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1

Separation of betalains by gradient high-speed counter-current chromatography

Spórna-Kucab A., Udziela D., Sobocińska M., Szot D., Wybraniec S.

Challenges of Modern Technology

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2016

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Vol. 7, no. 1

23--28

EN

A study on separation of betalain mixture obtained from red beet juice (Beta vulgaris L.) by analytical high-speed counter-current chromatography (HSCCC) was performed. The extract was obtained by thermal treatment of acidified red beet juice for 30 min in 85 °C. The pigment mixture consisted of betanin/isobetanin as well as their decarboxy- and dehydro-derivatives. The HSCCC process was accomplished in the ‘tail to head’ mode with two polar solvent systems containing salt: BuOH-EtOH-NaClsolution-H2OH3PO4 (1300:700-1000:1300:700:2.5-5.5 (system I), 1300:200-400:1300:700:2.5-4.5 (system II); v/v/v/v/v). The retention of the stationary phase was 73% (system I) and 79% (system II). The mobile phase was pumped at 2 ml/min flow rate. HPLC-DAD-ESI-MS analyses were performed in reversed phase mode for the obtained HSCCC fractions and crude extract. The solvent systems enabled separation of betanin and decarboxy-betanins (system I and II) as well as neobetanin (system II). Additionally, some pure fractions of 17-decarboxy-betanin and 2,17-bidecarboxy-betanin were obtained in system II.

2

Research on solvent systems with tetra-n-butylammonium bromide for counter-current chromatography of betalains

Spórna-Kucab A., Wysoczańska J., Bartel A., Świergosz T., Szot D., Wybraniec S.

Challenges of Modern Technology

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2016

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Vol. 7, no. 1

12--16

EN

In this study, new two-phase solvent systems for counter-current chromatography (CCC) consisting of n-butanol and water as well as various amounts of acetic acid, acetonitrile, ethanol, acetone or ethyl acetate were tested. Additionally, tetra-n-butylammonium bromide (TBAB) was introduced into the system in the form of aqueous solutions or phosphate-citrate buffer (pH 6.7) in order to form ion-pairs with betalains. The selection of buffer pH was based on their ability to create ion pairs by tetraalkylammonium salts, with selected betalains under these conditions. In this study, it is shown that the settling time of two phases is longer with the increase of acetic acid/acetonitrile/ethanol/acetone/ethyl acetate. For selected solvent systems with high amounts of acetonitrile, ethanol and acetone two phases were not observed. The systems with acetone have the largest increase of settling time. Ethyl acetate systems were characterized by a slow settling time increase. In systems containing additionally 2% aqueous TBAB, smaller changes in settling time than in similar systems without TBAB were observed. Addition of TBAB in the buffer resulted in a prolongation of settling time. Solvent systems in which the separation between the aqueous and organic phases was visually best, were selected from among all the tested systems and the betalain partition coefficients were measured by LC-DAD-ESI-MS. The best results were observed for systems: n-butanol-water-acetic acid (2:2.5:0.75, v/v/v), n-butanol-water-acetic acid (2:2.5:1, v/v/v) and n-butanol-TBAB in water-acetonitrile (2:2.5:0.5, v/v/v).

3

Enzymatic oxidation of neobetanin monitored by liquid chromatography with mass spectrometric detection

Starzak K., Szot D., Skopińska A., Swiergosz T., Wybraniec S.

Challenges of Modern Technology

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2016

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Vol. 7, no. 1

29--31

EN

The aim of this study was monitoring of enzymatic oxidation of neobetanin, an interesting type of betalains which is a partially oxidized betacyanin. As it belongs to betalains, it is water soluble and non-toxic, but a presence of a few functional groups makes it very reactive. Oxidation reactions were performed using horseradish peroxidase followed by spectrophotometric and mass spectrometric detection (LC-DAD-ESI-MS/MS) of obtained products. Enzymatic oxidation of neobetanin leads to a formation of new decarboxy- and dehydro-derivatives. The main identified oxidation product is 2-decarboxy-2,3-dehydroneobetanin. Searching for all formed oxidation products is extremely important for elucidation of the betalains oxidation mechanism.

4

The effect of ascorbic acid supplementation on betacyanin stability in purple pitaya (Hylocereus polyrhizus) juice

Skopińska A., Szot D., Starzak K., Mizrahi Y., Wybraniec S.

Challenges of Modern Technology

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2015

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Vol. 6, no. 4

14--18

EN

Current studies on betalains are focused on searching of new plant sources of these pigments. In this light, purple pitaya (Hylocereus polyrhizus) is a valuable batalain-containing fruit. Betalains are known to be sensitive at elevated temperatures conditions. Nevertheless, addition of specific food stabilizers (EDTA, ascorbic acid, citric acid) as well as natural matrix compounds may exert stabilizing effect on maintenance of pigments. Therefore, in presented study, the stability of betalains in Hylocereus polyrhizus juices with protective addition of ascorbic acid at 85 ̊C was examined. Spectrophotometric analyses enable monitoring of reaction during heating of solutions. The hypsochromic shift of λmax to 460 nm is observed in more acidic media without ascorbic acid, suggesting the generation of new reaction products. These absorption bands in solutions with ascorbic acid are not detected, indicating on retaining of the basic structure of betalain chromophore. Generally, pigment retention is diminishing with prolonged heating and depends on pH-values of tested solutions. The addition of ascorbic acid significantly affects the betalain stability. After 60 min of heating, more than 60% of the initial pigment is retained at pH 3-4 in samples of purple pitaya with ascorbic acid. In summary, betacyanins in purple pitaya juice are shown to exhibit proper heat stability, when are stabilized with ascorbic acid. However, a protective effect of natural juice matrix and ascorbic acid is more significant at acidic pH.

5

Profiles of products generated during betacyanins reduction by 2-methylpyridine borane

Szot D., Starzak K., Skopińska A., Wybraniec S.

Challenges of Modern Technology

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2015

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Vol. 6, no. 4

19--22

EN

Betanin and neobetanin (14,15-dehydrogenated betanin), natural betacyanin pigments were subjected to a gentle reduction by 2-methylpyridine borane. During each reduction reaction, the visible spectra were collected. The results were tentatively confirmed by LC-DAD-ESI-MS. In comparison to a previous study with sodium borohydride, various types of products were obtained. Depending on pH, one or two double bonds are reduced. Interestingly, a preliminary decarboxylation of the pigments is also noticed. Betacyanins are plant red-violet pigments applied as colorants in food and pharmaceutical industry which are proven to have an antioxidant activity. They are confirmed to be beneficial for human health as free radical scavengers. Therefore, betacyanins can prevent from many diseases which would be induced by an excessive accumulation of free radicals in the human body. There are more and more reports of their pro-health properties. However, the mechanism of betacyanins oxidation remains not completely elucidated. It is postulated that the rearrangement in the chromophoric system is a significant part of this mechanism. The products obtained as a result of reduction of betanin and neobetanin can be useful in oxidation investigations. Betacyanins with partially destroyed chromophoric system by reduction of the double bonds would by perfect models for further studies on their oxidation.

6

Effect of Cu (II) Cations on 2-Decarboxy-betanin Stability in Aqueous-Organic Solutions

Skopińska A., Szot D., Starzak K., Wybraniec S.

Challenges of Modern Technology

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2015

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Vol. 6, no. 3

24--29

EN

Several important factors, such us pH, exposure to light, oxygen or temperature affect significantly the betalain stability. In particular, the heavy metal ions exert negative effect on stability of betalain colorants and accelerate pigment decomposition. Trace amounts of metal ions may be present in the food products, as well as in food packaging, leading to undesirable colour changes or even discoloration of betalainic foodstuffs. Decarboxylated betalains, such as 2-decarboxy-betanin, generated, e.g. during betalain thermal treatment, may exhibit greater stability than parent pigments. Moreover, obtained derivatives retain attractive color, making them the promising material for study of pigment stability. An effect of Cu2+ cations as the most degradative metal ions on 2-decarboxy-betanin stability was investigated in ethanolic and methanolic solutions. The increase of the concentration of copper cations has a negative impact on stability of 2-decarboxy-betanin. The cations of Cu (II) in the presence of organic solvents induce the pigment degradation, while the increase of the concentration of organic solvents enhances the pigment decomposition. The main degradation products of the 2-decarboxy-betanin are compounds possessing absorption maxima at λmax ca. 430 nm. These studies allow obtaining the information needed for proper isolation and treatment of pigments, as well as proper storage of products containing betalains.

7

Chromatographic fractionation of betacyanins from flowers of Gomphrena globosa

Szot D., Starzak K., Skopińska A., Wybraniec S.

Challenges of Modern Technology

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2015

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Vol. 6, no. 2

19--23

EN

In this study, a chromatographic fractionation of betacyanin pigments from extract of purple Gomphrena globosa petals was performed by preparative high performance liquid chromatography (prep-HPLC). The particular betacyanins in each collected fractions were tentatively identified by chromatography with optical amd mass spetrometric detection (LC-DAD-ESI-MS). Betacyanins are natural pigments, which are confirmed to have an antioxidant activity. It was reported that betacyanins can prevent civilization diseases, because of an ability of free radicals scavenging. Betacyanins are present in tissue of plants from Amaranthaceae family, e.g. in petals of Gomphrena globosa. Preparative separation of these particular pigments is difficult, because they are structurally very similar to each other and tend to coelute. However, in this study, fractions, containing a dominant amount of the principal pigments of Gomphrena globosa inflorescences, were obtained successfully. In the first fractions, two isomeric pigments assigned to gomphrenin I and isogomphrenin I were detected. The fractions of significant amounts of gomphrenin III and isogomphrenin III were obtained separately with sufficient purity. The presence of other, minor gomphrenin-type betacyanins were also confirmed as: gomphrenin II, cis-isomer of gomphrenin II and sinapoyl-gomphrenin I as well as their 15S-diastereomers. Moreover, in other fractions, various unknown pigments were detected.

8

Thermal degradation of 17-decarboxy-betanin monitored by LC-MS

Szot D., Starzak K., Skopińska A., Tuwalska D., Wybraniec S.

Challenges of Modern Technology

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2014

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Vol. 5, no. 3

32--36

EN

A thermal stability study on 17-decarboxy-betanin depending on physicochemical process conditions was conducted. 17-decarboxy-betanin is one of betacyanin, natural origin pigments applied in food and pharmaceutical industry instead of artificial colorants. There is a need for searching of new non-toxic natural food components and this is a reason of increasing interest of betacyanins. As most derivatives of betanin obtained by decarboxylation, 17-decarboxy-betanin tends to degrade in the presence of some factors such as increased temperature or other conditions of the reaction environment [1]. This subject was investigated in this study because the stability of the pigments is still a significant issue limiting their wide application. The degradation of 17-decarboxy-betanin during heating in selected solutions: water as well as aqueous solutions of ethanol 50% (v/v), methanol 50% (v/v) and acetonitrile 50% (v/v) at pH in the range 3-8 was tested. As UV-Vis spectra indicate, 17-decarboxy-betanin tends to degrade mostly at pH 3, notwithstanding a type of solution. The products of degradation were identified by LC-DAD-ESI-MS. As a result of incubation at 85°C in different solutions, various mono-, bi- and tridecarboxylated as well as dehydrogenated derivatives were obtained. The dominant product of 17-de-carboxy-betanin degradation is 2,15,17-tridecarboxy-2,3-dehydro-betanin.

9

The effect of citric acid on stabilization of betanin solutions

Skopińska A., Szot D., Tuwalska D., Starzak K., Osiadło M., Wybraniec S.

Challenges of Modern Technology

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2014

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Vol. 5, no. 2

19--24

EN

Elevated temperature is known to be the most crucial factor influencing betanin integrity during food processing and storage. Nevertheless, certain chelating agents or antioxidants, such as acetic acid, may act as stabilizers. Therefore, the effect of citric acid on the stability of betanin – basic betacyanin – was investigated during the heating experiments in water as well as 50% and 95% (v/v) aqueous-organic solutions of methanol, ethanol and acetonitrile. The presence of citric acid in tested solutions affects betanin stability, especially in the 95 % solutions, in which the stabilizing effect of this compound is significantly higher than in aqueous solutions. However, some decrease of retention in 50% methanol was also observed. The main products of betanin thermal degradation in aqueous and aqueous-organic solutions were compounds characterized by absorption bands around the wavelength at 420 nm.

10

Semi-synthesis of red beet betacyanin ethyl-esters by esterification

Tuwalska D., Starzak K., Szot D., Wybraniec S., Winterhalter P., Jerz G.

Challenges of Modern Technology

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2014

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Vol. 5, no. 3

27--31

EN

Red beet pigments recovered from Beta vulgaris L. (Chenopodiaceae), mainly consist of purple betacyanins, such as 15R/15S-betanin. Currently,this pigment class is intensively investigated in respect of stability in food systems. Quite popular is the use of red beet extracts in dairy products such as yoghurts, and ice-creams. So far, there are no issues of toxicity using these natural pigments in food – so they seem to be a very good alternative to replace synthetic dyes which had been frequently discussed for negative side-effects to human health. Betacyanins, the glycosidation products of betanidin are very polar and therefore water soluble pigments, which are biosynthetically derived from condensation of cyclo-DOPA and betalamic acid. The significant polarity of betacyanins requires the use of ion-pair reagents such as trifluoro-acetic acid (TFA) or other homologue fluorinated additives under standard separation conditions on larger scale in spiral coil countercurrent chromatography (spCCC). In our synthetic experiment, the polarity of the natural betacyanins was reduced. This omitted the use of toxic per-fluoro ion-pair reagents. The three carboxylic groups of the betanin pigment backbone were the target functional groups of the derivatisation. In semi-synthesis, fortified red beet pigment extract was esterified with water-free ethanolic hydrogen chloride solution. The reaction was carried out at ambient temperature (7 days) to yield a mixture of betacyanin mono-, di- and tri-ethyl-esters, and their epimeric forms, as well as the respective betanidin-ethyl-esters. The time course of the reaction mixture was monitored by analytical C18-HPLC with ESI-DAD-MS/MS detection. The detected molecular weights of the pigments confirmed the presence of expected products. To the best of our knowledge, the presence of betalains containing ethyl-ester groups in biological samples have not been studied before.

11

Reactions of decarboxylated betanins under influence of halogen light

Skopinska A., Szot D., Starzak K., Wybraniec S.

Challenges of Modern Technology

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2014

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Vol. 5, no. 3

37--43

EN

Betalains as natural plant pigments are very sensitive to several factors, including light, therefore, studies on stability of their derivatives which retain their attractive color, are crucial for their potential application in pharmaceutical, food or cosmetic industries. An effect of irradiation by halogen light on reactivity of main decarboxylated derivatives of betacyanins (2-decarboxy-betanin, 17-decarboxy-betanin and 2,17-decarboxy-betanin) was investigated in aqueous and organic-aqueous solutions: 50% (v/v) acetonitrile, 50% (v/v) methanol and 50% (v/v) ethanol, at pH range 3-8. The double decarboxylation of betanin (at C-2 and C-17 position of carbon) significantly increases the stability of this pigment in the acidic media. As a result of pigments reactions induced by light, some betacyanin derivatives with different decarboxylation and dehydrogenation levels were identified by chromatography with tandem mass spectrometry (LC-MS/MS) and diode array detection (LC-DAD).

12

Mass spectrometric detection of products of decarboxy-betanins UV-irradiation

Starzak K., Skopińska A., Szot D., Wybraniec S.

Challenges of Modern Technology

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2014

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Vol. 5, no. 3

22--26

EN

Betalains are a group of natural plant pigments, responsible for red-violet colour of numerous flowers, fruits and roots. They commonly occur in order of Caryophyllales (Caryophyllales). Recent studies revealed great health promoting properties of these pigments, but their natural origin make them very labile under various physicochemical conditions. In the case of betanin degradation, many new, decarboxy-betanins are formed. Latest study showed that UV-irradiation had significant impact on betanin stability, however, no deeper studies were performed in this field. In order to know an impact of the process conditions on degradation of betanin and its decarboxylated derivatives, series of experiments with UV-photo-irradiation was performed in aqueous solutions as well as in various organic solvents at wide range of pH. The highest pigment stability was observed in aqueous solutions for betanin and 17-decarboxybetanin at pH 4.5–7, and for 2,17-didecarboxybetanin at low pH. In all investigated organic solutions, all of the studied compounds have been degraded. The LC-DAD-ESI-MS analysis identified new reaction products. The results showed that UV-photo-irradiation leads mainly to a formation of many new and various 14,15-dehydrobetanins.

13

Stability of Betanidin Influenced by Copper Cations

Tuwalska D., Starzak K., Szot D., Skopińska A., Wybraniec S.

Logistyka

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2013

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nr 4

EN

Latest studies revealed some antioxidant properties of betalain pigments that contribute to the growth of interest in these compounds. During the isolation and storage, betalains are exposed to many different chemical and physical degradative factors, which often lead to their decomposition and discoloration. An effect of copper cations on betanidin stability was investigated in aqueous and organic-aqueous solutions. Betanidin stability experiments were performed at different pH’s, with different concentrations of metal cations and in different solvents. Using the modern spectrophotometric microplate reader it was possible to monitor changes of absorption spectra of the pigment samples during their incubation with copper ions in parallel at various physico-chemical conditions. Incubation with copper cations leads to betanidin degradation in all investigated solvents in the whole range of pH. The formed products are its new oxidized derivatives. A possibility of a formation of metal-pigment complexes is also considered. Recognition of betalains degradation paths is important because of their not yet fully recognized antioxidant and chemopreventive properties.

PL

Ostatnie badania wykazały przeciwutleniające właściwości betalain, co przyczyniło się do wzrostu zainteresowania tą grupą związków. Podczas izolacji i przechowywania, betalainy są narażone na wiele różnych degradujących czynników, chemicznych i fizycznych, które często powodują ich rozkład i odbarwienie. Zbadano wpływ kationów miedzi na stabilność betanidyny w roztworach wodnych i wodno-organicznych. Eksperymenty były przeprowadzane w różnym pH, z różnymi stężeniami kationów metalu i rozpuszczalników organicznych. Zastosowanie nowoczesnego spektrofotometrycznego czytnika mikropłytek pozwoliło na monitorowanie zmian w widmie absorpcyjnym pigmentu w różnych warunkach fizyko-chemicznych. Inkubacja betanidyny z jonami miedzi prowadzi do jej degradacji we wszystkich badanych rozpuszczalnikach w całym zakresie pH. Powstałe produkty są jej nowymi, utlenionymi pochodnymi. Rozważano również powstawanie kompleksu metal-pigment.Poznanie szlaków utleniania betalain jest ważne z powodu nie w pełni jeszcze poznanych ich antyutleniających i chemoprewencyjnych właściwości.

14

Protective Activity of EDTA Towards Betanin Degradation

Szot D., Skopińska A., Starzak K., Wybraniec S.

Logistyka

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2013

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nr 4

EN

Betanin is the most common betalain pigment, which provides colors to a wide variety of flowers and fruits. Current studies indicate on very interesting properties of these compounds, such as an ability to scavenge free radicals or chemopreventive and antiinflammatory effects. All natural pigments are affected by inferior stability in comparison to artificial colorants. However, the addition of EDTA (ethylenediaminetetraacetic acid) – a very popular chelating agent – improves their stability. In order to determine the protective activity of EDTA during the thermal treatment, a set of degradation experiments of betanin at pH 3–8 was performed. The pH of the greatest compound stability was found at pH 3.5–5.5. The addition of EDTA to the tested solutions increases the pigment stability, especially in acidic media. The analysis of obtained absorption spectra indicated that the reaction products retained the basic chromophoric skeleton.

PL

Betanina jest najczęściej występującym w przyrodzie pigmentem betalainowym odpowiedzialnym za barwę wielu rodzajom kwiatów i owoców. Aktualnie prowadzone badania wskazują również na inne ciekawe właściwości betalain, takie jak zdolność do zmiatania wolnych rodników, jak również działanie chemoprewencyjne i przeciwzapalne. Niestety naturalne barwniki wykazują mniejszą stabilność niż barwniki syntetyczne. Jednak dodatek EDTA (kwasu etyelenodiaminotetraoctowego) – bardzo popularnego czynnika chelatującego, może zwiększać ich stabilność. W celu określenia ochronnego działania EDTA podczas obróbki termicznej, przeprowadzono eksperymenty degradacji betaniny przy pH 3- 8. Największą stabilność betanina wykazuje przy zakresie pH między 3.5 a 5.5. Obecność EDTA zwiększa stabilność pigmentu, zwłaszcza w kwaśnym środowisku. Analizy uzyskanych widm absorpcji wskazują, że produkty reakcji zachowują podstawowy szkielet chromoforowy.

15

Halogen light influence on stability of selected betacyanins

Szot D., Starzak K., Skopińska A., Tuwalska D., Wybraniec S.

Logistyka

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2013

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nr 4

EN

Betacyanins are a group of plant pigments, widely applied as colorants in food industry. Betacyanins have gained importance since studies on their beneficial influence on health, confirmed by their antioxidant activity. Because of their inferior stability under some conditions, e.g. high temperature, presence of heavy metals and exposure to light, their industrial application is limited. Hence, this report presents evaluation of halogen light impact on the stability of betanin (a prominent betacyanin) in relation to stability of its decarboxylated derivatives in water and aqueous-organic solutions in pH range 3–8. During the experiments, UV-VIS spectra were collected and a decrease in concentration of particular pigments was monitored. This study shows that betacyanins degradation under the influence of halogen light is different in various solutions and pH’s. Generally, for betanin, in a comparison to its derivatives, the lowest stability irrespective of conditions was indicated. In most cases, pigments indicate higher stability in water than in aqueous ethanolic 50% (v/v) solutions, similarly at pH above 3.5 and below 7. Relatively high stability of decarboxy-betanins during the light treatment can suggest that decarboxylation can be a pathway to increasing stability of these food colorants. This investigation is important for optimizing conditions of storage of food coloured by betacyanins.

PL

Betacyjaniny należą do grupy roślinnych pigmentów, szeroko stosowanych jako barwniki w przemyśle spożywczym. Zyskały na znaczeniu odkąd potwierdzono ich antyoksydacyjne właściwości, korzystne dla zdrowia. Z powodu niskiej stabilności wobec czynników takich jak na przykład: wysoka temperatura, obecność metali ciężkich i światła, ich stosowanie jest ograniczone. Stąd też raport ten prezentuje ocenę wpływu rodzaju rozpuszczalnika i pH roztworu (w zakresie 3–8) na indukowaną światłem lampy halogenowej degradację betaniny (podstawowej betacyjaniny) w porównaniu do jej dekarboksylowanych pochodnych. Spadek retencji pigmentów spowodowany degradacją był monitorowany przez pomiary widm UV-Vis. Badania wykazały różnice w szybkości degradacji poszczególnych pigmentów w zależności od pH i rodzaju roztworu. Generalnie, niezależnie od warunków, betanina wykazuje niższą stabilność niż jej dekarboksylowane pochodne. W większości przypadków betacyjaniny są stabilniejsze w wodzie niż w 50% roztworze etanolu. Podobnie w zakresie pH 3,5-7. Relatywnie wyższa stabilność dekarboksylowanych pochodnych betaniny potwierdzona w trakcie eksperymentów może być wykorzystana jako sposób zwiększenia stabilności naturalnych barwników żywności. Te badania mają również znaczenie dla optymalizacji warunków przechowywania żywności barwionej za pomocą betacyjanin.

16

Spiral-coil countercurrent chromatographic separation of betanin and betanidin ethyl-esters monitored by continuous ESI-MS/MS injection and LC-ESI-MS/MS

Tuwalska D., Szot D., Wybraniec S., Winterhalter P., Jerz G.

Challenges of Modern Technology

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2013

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Vol. 4, no. 4

21--25

EN

Betalains present in Beta vulgaris L. (Chenopodiaceae) have a high potential as natural pigments for food applications. They have been used as a substitute of synthetic dyes in processing of gelatine, strawberry yogurts, ice creams, fruits cocktails, candies and biscuits. The stability of betalains is strongly influenced by numerous factors – sugar content, and impact of light, oxygen, water activity, pH and temperature. A mixture of betacyanin mono-, di- and tri-ethyl-esters was separated by a large-volume spiral-coil countercurrent chromatography (spCCC) prototype which yielded fractions continuously off-line injected to anÂ ESI-MS/MS device. This method yielded a reconstituted pigment profile and finally enabled the MS-target-guided isolation procedure for the whole spCCC-experiment. The individual fractions were analyzed by LC/ESI-MS/MS. Betanin and betanidin mono-, di- and tri-ethyl-esters as well as other decarboxylated derivatives were detected in the chromatograms. The ethyl-esters of betanin were only partially fractionated, but clearly fractionated from the ethyl-esters of betanidin which will be of great value for biological evaluations.