1. RESULTS and DISCUSSION CONCLUSIONS and FUTURE APPROACH
Effect of gastrointestinal digestion according to Infogest Consensus on milk proteins in the presence of prebiotic carbohydrates
Mar Villamiel1, Anabel Mulet2, Neil Rigby2, Antonia Montilla1, Nieves Corzo1, Alan Mackie2
1Instituto de Investigación en Ciencias de la Alimentación, CIAL (CSIC-UAM).
C/Nicolás Cabrera, 9, Campus de la Universidad Autónoma de Madrid, Madrid, Spain
2Institute of Food Research, Norwich Research Park, Norwich, NR4 7UA, United Kingdom
INTRODUCTION
The susceptibility of prebiotic oligosaccharides to their passage through the gastrointestinal tract is an important feature, since this can affect their final state when
they reach the colon to be fermented by the microbiota. In vitro gastrointestinal assays under simulated physiological conditions have shown the importance of the
type of oligosaccharide and different results can be achieved depending on its molecular weight and structure (1). In addition, during the last years, a lot of attention
has been paid on the necessity of supported digestion methods which can simulate the physiological human conditions and allow the comparison of results. In this
sense, an international consensus method was reported by the Infogest Cost Action (2).
On the other hand, new structures of prebiotics derived from lactulose (OsLu) have been exhaustively characterized and evaluated in recent years (3).
To our knowledge, limited studies have been carried out on the digestibility of OsLu. In assays with rats, the ileal digestibility of OsLu seemed to be
4-fold lower than that of galactooligosacharides (GOS), probably due to the terminal monosaccharide (4). However, no in vitro studies have been done
on the susceptibility of OsLu to the digestion when they are within a real food neither on the impact of these compounds on the digestion of other food
components.
OBJECTIVES
The aims of the present work have been: i) to study the effect of the presence of OsLu on the digestion of milk proteins in a semi-dynamic gastric model and ii) to evaluate the changes in the carbohydrate fraction during the gastrointestinal digestion; in both cases following Infogest protocol.
MATERIALS and METHODS
SAMPLES
Dilution of milk Skim Milk Powder (SMP) at 10% in
distilled water
Addition of ingredients (OsLu, lactulose (Duphalac®),
GOS (Vivinal®) to SMP at 5% (required for prebiotic
effect)
SEMI-DYNAMIC
GASTRIC MODEL
12% Bis-Tris-Gel (Novex, NuPage)
200 V, 350 mA, 35 min and RunBlue
Precast gels (180 V, 400 mA, 60 min)
Sampling 0, 1 and 2 h for testing
milk proteins by SDS-PAGE
DIGESTION
37°C +
INCUBATION
Sampling 0, 1 and 2 h for testing
carbohydrates by GC-FID
GC-FID ANALYSIS:
TMS Oximes
Column: HT5 (12 m x 0.32 mm x 0.10 µm)
Temperature program: Ti 150 ºC, temperature gradient 3 ºC/min, Tf 380ºC, holding time 20 min
INFOGEST PROTOCOL (2)
Fig. 1. Scheme used was a semi-dynamic gastric model based on the static protocol from Infogest. Simulated Salivary Fluid (SSF), Simulated Gastric Fluid (SGF) and Simulated Intestinal Fluid (SIF). Enzyme activities are in units per mL of final digestion mixture at each corresponding digestion phase.
RESULTS and DISCUSSION
A) EFFECT ON PROTEIN DIGESTION
BSA
BSA
pepsin
caseins
pepsin
β-Lg
α-La
caseins
β-Lg
α-La
0.54*
0.61
0.61
0.62
0.64
0.75
0.68
0.68
Fig. 2 . Profile of pH of milk samples during gastric digestion.
►All samples presented similar pH (close to 7) at the beginning
of the assay and hardly any change was observed, mainly after
1500 s of digestion.
Fig. 3. Electrophoretic profiles of milk protein fractions (caseins, β-Lg, α-La, BSA) before and after 2 h of digestion (Bis-Tris-Gel, Novex, NuPage)
M: Marker, 1 SMP 0 h, 2 SMP 2 h, 3 SMP+OsLu 0, 4 SMP+OSLu 2 h, 5 SMP+ Duphalac 0 h, 6 SMP+Duphalac 2 h, 7 SMP+Vivinal 0 h, 8 SMP + Vivinal 2 h, 9 blank
Fig. 4. Electrophoretic profiles of milk protein fractions (caseins, β-Lg, α-La, BSA) during
0, 1 and 2 h of digestion (RunBlue Precast gels).
►Pepsin is shown in samples after digestion and increases with the time (Fig. 3 and 4).
►Complexes between α-La and carbohydrates in samples with OsLu and Vivinal GOS could be formed (Fig. 3).
►After 1 and 2 h of digestion all samples presented similar pattern of proteins (Fig. 3 and 4).
M: Marker; 1, 5 and 9 SMP; 2, 6 and 10 SMP+OsLu, 3, 7 and 11 SMP+Vivinal; 4, 8 and 12
SMP+Duphalac. *Values of optical density measured in the maximum of the peak in the profile
obtained with the Software Quantity One.
B) EFFECT ON CARBOHYDRATE FRACTION
Table 1. Carbohydrate evolution of milk samples during gastric digestion. *Oligosaccharides: Each value is the sum of other disaccharides, tri- and tetrasaccharides. ** Mean ± standard deviation
Carbohydrates (mg/g)
Galactose
Glucose
Lactulose
Lactose
Other disaccharides
Trisaccharides
Tetrasaccharides
Oligosaccharides*
Total carbohydrates
Milk-SMP 0 h
0.5±0.2**
0.8±0.5
180.0±24.6
5.0±0.8
5.0
186.3
Milk-SMP 1 h
0.5±0.2
0.9±0.1
189.8±41.8
5.6±1.0
5.6
196.8
Milk-SMP 2 h
0.4±0.1
0.7±0.3
170.5±5.3
5.2±0.2
5.2
176.8
Milk+Vivinal 0 h
1.7±0.6
22.8±3.7
195.7±17.9
33.2±5.8
39.0±8.8
7.2±1.9
79.4
299.5
Milk+Vivinal 1 h
1.4±0.2
21.1±4.1
179.8±11.6
32.5±5.5
33.2±3.7
8.9±1.6
74.7
277.0
Milk+Vivinal 2 h
1.6±0.2
20.0±1.2
197.8±9.7
31.3±4.6
31.5±3.1
9.6±2.1
72.4
291.8
Milk+Duphalac 0 h
9.2±1.6
1.0±0.8
58.9±16.0
197.8±21.2
8.4±1.2
8.4
275.3
Milk+Duphalac 1 h
8.4±0.9
0.8±0.8
50.3±7.2
186.6±24.2
7.6±1.2
7.6
253.8
Milk+Duphalac 2 h
8.2±0.6
1.0±0.6
52.1±7.8
188.2±23.9
7.9±0.8
7.9
257.4
Milk+OsLu 0 h
16.7±2.7
1.0±0.5
20.5±5.4
227.0±26.8
32.6±2.2
31.0±2.6
3.0±0.6
66.6
331.8
Milk+OsLu 1 h
15.1±1.4
1.3±0.7
21.6±4.8
205.9±14.6
30.1±3.3
28.9±1.9
2.4±0.7
61.5
305.4
Milk+OsLu 2 h
17.8±0.2
1.0±0.1
20.5±0.2
234.0±9.7
34.1±0.7
28.7±4.2
2.6±0.5
65.4
338.7
Fig. 5. GC-FID profile of TMSO derivatives of carbohydrates present in milk samples with OsLu after 1 h of gastric digestion. Peak 1 Galactose; 2 Glucose; 3 Galactose + Glucose; I.S. Internal Standard; 4 Lactose; 5 Other disaccharides. * Matrix effect, DP: Degree of Polymerization.
CONCLUSIONS and FUTURE APPROACH
*According to the electrophoretic profile of the protein fraction, the presence of carbohydrates in milk in the prebiotic concentration, does not affect the gastric digestion of milk protein.
*After the gastrointestinal digestion, following the Infogest protocol, hardly any change in the fraction of carbohydrates, including OsLu, was observed.
*Due to the resistance of OsLu to gastric and pancreatic fluids and bile salts, the digested samples will be subjected to the action of an extract of rat intestine, in order to compare the
behaviour of the different prebiotics versus the β-galactosidase activity derived from mammals.
Bibliografía
References: (1) Playne and Crittenden, Advances Dairy Chemistry vol 3 Ed. McSweeney and Fox, Springer Science Business Media LLC, 2009; (2) Minekus et al. 2014, Food Function 5, ;
Acknowledgements :This work has been funded by MINECO of Spain Project AGL R and ALIBIRD-CM S-2013/ABI MV thanks Salvador de Madariaga Program for the economical support of this project. Authors thank J. Megino for technical assistance.
(3) Moreno et al. 2014, Electrophoresis 35, ; (4) Hernández-Hernández et al. 2012, J. Nutr. June,