Kristina Golling
Phenolic compounds in oak acorns (Quercus spp.) and in urine from pigs
fed with oak acorns
The present dissertation shows an overview of the phenolic compounds of Quercus rubor, Q. petraea and Q. rubra. The reason for this paper is the renaissance of acorn fattening, where pigs are fed with acorns in autumn. After slaughtering and maturation of the meat, the acorn feeding results in a unique flavour of the meat.
Consumers are willing to pay a higher price for this meat. In order to ensure this way of feeding in the stable, high standards are required for storing the acorns. Storing can be optimised by processing the acorn raw material. For this reason, untreated acorns as well as thermally dried acorns and acorn silage were investigated.
By analysing processed samples of acorn feed new knowledge was gained. The polyphenolic content after thermal drying is very low. Also, the silage production reduces the total phenolic content. In processed samples of acorn feed free phenols were detected that were not detectable in untreated acorns. So it can be concluded that the use of processed acorns will not lead to the same results in the acorn meat products that are achieved by the feeding (in stable or grazing land) untreated acorns.
For the first time different ripening phases of acorns were analysed. It was possible to show that the polyphenolic content of unripe acorns is significantly lower than in ripe or overripe acorns. In ripe acorns substances could be detected by
HLPC-MS/MS that were not detectable in unripe acorns. For example, valoneic acid dilactone was not detectable in green oak acorns.
For analysing the crucial polyphenols, different analytical methods were used and combined with each other. For the analysis by HLPC-UV a Chromolith® Performance RP 18 ec column was used. This made it possible to separate the extractions of the acorn samples and to detect several polyphenols (e.g. chlorogenic acid, gallic acid, ellagic acid) in untreated acorns. These polyphenols can also be detected in unripe and overripe acorns. Therefore, it can be concluded that these polyphenolic substances are entirely persistent during the growth of the acorns and there is no difference, whether the pigs are fed with unripe, ripe or overripe acorns. The high toxicity of unripe acorns cannot be explained clearly by the content of polyphenols.
The results of all applied analytical methods (UV and –DAD, GC-MS, HPLC-MS/MS) significantly show that the thermal drying of acorns results in the lowest total polyphenolic content and the lowest single polyphenolic content. For this reason the feeding with thermally treated acorns is not sufficient considering the polyphenolic content necessary to produce high-value meat products. The amount of fed acorns needs to be increased significantly in order to feed the same amount of polyphenols.
By feeding high amounts of acorns, the acceptance of the pigs for this feed decreases.
In the acorn silage free epicatechine and cholorogenic acid were not detectable, but high amounts of ellagic acid were detected. In the untreated acorns, beside gallic acid and ellagic acid, quercetine-galactosid dominated also. These substances are influenced in different ways by thermal treatment and fermentation. A lot of substances are oxidised and/or split during processing. The amount of gallic acid detectable by HPLC drops about 50 % and the contents of quercitine-galactosid and epicatechine decrease as well. On the other hand, the content of ellagic acid in thermally dried acorns increases fourfold, the content of detectable ellagic acid in acorn silage is fivefold higher in comparison with untreated acorns.
Therefore, the feeding of acorn silage results in an enhanced supply of especially ellagic acid, chlorogenic acid and caffeic acid. Due to fermentative processes degradation during silaging, other ingredients of untreated acorns are not detectable any more. The total polyphenolic content of acorn silage is comparable with the content of untreated acorns.
The present paper also shows that various characteristic metabolites of the polyphenols can be detected in the urine a few hours after the first feeding of acorns.
Comparatively low amounts of acorn feed are sufficient. By using the HPLC-MS/MS it was possible to show the dependence of quercetinsulfat, quercetin-3-glucoside, quercetagetin-7-O-glucoside and urolithin A-diglucuronid to the acorn fattening.
These investigations are only the basis for the analysis of the metabolism of acorn polyphenols and their influence on animals. Further analyses are absolutely necessary.
As a result of this dissertation, it has to be advised to investigate the acorns intended for feeding (differences in acorn variety, ripening and processing) considering their total polyphenolic content or even better their polyphenolic spectra in order to rule out the occurance of intolerances during the fattening period.
Further investigation of the acorn ingredients can contribute to the evaluation of tannins and polyphenols in a lot of plants and pieces of them based on several specific substances, considering whether they show beneficial and/or adverse health effects. This knowledge leads to further information that can be transferable to human nutrition and health.
8.1. Liste der verwendeten Chemikalien
8.2. Liste der verwendeten Vergleichssubstanzen Kaffeesäure 3,4-Dihydroxyzimtsäure Fluka 95,0 % C9H8O4
Morin Fluka 98,0 %
Myrecitin Fluka 98,0 %
p-Cumarsäure Fluka 98,0 % C9H8O3
Quercetin 3,3’,4’,5,7-Pentahydroxyflavon Fluka 99,0 % C15H10O7
Resorcin 1,3-Dihydroxybenzol Merck 99,0 % C6H6O2
Rutin Rutosid
Vitamin P
Quercetin-3-rutosid
Fluka 98,0 % C27H30O16
Salicylsäure 2-Hydroxybenzoesäure Fluka 98,0 % C7H6O3
Vanillin
4-Hydroxy-3-methoxybenzaldehyd
Fluka 97,0 % C8H8O3
Zimtsäure 3-Phenylacrylsäure Fluka 99,0 % C9H8O2
8.3. Geräteliste HPLC-UV
Name des Gerätes Bezugsquelle
Feinwaage Kern 770 G. Kern & Sohn GmbH, Albstadt, Deutschland Festphasensäule SiOH 6ml, Nr. 102022, Merck KGaA, Darmstadt, Deutschland UV-Detektor Knauer GmbH, Berlin, Deutschland
HPLC-Vorsäule Chromolith®-Vorsäule (5 x 4,6 mm) Merck, Darmstadt, Deutschland
HPLC-UV-Säule
Deutschland
HPLC-Mischkammer Mixing chamber 64, Knauer GmbH, Berlin, Deutschland HPLC-Probenschleife
HPLC-Pumpen Modell 64, Knauer GmbH, Berlin, Deutschland
Stabrührer Ultra Turrax, TP 18-10 Janke-Kunkel GmbH & Co. KG-IKA, Labortechnik, Staufen, Breisgau
Tischzentrifuge Hettich Universal, 1200 Andreas Hettich GmbH & Co KG, Tuttlingen, Deutschland
Zentrifuge 3200 Eppendorf, Eppendorf Vetrieb Deutschland GmbH, Wesseling-Berzdorf, Deutschland
Software Eurochrom 2000, Knauer, Berlin, Deutschland
Gaschromatograph HP 5890 Series II, Hewlett-Packard, Böblingen, Deutschland Massenspektrometer (GC-MS)
HP 5989 A mit Quadropol, Fa. Agilent, Böblingen, Deutschland GC-MS-Säule CP-Wax Kapillarsäule (60 m x 0,25 mm, 0,25 m) Fa. Varian,
Palo Alto, CA, USA
DB-5 Kapillarsäule (30 m x 0,320 mm, 1 Scientific, Köln, Deutschland
Software MS ChemStation, Fa. Agilent, Böblingen, Deutschland Massenspektrometer (HPLC-MS/MS)
Quadrupol-Tandem-Massenspektrometer mit ESI, Fa. Thermo Finnigan, San Jose, CA, USA
HPLC-MS/MS-Säule Waters Symmetry C18 (150 x 2,1 mm, 5 m), Waters Corporation, Milford, MA, USA
Software Xcallibur, Thermo Electron Corporation, USA
Anhang 8.4. Übersicht der wichtigsten Literaturstellen über Polyphenolanalysen
Anhang 8.5. Berechnungsgrundlagen für den Gesamtphenolgehalt
Extinktion der Standardlösung aus Gallussäure-Monohydrat
Gemessen am Photospektrometer, Wellenlänge 720 nm, korrigiert um den Blindwert von Wasser 5 0,0148 0,0140 0,0120 0,0128 0,0127 0,0140 0,0138 0,0134 7,2289 10 0,0294 0,0301 0,0296 0,0342 0,0345 0,0314 0,0304 0,0314 6,8012 50 0,2008 0,2307 0,2419 0,2345 0,2348 0,2183 0,2683 0,2328 8,9239 100 0,7558 0,6073 0,6150 0,6511 0,6515 0,5998 0,6007 0,6402 8,6785
Extinktion der Eichelfutterproben; der Gesamtphenolgehalt wird berechnet anhand der Extinktion der Standardlösung, 1 mg/100 mL Gallussäure-Monohydrat ergeben eine durchschnittliche Extinktion von 0,0042
Gemessen am Photospektrometer, Wellenlänge 720 nm, korrigiert um den Blindwert von Wasser
Probe Nr. 1 Nr. 2 Nr. 3 Nr. 4 Nr. 5 Nr. 6 Nr. 7 Mittelwert Standard-Abweichung
(%) Eicheln
normal 0,467 0,423 0,437 0,507 0,511 0,514 0,515 0,4819 8,1758 Eicheln
therm. getr. 3,775 3,946 3,956 3,981 3,973 3,267 3,265 3,7376 8,8226 Eichelsilage 0,975 0,873 0,886 0,978 0,979 0,978 0,982 0,9501 5,0998 Grüne
Eicheln 2,556 2,827 2,821 3,096 3,099 2,584 2,587 2,7959 8,3837 Roteicheln 0,693 0,645 0,659 0,642 0,643 0,551 0,554 0,6267 8,5342 Überreife
Eicheln 0,555 0,539 0,552 0,628 0,628 0,661 0,616 0,5969 7,9841
Anhang 8.6. Berechnung der Polarität
Das eingesetzte Fließmittel für die Dünnschichtchromatographie hat folgende Polarität:
Anhang 8.7. Kalibration der Vergleichssubstanzen
Die Kalibration der Vergleichssubstanzen erfolgte nach Festphasenextraktion (Kap.
3.3.1.2.). Sie wurde unter Verwendung der HPLC-UV durchgeführt.
Substanz Konzentration Peakfläche
Substanz Konzentration Peakfläche
Substanz Konzentration Peakfläche
Substanz Konzentration Peakfläche
Wieder-findung Wiederfindung Durchschnitt
Standard-abweichung
(ppm) (mV*min) % % %
Quercetin-glykosid 10 6,75 95,50 92,34 8,57
Quercetin-glykosid 50 33,62 95,11
Quercetin-glykosid 100 54,50 77,09
Quercetin-glykosid 100 54,50 77,09
Quercetin-glykosid 100 68,71 97,20
Quercetin-glykosid 500 342,14 96,80
Salicylsäure 10 12,63 74,13 90,76 9,98
Salicylsäure 10 16,30 95,64
Salicylsäure 50 82,83 97,20
Salicylsäure 100 167,84 98,47
Salicylsäure 100 140,99 82,72
Salicylsäure 500 821,65 96,42
Vanillinsäure 10 17,84 81,48 83,00 2,04
Vanillinsäure 50 91,61 83,66
Vanillinsäure 100 189,39 86,48
Vanillinsäure 100 183,21 83,66
Vanillinsäure 100 178,44 81,48
Vanillinsäure 500 889,15 81,21
Zimtsäure 50 21,77 90,46 91,97 2,76
Zimtsäure 100 46,38 96,36
Zimtsäure 100 43,54 90,46
Zimtsäure 100 43,88 91,17
Zimtsäure 500 214,42 89,09
Zimtsäure 1000 453,88 94,29
Anhang 8.8. Berechnungsgrundlagen für die HPLC-UV
Retentionszeiten der unbehandelten, normalen, reifen Eicheln En = normale, reife Eicheln, Lauf 1 - 6; Rt = Retentionszeit
Retentionszeiten der thermetisch getrockneten Eicheln
Eg = thermetisch getrocknete Eicheln, Lauf 1 - 6; Rt = Retentionszeit Eg 1 EG 2 Eg 3 Eg 4 Eg 5 Eg 6
Rt Rt Rt Rt Rt Rt Mittelwert Standardabweichung
(min) (min) (min) (min) (min) (min) (min) in %
Rt Rt Rt Rt Rt Rt Mittelwert Standardabweichung
(min) (min) (min) (min) (min) (min) (min) in %
Retentionszeiten der Eichelsilage
ES = Eichelsilage, Lauf 1 - 6; Rt = Retentionszeit ES 1 ES 2 ES 3 ES 4 ES 5 ES 6
Rt Rt Rt Rt Rt Rt Mittelwert Standardabweichung
(min) (min) (min) (min) (min) (min) (min) in %
Anhang 8.9. Berechnungsgrundlagen für die HPLC-UV, Flächenanteile Flächenanteile der Peaks von unbehandelten, normalen, reifen Eicheln En = normale, reife Eicheln; Rt = Retentionszeit
En Rt En 1 En 2 En 3 En 4 En 5 En 6
Flächen-anteil Stand.abw.
(min) Mittelwert In %
1 2,261 23,541 23,934 25,252 15,824 11,919 13,816 18,890 2 9,971 1,288 1,162 4,996 3,756 3,720 2,421 62,942 3 12,228 5,741 4,938 4,095 2,868 1,876 2,774 2,632 24,603
4 13,310 0,435 0,693 2,408 1,698 1,013 74,778
5 15,881 1,571 1,464 4,536 0,525 4,057 4,277 2,158 72,547
6 16,529 2,810 0,267 2,200 1,436 71,701
7 16,922 7,358 7,939 5,441 1,898 2,110 3,496 3,284 42,542
8 17,455 0,319 0,218 4,599 0,173 0,993 165,880
9 19,497 1,531 1,996 0,205 1,276 0,291 0,670 0,693 62,386 10 21,500 4,188 4,278 3,893 0,600 0,369 0,735 1,559 75,912
11 21,563 0,716 0,396 0,595 0,479 19,260
12 23,177 5,652 3,105 4,030 45,385
13 24,940 9,095 12,056 15,999 0,614 2,453 5,726 80,575 14 25,070 36,721 26,416 20,432 9,530 0,557 12,517 66,879 15 26,069 1,177 0,203 0,452 0,513 0,602 12,387 2,173 197,453 16 27,419 3,026 3,173 2,975 8,583 0,486 0,806 2,251 96,537 17 30,156 2,137 2,264 2,127 1,657 0,421 2,424 1,327 41,926 18 31,314 2,585 2,873 2,933 19,009 15,848 19,979 8,706 87,902 19 31,958 3,185 3,046 1,736 1,040 1,767 1,545 26,891
20 33,653 14,041 6,177 9,335 58,934
21 36,018 2,490 0,968 1,662 1,417 34,120
22 36,552 0,605 1,423 0,912 53,010
23 37,036 23,541 23,934 25,252 15,824 3,284 4,063 3,337 10,351
Flächenanteile der Peaks von thermetisch getrockneten Eicheln Eg = thermetisch getrocknete Eicheln; Rt = Retentionszeit
Eg Rt Eg 1 Eg 2 Eg 3 Eg 4 Eg 5 Eg 6
Flächen-anteil Stand.abw.
(min) Mittelwert In %
1 0,242 53,63 49,97 38,11 37,98 49,33 33,76 43,796 18,613 2 1,947 0,50 0,57 0,65 0,71 1,12 1,67 0,870 51,281 3 11,587 1,59 1,36 1,51 1,86 0,00 1,82 1,630 12,886 4 11,975 0,00 0,00 3,58 0,00 0,00 1,45 2,511 59,930 5 12,167 0,00 0,00 0,00 0,00 0,00 1,97 1,967
6 16,689 0,00 0,00 5,65 0,00 5,79 1,72 4,388 52,711 7 18,575 2,33 2,51 2,54 2,76 2,36 4,50 2,833 29,382 8 23,813 1,24 1,58 1,92 1,20 0,00 1,48 1,484 19,704 9 28,889 0,00 0,00 1,64 2,13 0,00 1,43 1,736 20,626 10 32,142 40,72 44,01 44,40 53,34 41,40 50,21 45,679 11,017
Flächenanteile der Peaks von Eichelsilage ES = Eichelsilage; Rt = Retentionszeit
ES Rt ES 1 ES 2 ES 3 ES 4 ES 5 ES 6
Flächen
-anteil Stand.abw,
(min) Mittelwert In %
1 2,178 32,46 29,53 22,51 21,36 19,18 19,83 8,081 68,798
2 10,861 6,92 13,23 14,52 3,01 2,73 3,555 39,369
3 12,153 5,62 4,06 4,35 1,81 2,37 3,12 1,932 56,769
4 13,720 0,70 2,96 3,21 1,53 1,26 13,362 32,020
5 14,843 17,02 16,56 18,13 9,64 9,67 9,15 1,424 61,932
6 16,083 2,07 1,96 2,17 0,54 0,39 19,209 40,723
7 18,553 14,08 10,81 12,03 23,22 28,60 26,51 2,864 28,233 8 22,436 2,65 3,58 3,96 1,68 2,79 2,54 3,069 21,922 9 26,819 3,23 3,49 3,64 3,05 1,76 3,25 7,536 51,172
10 28,597 6,28 5,70 6,34 14,37 5,00 2,514 38,573
11 32,042 2,52 2,20 2,49 1,27 4,26 2,35 14,763 63,249
Anhang 8.10. DAD-Spektren der einzelnen Eichelfutterproben
Aufgrund der gewählten Darstellung mit unterschiedlichen Absorbance-Bereichen je nach
Wellenlänge sind nicht alle Peaks darstellbar. Die in dieser Abbildung nur undeutlichen Peaks sind mit Pfeilen gekennzeichnet.
Peak 4 Peak 3
Peak 2 Peak 1
Normale Eicheln
1 4
6 7
9
13 10 11
21 17
20
23
16 14
5 2
3
8
12 15 19
18
nm->
nm->
Peak 9 Peak 5
Peak 10 Peak 8 Peak 7
Peak 6
Peak 14 Peak 13
Peak 12 Peak 11
nm->
nm->
nm->
nm->
nm->
nm->
nm->
nm->
Peak 16
Peak 17 Peak 15
Peak 18
Peak 23
Peak 20 Peak 19
Peak 22 Peak 21
nm->
nm->
nm->
nm->
nm->
nm->
nm->
nm->
Aufgrund der gewählten Darstellung mit unterschiedlichen Absorbance-Bereichen je nach Wellenlänge sind nicht alle Peaks darstellbar.
Therm. getr. Eicheln
Peak 4 Peak 3
Peak 2 Peak 1
10 4
3 2
1
8 5
7 9
nm->
nm->
nm->
nm->