The impact of distillation process on prebiotic activity of different oligosaccharidic fractions extracted from grape seeds.

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The impact of distillation process on prebiotic activity of different oligosaccharidic fractions extracted from

grape seeds.

Matteo Bordiga, Emmanuelle Meudec, Pascale Williams, Thierry Doco, Rosa Montella, Jean Daniel Coïsson, Fabiano Travaglia

To cite this version:

Matteo Bordiga, Emmanuelle Meudec, Pascale Williams, Thierry Doco, Rosa Montella, et al.. The impact of distillation process on prebiotic activity of different oligosaccharidic fractions extracted from grape seeds.. 7. International Symposium on Macromolecules and Secondary Metabolites of Grapevine and Wine, MACROWINE2018, 2018, Saragosse, Spain. 2018. �hal-01982721�

Acknowledgements: The research was conducted with the financial support of Progetto Ricerca Locale DSF 2016.

The impact of distillation process on prebiotic activity of different oligosaccharidic fractions extracted from grape seeds

Matteo Bordiga ¹ , Emmanuelle Meudec ² , Pascale Williams ³ , Thierry Doco ³ , Rosa Montella ⁴ , Jean Daniel Coïsson ¹ , Fabiano Travaglia ¹

1 Dipartimento di Scienze del Farmaco and Drug and Food Biotechnology Center, Università degli Studi del Piemonte Orientale, Novara, Italy.

2 UMR 1083 Sciences Pour l’Œnologie, Polyphenols Platform, Montpellier SupAgro, INRA, Université de Montpellier2, Montpellier, France.

3 UMR 1083 Sciences Pour l’Œnologie, Montpellier SupAgro, INRA, Université de Montpellier2, Montpellier, France.

4 Proge Farm s.r.l., Largo Donegani 4/A, Novara, Italy.

matteo.bordiga@uniupo.it

OVERVIEW AND AIM OF THE WORK

EXPERIMENTAL

CONCLUSIONS

Grape pomace, a remnant of the wine making process, is one of the most important residues of the wine industry. It consists of different amounts of grape, skin, pulp, seeds, and, if not removed, stems. Some industrial uses currently under investigation for wine pomace waste include use as animal feed, as possible nutritive ingredients for value added products, in the production of citric acid, and the use of anthocyanins from grape skins as colorants (Bordiga et al., 2013). Spirits from fermented grape pomace are very popular in Mediterranean countries. Several countries produce traditional distilled alcoholic beverages from different raw materials. Grappa is a typical Italian spirit of commercial, cultural and historical importance, which is obtained by processing grape pomace or marcs from must or wine at the end of alcoholic fermentation. The high concentrations of bioactive compounds and the variation of their concentrations among different parts of grape pomace show the importance of analyzing in depth this winemaking by-product as a good and economically viable source of natural molecules (Bordiga et al., 2011). Despite the degradation caused by the drastic conditions of the distillation process, many interesting compounds still exist in this spent matrix (Bordiga et al., 2015). Together with a high content in phenols, grape seeds also contain a certain amount of oligosaccharides (Bordiga, 2015). One of the most well-demonstrated actions of oligosaccharides is the prebiotic activity.

Aims of this study was to evaluate different oligosaccharidic fractions, extracted from PRE and POST distilled grape seed pomace, as potential functional ingredients with prebiotic activity toward well-known probiotic bacteria, allowing to improve the growth during in vitro fermentation.

The final conclusion from this study is that despite the degradation caused by the drastic conditions of the distillation process, many interesting, valuable compounds are found in the extract from the waste of this process, which make it a useful matter for a better exploitation than as a heating source. Oligosaccharides still contained (yield of about 1% from degreased grape seeds) may be considered a novel ‘‘functional

ingredient’’ with potential prebiotic activity towards L. plantarum P17630 and L. acidophilus P18806, allowing to improve the growth during in vitro fermentation.

REFERENCES

Bordiga, M., Coïsson, J. D., Locatelli, M., Arlorio, M., & Travaglia, F. Food Analytical Methods, 2013, 6, 148-156.

Bordiga, M., Travaglia, F., Locatelli, M., Coïsson, J. D., & Arlorio, M. Food Chemistry, 2011, 127, 180-187.

Bordiga, M.; Travaglia, F.; Locatelli, M.; Arlorio, M.; Coïsson, J.D., International Journal of Food Science and Technology, 2015, 50, 2022-2031.

Bordiga, M. Valorization of wine making by-products, 2015, CRC Press, Boca Raton (FL), USA.

Qiang, X., YongLie, C., QianBing, W. Carbohydrate Polymers, 2009, 77(3), 435-441.

Bordiga, M.; Travaglia, F.; Meyrand, M.; German, J. B.; Lebrilla, C. B.; Coisson, J. D.; Arlorio, M.; Barile, D. Journal of Agricultural and Food Chemistry, 2012, 60(14), 3700-3707.

Doco, T., Williams, P., Meudec, E., Cheynier, V., Sommerer. N. Journal of Agricultural and Food Chemistry, 2015, 63, 671-682.

RESULTS AND DISCUSSION

The glycosyl residue composition determined by GC after acidic hydrolysis of oligosaccharides is summarized in Table 1. Galactose (Gal), arabinose (Ara), mannose (Man) and glucose (Glc) represent the major constituents of oligosaccharides from grape seeds. The composition of the PRE and POST extracts is very similar.

DISTILLATION PROCESS 95 ° C

10 minutes

preH2O-neg-frag_8339-All MS

postH2O-neg-frag_8340-All MS

pre40-neg-frag_8341-All MS

post40-neg-frag_8342-All MS

preH80-neg-frag_8343-All MS

post80-neg-frag_8344-All MS

0.50 1.00

0.50 1.00

0.50 1.00

0.50 1.00

0.50 1.00

0.00 0.50 1.00

4 6 8 10 12 14 16 18 Time [min]

A B C D E F

Intensityx106

0.00 0.00 0.00 0.00 0.00 1.50

1.50

1.50

1.50

Figure 3 : Full-scan mass spectrum of Oligosaccharides from PreH₂O (A), PostH₂O (B), Pre40 (C), Post40 (D), Pre80 (E) and Post80 (F) fractions after purification by SEC on a Superdex Peptide column.

Figure 2 : Total ion profiles obtained by UPLC-MS analysis on a Nucleodur HILIC column of Oligosaccharides from PreH2O (A), PostH2O (B), Pre40 (C), Post40 (D), Pre80 (E) and Post80 (F) fractions after purification by SEC on a Superdex Peptide column.

% Mol PREH₂O POSTH₂O PRE40 POST40 PRE80 POST80

2-O-MeFuc^a 1.1

Rha 2.9 11.6

Fuc 1.4

Ara 84.4 82.5 73.4 62.1 24.4 23.1

Xyl 1.6 Tr. 2.8

Api 1.2

Man 2.8 3.2 2.6 4.0 11.3 19.6

Gal 2.4 4.5 12.6 10.2 10.5 13.5

Glc 10.4 8.2 7.8 5.0 53.8 43.8

Table 1. Glycosyl residue composition (% Mol) of Oligosaccharides from PreH₂O, PostH₂O, Pre40, Post40, Pre80 and Post80 fractions after purification by high-resolution size exclusion chromatography on a Superdex Peptide column.

a2-O-MeFuc, 2-O-CH₃-fucose; Rha, rhamnose; Fuc, fucose; 2-O- MeXyl, 2-O-CH₃-xylose; Ara, arabinose; Api, apiose; Xyl, xylose;

Man, mannose; Gal, galactose; Glc, glucose.

Figure 4 : Spectra of MS3 fragmentation by ESI-TI in negative mode of ion parent at m/z 989.41 (A). MS spectrum of ion at m/z 989.41 [M-H]1-.

(B). MS2 spectrum of the ion at m/z 989.41 [M-H]1-.

(C). MS3 spectrum of the ion at m/z 826.66 [M-H]1- from the m/z 989.41 [M-H]1-.

→ Loss of a fragment of m/z 162 Da.

Figure 1 : A) ground defatted seeds, PRE (left) and POST (right); B) example of a lyophilized oligosaccharidic fraction.

EXTRACTION PROCESS

Grape seeds were ground to a powder using liquid N₂. Seeds powder was extracted with Soxhlet apparatus using hexane for 9 h to remove oil (Figure 1A). Subsequently, ground defatted seeds were extracted using EtOH/water (85:15, v/v) to obtain crude extract. Proper oligosaccharides purification has been realized by using an appropriate multistep solid-phase extraction (using C-18 cartridge followed by carbograph cartridge) (Figure 1B).

Elution by the latter cartridge was carried out with H₂O to obtain the first fractions (PreH₂O and PostH₂O). The second oligosaccharidic fractions were then eluted with a 60:40 deionized H₂O/ACN solution containing 0.1%

trifluoroacetic acid (Pre40 and Post40). Finally, the third fractions released with a 20:80 deionized H₂O/ACN solution containing 0.05% trifluoroacetic acid (Pre80 and Post80) .

A B

Chemical characterization of the complex oligosaccharides from grape seeds extract was performed using a UPLC-ESI-MSⁿ (Qiang et al., 2009). Figure 2 (A to F) show the total ion profiles obtained by UPLC-MS analysis on a Nucleodur HILIC column of Oligosaccharides from PreH₂O (Fig 1-A), PostH₂O (Fig 1-B), Pre40 (Fig 1-C), Post40 (Fig 1-D), Pre80 (Fig 1-E) and Post80 (Fig 1-F) fractions. Analysis of the full- scan mass spectra of Oligosaccharides from PreH₂O and PostH₂O, Pre40 and Post40, Pre80 and Post80 fractions (Figure 3) showed a multitude of masses between m/z 400 and m/z 1600 Da. The full- scan mass spectrum analysis also shows the presence of mono- and di-charged ions.

Complex oligosaccharides from grape seeds extract purified by size exclusion chromatography consist mainly on a neutral oligosaccharides rich in arabinose and certainly in glucose. These neutral oligosaccharides identified were really similar to those identify from Carignan wines (oligo-arabinans, oligorhamno-arabinans, and different arabinorhamnogalacturonan- oligosaccharides, Doco et al., 2015) and from Grignolino and Chardonnay wines (hexose-oligosaccharides, oligoxyloglucans, and oligoarabinogalactans, Bordiga et al., 2012). They represent the degraded structures of polysaccharides originating from the cell wall degraded by enzyme activities.

In Figure 3A, the full-scan mass spectra of oligosaccharides from PreH₂O, a series of ions was observed at m/z 503.15, 665.23, 827.29, 989.37 and 1151.38, with a difference of 162 Da between the different ions. The ions of the series correspond to oligosaccharide molecules as single deprotonated [M-H]^- ions.

The difference find between the ions of the series corresponds to the presence of a hexose (m/z 162 Da), likely a glucose residue. Figure 4A shows, as an example, the MSⁿ spectra of the [M-H]^- ion at m/z 989.41 obtained from the oligosaccharides of PreH₂O fraction.

The fragment ions observed in the MSⁿ spectra are usually named according to the nomenclature of Domon and Costello (1988). MSⁿ spectra and the fragment ions obtained of deprotonated oligosaccharides can be read from

“right to left”. The MS² fragmentation (figure 4B) of the parent ion at m/z 989.41 showed the presence of a fragment ion at m/z 827.66, due to the loss of a hexose residue (m/z 162 Da).

This fragment ion at m/z 827.66 loses a second hexose residue to give the fragment ion at m/z 665.24, and a successive loss of hexose residue yield fragment ion at m/z 503.04. MS³ fragmentation (figure 4C) of the fragment ion at m/z 827.66 showed the presence of fragment ions arising from successive losses of hexose residues, fragment ions at m/z 665.15 and m/z 503.09.

443.08^2-503.15

545.201- 1-

631.08^2-

665.23

707.22^1-

767.26 785.80

1- 2-

827.29^1-

851.78 869.34^1-

899.36 929.31

2-

961.35^2-

989.37^1-

1031.39 1093.35^1-

1151.38^1-

1193.54^1- 1315.67^2-

preH2O-neg-frag, 6.7-19.9 min

0 1000 2000 3000 4000 Intens.

400 600 800 1000 1200 1400 m/z

1-

1- 1-

737.24^1-

1227.44 386.44^2-

455.06^2- 557.10^1- 667.73^1- 697.18^2-

735.15^2- 763.33^2-

805.94^2- 829.30^2-

895.31^2-961.31^1-

998.87^2- 1028.00^1-

1048.71^2- 1093.87^2-

1121.28^2-

2-1359.38^2- 1468.23 postH2O-neg-frag, 8.4-17.3min

0 200 400 600 Intens.

400 600 800 1000 1200 1400 m/z

324.082- 454.991-

503.191-587.09^2- 631.17697.22^{2- 2-}

719.23^2- 763.22^2- 785.29^2-

829.67^2- 851.29^2-

869.35 2- 895.79 2-

917.30^2-

961.74^2- 983.34^2-

1027.37^2- 1049.81^2-

1093.34^2- 1115.39^2-

1187.521- 1226.921-

1307.51^1-

1396.491440.61^1-1527.47^1- pre40-neg-frag, 8.0-22.3min

0 500 1000 1500 2000 Intens.

400 600 800 1000 1200 1400 m/z

1-

434.80^2- 554.75^2-

684.74^2-

827.31^1- 902.91^2-

989.36^1- 1250.55^2-

preH80-neg-frag, 5.9-20.6min

0.0 0.5 1.0 1.5 2.0 x104 Intens.

400 600 800 1000 1200 1400 m/z

1308.47 1395.55 983.41^2-

455.10^2-521.11587.16^{2- 2-} 653.22^2-

719.22^2- 763.23^2-

785.29^2-

829.26^2- 851.31^2-

895.32^2- 917.36^2-

961.34^2- 1049.86^2-

1115.40^2- 1181.90^2-

1247.91^2-

1- 1-

1527.531572.49^1-

1-

post40-neg-frag, 5.9-16.7min

0 1 2 3 x104

Intens.

400 600 800 1000 1200 1400 m/z

1440.48^1- 1027.36^2-

1093.38^2-

437.21^2-537.24^2- 584.22^1-

665.24^1- 712.10^2-

806.14^1- 902.90^2-

974.51^2-1088.14

1250.54^1- 1270.61^2-

1299.64^2- 1384.59^2- post80-neg-frag, 5.8-23.3min

0 1000 2000 3000 4000 Intens.

400 600 800 1000 1200 1400 m/z

1232.58^1- 1202.59^1- 893.91^2-

A

D B

C

E F

830.70^2- 902.56^1- 956.59^2- 1071.41^1-

[M-H]^- = 989.41

preH2O-neg-frag : -MS, 12.5min

503.04 _546.33 665.24_707.28^{1- 737.34}_767.32^{2- 1-}

869.35^1- 899.36^1-

929.31^2- 1065.07

827.66^1-

preH2O-neg-frag : -MS²(989.41), 12.5min

604.34

646.45

796.52

855.22

913.72 992.83 1033.22 966.51

preH2O-neg-frag : -MS²(1121.37), 12.5min

503.09^1- 545.28 606.31

665.15^1-

707.602- 737.181-

768.51

809.12 preH2O-neg-frag : -MS³(989.85->827.66), 12.5min

preH2O-neg-frag : -MS³(1121.00->966.51), 12.6min

0 2 x10Intens.⁵ 4

0.0 0.5 1.0

0.00.5 1.0

0.00.5 1.0

-101

300 400 500 600 700 800 900 1000 m/z

x10⁵

x10⁴ x10⁴

1- 1-

830.70^2- 902.56^1- 956.59^2- 1071.41^1-

[M-H]^- = 989.41

preH2O-neg-frag : -MS, 12.5min

503.04 _546.33 665.24_707.28^{1- 737.34}_767.32^{2- 1-}

869.35^1- 899.36^1-

929.31^2- 1065.07

827.66^1-

preH2O-neg-frag : -MS²(989.41), 12.5min

604.34

646.45

796.52

855.22

913.72 992.83 1033.22 966.51

preH2O-neg-frag : -MS²(1121.37), 12.5min

503.09^1- 545.28 606.31

665.15^1-

707.602- 737.181-

768.51

809.12 preH2O-neg-frag : -MS³(989.85->827.66), 12.5min

preH2O-neg-frag : -MS³(1121.00->966.51), 12.6min

0 2 x10Intens.⁵ 4

0.0 0.5 1.0

0.00.5 1.0

0.00.5 1.0

-101

300 400 500 600 700 800 900 1000 m/z

x10⁵

x10⁴ x10⁴

1- 1-

830.70^2- 902.56^1- 956.59^2- 1071.41^1-

[M-H]^- = 989.41

preH2O-neg-frag : -MS, 12.5min

503.04 _546.33 665.24 ^1-

707.28737.34^2- 767.32^1-

869.35^1- 899.36^1-

929.31^2- 1065.07

827.66^1-

preH2O-neg-frag : -MS²(989.41), 12.5min

604.34

646.45

796.52

855.22

913.72 992.83 1033.22 966.51

preH2O-neg-frag : -MS²(1121.37), 12.5min

503.09^1- 545.28 606.31

665.15^1-

707.602- 737.181-

768.51

809.12 preH2O-neg-frag : -MS³(989.85->827.66), 12.5min

preH2O-neg-frag : -MS³(1121.00->966.51), 12.6min

0 2 x10Intens.⁵ 4

0.0 0.5 1.0

0.00.5 1.0

0.00.5 1.0

-101

300 400 500 600 700 800 900 1000 m/z

x10⁵

x10⁴ x10⁴

1- 1-

A B C

The probiotic strains L. plantarum P17630 and L. acidophilus P18806 were cultured in an industrial medium (specifically optimized for each strain) added with two different concentrations of oligosaccharidic fractions (OF) (1.0% and 0.1%, respectively). Results have been compared to control, representing the growth of the strain using exclusively their specific industrial medium. Considering the L. plantarum P17630 , any significant additional improvement on bacterial growth was observed when the OF were added (Figures 5A and 5B). Especially when the addition was 1.0%, all the results obtained with the different OF were significantly lower if compared to control. Results (substantially) comparable have been obtained instead when the addition was 0.1%. Conversely, considering the L. acidophilus P18806, novel and interesting results have been obtained. Post80 and Pre80 showed significantly higher values when the addition was 1.0% (Figure 5C). However, the best performance has been obtained when the addition of oligosaccharidic fractions was 0.1%. All the fractions (except for PreH

₂

0) showed significantly higher values during growth test. These results demonstrate that, under these conditions, the addition of these bioactive compounds are able to generate a sort of “booster effect”. However, looking at the results, it appears clear that each strain responds differently to the addition of these compounds. A possible explanation for this effect could be correlated to the high specie-specificity (and strain-specificity) of each carbohydrate substrate, because of the need of a specific and appropriate enzymatic pattern for the linkage degradation typical for each single microorganism, as well as the existence of specific pathways for the uptake of the substrate from the medium.

CHARACTERIZATION OF OLIGOSACCHARIDES

GROWTH TEST (IN VITRO)

*

*

A

*

*

B

*

*

C

*

*

* hidden behind orange line

D