutinoside 0.02c _{± 0.001 0.19}a _{± 0.024 0.11}b _{± 0.017 0.02}c _{± 0.003 ˂0.001} 14.6 Kaempferol-3-O-rutinoside 0.03 ± 0.018 0.01 ± 0.005 0.01 ± 0.002 0.01 ± 0.002 NS 14.8 Quercetin-3-O-glucoside 0.23b _{± 0.011 1.18}a _{± 0.227 0.63}b _{± 0.135 0.30}b _{± 0.031 0.004} 15.7 Quercetin-3-O-glucuronide 0.18b _{± 0.023 0.74}a _{± 0.120 0.39}b _{± 0.078 0.16}b _{± 0.025 0.002} 16.6 Kaempferol-3-O-glucoside 0.02b _{± 0.005 0.41}a _{± 0.113 0.11}b _{± 0.024 0.02}b _{± 0.006 0.004} 75

16.8 Isorhamnetin-3-O-glucoside 0.02b _{± 0.007 0.12}a _{± 0.026 0.06}b _{± 0.013 0.03}b _{± 0.015 0.009}

17.2 Kaempferol-3-O-glucuronide 0.01b _{± 0.003 0.04}a _{± 0.006 0.01}b _{± 0.006 0.01}b _{± 0.004 0.004}

Figure 4.9 The effects of leaf removal and UV radiation on flavonoid composition in the skin of Sauvignon blanc berries at harvest in 2010 (a) and 2011 (b)

Data shown are the average mean ± standard error of three replicates (n=3). Different letters indicate statistical significance (P<0.05) among different treatments according to One-way ANOVA and a Fisher’s LSD test at the 5% level. The treatments are: vine with leaves maintained and no screen applied (Control); leaves removed and maintained to harvest (6 weeks post-veraison) (LR); leaves removed and bunches covered with an acrylic screen (ACRYLIC); leaves removed and bunches covered with a PETG screen until harvest (PETG); leaves removed and bunches covered with a polycarbonate screen until harvest (POLYCARB); leaves removed and bunches covered with a PETG screen from 4 weeks pre-veraison to veraison, then removed screen and bunches exposed until harvest (PETG-BV); and leaves removed and bunches fully exposed from 4 weeks pre-veraison to 2 weeks post- veraison, then covered with a PETG screen until harvest (PETG-AV). The leaf removal and screens were applied to the vines at 5 and 4 weeks pre-veraison in 2010 and 2011, respectively.