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* Laboratoire des Sciences Animales, ENSAIA-INPL, USC INRA, F-54505 Vandoeuvre-lès-Nancy Cedex 5, France
Laboratoire de Physico-Chimie et Génie Alimentaires, ENSAIA-INPL, USC INRA, F-54505 Vandoeuvre-lès-Nancy Cedex 5, France
Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition, EMI 0014 INSERM and URM 20 IFREMER, Faculté de Médecine,54500 Vandoeuvre-lès-Nancy, France
Laboratoire de Biochimie Médicale et Pédiatrique, Faculté de Médecine,54500 Vandoeuvre-lès-Nancy, France
Corresponding author: J. Mouécoucou; e-mail: justinemouecoucou{at}yahoo.fr.
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ABSTRACT |
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Key Words: ß-lactoglobulin • gum arabic • protein-polysaccharide interaction • protein digestibility
Abbreviation key: ß-lg = ß-lactoglobulin, LM = low methylated, PD = peptic digestibility, pHi = isoelectric point, TPD = total protein digestibility
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONCONCLUSIONSREFERENCES |
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An increasing number of dairy products such as yogurt drinks, flavored milk, flans, custards, cream, ice cream, and puddings contain plant soluble polysaccharides such as alginates, carrageenans, pectins, gum arabic, guar gum, or locust bean gum. Interactions between polysaccharides and milk proteins, depending on the aqueous environmental conditions (pH and ionic strength), charge density of the two macromolecules and molecular weight of polysaccharides (Dickinson, 1998; Schmitt et al., 1998; Syrbe et al., 1998), improve the texture and stability of dairy products. When these two macromolecules are mixed together in water, phase separation generally occurs, leading either to the segragation of each macromolecule in separated phases (segregative phase separation) or the concentration of both macromolecules in one phase with the appearance of an equilibrium dilute phase (associative phase separation) (Albertsson, 1995; Schmitt et al., 1998). In this latter phase, the formation of protein-polysaccharide complexes is due to strong interactions (covalent bonding) or to several weak interactions (electrostatic, Van der Waals, hydrogen, or hydrophobic bonding).
The ability of proteins and polysaccharides to induce the formation of complexes could have consequences on the physiological fate of proteins. Low protein hydrolysis, decrease of hydrolyzed protein absorption, and decrease of animal growth are generally observed with addition of soluble polysaccharides to foods at low or high levels (Jenkins et al., 1978, Shah et al., 1982; Low and Rainbird, 1984; Schneeman and Gallaher, 1985; Mouécoucou et al., 1990, 1995; Astwood and Morris, 1992; Larsen et al., 1994; Eggum, 1995; Ellis et al., 1995; Hsu et al., 1996; Grala et al., 1999; El Kossori et al., 2000).
The main objective of this study was to determine the effects of gum arabic, low methylated pectin, and xylan on the in vitro digestibility of ß-lg. Gum arabic, also called acacia gum, the natural exudate from the Acacia senegal, is a high molecular weight polysaccharide consisting of branched arabinogalactan heteropolymers (Anderson et al., 1983; Street and Anderson, 1983). Gum arabic also contains proteins (2%) covalently attached to the polysaccharide moiety. Low methylated pectin, is a biopolymer essentially made up of D-galacturonic residues. The presence of uronic acids is responsible for the polyanionic character of gum arabic and pectins, with a pHi around 3 (Kravtchenko, 1997; Wang and Qvist, 2000). They are both used in food industry. Xylan extracted from Palmaria palmata, a red seaweed authorized in human nutrition, is a linear oligomer composed of 1-3/1-4 linked xylose in proportion of 1/4 and 1/3, respectively, with a low molecular weight, and a mean degree of polymerization around 8 to 12.
Hydrolysis was done using two models. In the first model, ß-lg was hydrolyzed by pepsin, mimicking the in vivo gastric digestion, by reducing progressively the pH (Bernier et al., 1988). In the second model according to Savoie and Gauthier (1986), ß-lg was hydrolyzed by a mixture of trypsin and chymotrypsin into a dialysis cell. Dialysis bags with two different MW cutoffs were used to estimate the ß-lg digestibility. The aim of using two MW cutoffs was to assess the effects of polysaccharides on ß-lg digestibility in the physiologic situation (bag with a MW cutoff of 1000 Da), and in nonphysiological, when high MW peptides diffused through the intestinal barrier as in the case of some allergenic proteins (bag with a MW cutoff of 8000 Da).
MATERIALS AND METHODS
Materials
Acid-processed bovine ß-lg powder (lot no. 838) was kindly provided by Lactalis (Retiers, France). Its composition was (g/100 g): 89.75% protein (N x 6.38). Powdered gum arabic (lot no. 97 J 716) was a gift from the Colloïdes Naturels International Company (CNI, Rouen, France). It contained 89.95% polysaccharide. Xylan (lot no. PP28-4-00) was extracted from a Rodophyceae, Palmaria palmata or dulse by the Laboratoire de Pathologie Cellulaire et Moléculaire en Nutrition (Faculté de Médecine Vandoeuvre-lès-Nancy, France). It contained 80.40% pentose, 13.20% hexose. Low methylated (LM) pectin (lot no. 0B800), with 87% polysaccharide, was provided by Degussa Texturant Systems (Boulogne-Billancourt, France).
Pepsin (3800 U/mg protein, 1:60,000, EC 3.4.23.1), bovine trypsin (13,800 U/mg, EC 3.4.21.4), porcine chymotrypsin (51 U/mg, EC 3.4.4.5), and thimerosal were purchased from Sigma (France).
Sodium hydroxide, hydrochloride, and trichloroacetic acid were of analytical grade.
Methods
Preparation of ß-lg/polysaccharide mixtures.
ß-Lactoglobulin/polysaccharide mixtures were measured in percent by weight (%wt). ß-Lactoglobulin powder containing 40 mg of N (1.7% wt of protein, N x 6.38) was taken in triplicate and dissolved in 15 ml of sodium phosphate buffer 0.17 M pH 7 under gentle mixing. Polysaccharides were dissolved in the same buffer at the same concentration (1.7% wt). Dispersions were left overnight at 4°C to allow complete hydration of macromolecules. The ß-lg and polysaccharide stock were blended so as to obtain 0 (ß-lg without polysaccharide), 1, 10, 20, 30, and 50% weight of relative polysaccharide concentration (as compared to the ß-lg concentration). Thimerosal was added to ß-lg/polysaccharide mixtures at 50 mg/L to avoid bacterial growth.
Enzymatic digestions.
Two different models of enzymatic digestions of ß-lg/polysaccharide mixtures were performed:
Peptic digestion.
One millilliter of pepsin in 0.02 N HCl (1 mg/ml) was added to 15 ml of ß-lg/polysaccharide mixtures (E/S: 1/250). For simulating the in vivo gastric digestion, the pH of dispersions was progressively reduced from pH 7 to 2 within 2 h by adding HCl 0.02 N with a peristaltic pump (flow rate: 80 µl/min) at 37°C. The digestion was stopped at pH 5, 4, 3, and 2 by adding 30% (vol/vol) TCA. Samples were centrifuged at 5000 rpm for 20 min (Beckman Coulter; Villepinte, France). Pellets were discarded and 10 ml of supernatant were taken for soluble N analysis. Experiments were made in triplicate and assays in duplicate.
Total proteic digestion.
The in vitro total digestion of ß-lg/polysaccharide mixtures was carried out at 37°C in a dialysis cell (Serna, Laval, Quebec) according to the two-step hydrolysis method developed by Savoie and Gauthier (1986), with minor modifications of the first step. The dialysis cell consisted of an inner compartment (a dialysis bag), where digestion occurs, fixed into a double-wall cylindrical outer compartment with buffer circulation.
In the first step, peptic digestion of ß-lg/polysaccharide mixtures was made as described in the previous section. Peptic digestion was stopped by raising the pH to 8 with NaOH 2 N. In the second step, the samples previously submitted to peptic digestion were transferred to dialysis bags with MW cutoff of 1000 or 8000 Da (SpectraPor 6, Interchim, Montlucon, France). Then, 1 ml of a mixture of trypsin/chymotrypsin (1/2.3, wt/wt) at a weight concentration of 2.5 mg/ml (enzyme/substrate 1/50) was added. Digestion products diffusing through the dialysis bag were collected every hour during 6 h by a circulating (1.6 ml/min) sodium phosphate buffer 0.01 M pH 8, and the fractions were taken for the N analysis. Experiments were made in triplicate and assays in duplicate.
Nitrogen Analysis
The total N content in supernatants (peptic digestion) or dialysates (total proteic digestion) was determined by the micro Kjeldhal procedure, according to AOAC methods (47.021 and 47.023). The protein digestibility was calculated as follows:
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Statistical Analysis
The results are given as mean ± standard deviation of samples and were analyzed by a one-way analysis of variance (ANOVA, Statview V). Differences between means at P < 0.05 were analyzed using the Fisher test.
RESULTS
Peptic digestion.
The effect of plant hydrocolloids on the in vitro ß-lg peptic digestibility (PD) is showed in Table 1
. The digestibility of ß-lg alone was very low at all pH considered with PD values comprised between 1 and 2%.
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), LM pectin (Table 1B) and xylan (Table 1C) at levels 1 and 10 %wt did not change significantly the ß-lg peptic digestibility. As a general trend, high levels of polysaccharides (50% wt for gum arabic and LM pectin; 30 and 50% wt for xylan) resulted in a low but significant increase of ß-lg peptic digestibility at all pH values compared with the protein alone.
Total proteic digestion (dialysis membrane with cutoff of 1000 Da).
After 6 h of hydrolysis with a mixture of trypsin and chymotrypsin at pH 8, the total proteic digestibility (TPD) in the control ß-lg was 27.5% ± 3.5 (Figure 1). This value was significantly lower in presence of gum arabic, LM pectin, or xylan.
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) decreased significantly more ß-lg digestibility than both gum arabic (Figure 1A) and LM pectin (Figure 1B). There was no significant difference between the ability of LM pectin and gum arabic to decrease digestibility.
Total proteic digestion (dialysis membrane with a MW cutoff of 8000 Da).
Proteolysis in a dialysis bag with a MW cutoff of 8000 Da was made under the same experimental conditions as in the dialysis bag with a MW cutoff of 1000 Da (Figure 2). No differences in the calculated TPD were observed. The TPD in the control ß-lg after 6 h of digestion was 32.05% ± 5.95. The presence of the three polysaccharides did not significantly modify this TPD value during the time of hydrolysis, whatever the level of polysaccharides in the ß-lg dispersions.
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The effect of gum arabic, LM pectin, and xylan on the in vitro digestibility of ß-lg was estimated using gastric peptic and peptic followed by trypsic/chymotrypsic digestion models.
In the peptic model, the pH was progressively reduced from 7 to 2 within 2 h as is generally observed in vivo. The products of peptic digestion are high molecular weight peptides, which mainly precipitate with TCA, leading to small amounts of soluble N. The digestibility of ß-lg by pepsin was very low at pH 2, where ß-lg is in a monomeric form (Mc Kenzie, 1971). Reddy et al. (1988) and Schmidt and Poll (1991) have already reported a low in vitro ß-lg hydrolysis by pepsin using different methods. Our results also agree with in vivo studies demonstrating the presence of intact ß-lg after its passage through the stomach (Miranda and Pelissier, 1983; Yvon et al., 1984). Kella and Kinsella (1988) suggested that the acid stability of ß-lg could result from increased internal H bonding arising from either two titrated carboxyl groups or from one amide and one carboxyl group. The increase in PD observed with 30 and 50% wt of polysaccharides is not due to an increased ability of pepsin to hydrolyze ß-lg, but is due rather to an incomplete TCA precipitation of protein resulting of protein/polysaccharide interactions. Electrostatic interactions between ß-lg and polysaccharides likely occur in the pH range 3 to 5, where the two macromolecules carry opposite charge. The formation of protein/polysaccharide macromolecular complexes or aggregates of complexes could decrease the protein precipitation by TCA, resulting in the erroneous conclusion of a stronger ß-lg proteolysis. This hypothesis is reinforced by the observation that the effect is stronger in mixtures containing the higher polysaccharide concentrations.
The ß-lg digestibility calculated after hydrolysis of peptic and pancreatic enzymes hydrolysis in a dialysis bag with a MW cutoff of 1000 Da was about 27%. The main role of pepsin is to hydrolyze bonds between aromatic amino acids (tyrosine and phenylalanine) and to facilitate the digestion by pancreatic enzymes (Savoie and Gauthier, 1986; Reddy et al., 1988; Mullaly et al., 1998). Some authors have shown that pepsin makes the molecular structure of ß-lg more susceptible to proteolytic attack by other proteinases (Porter et al., 1984; Antila et al., 1991). Trypsin and chymotrypsin cleave bonds between basic amino acids (lysine and arginine) and aromatic amino acids (tryptophan, phenylalanine, tyrosine), respectively. At pH 8, ß-lg undergoes a conformational change (Tanford transition) and is in a monomeric form, which makes the access of enzymes to a few strategic peptide bonds easier (Kella and Kinsella; 1988; Reddy et al., 1988; Chobert et al., 1991).
The addition of gum arabic, LM pectin, or xylan decreased the digestibility of ß-lg and the decrease was dependent on the level and the type of polysaccharide. Among the three polysaccharides used, xylan produced the most remarkable effect. Scince three polysaccharides have different chemical structures and different physico-chemical properties, their aptitudes to alter ß-lg digestibility are also different. One possible hypothesis to explain the decrease of ß-lg digestibility is the ability of most polysaccharides to increase the apparent viscosity of mixtures. Increasing the apparent viscosity of protein/polysaccharide mixtures is usually related to low protein digestibility and low nutrient absorption (Schneeman and Gallaher, 1985; Edwards et al. 1988; Häglund et al. 1988). Gum arabic (Sanchez et al., 2002) and xylan (data not shown) dispersions displayed low apparent viscosities at low polysaccharide concentrations, as was the case in our experiment. So, in these cases the apparent viscosity cannot explain the reduction of trypsin/chymotrypsin protein digestibility.
Another possible hypothesis is the existence of nonspecific interactions between molecular species in protein/polysaccharides mixture. The three polysaccharides and ß-lg have negative charges at pH 8, and electrostatic interactions between biopolymers are therefore unlikely. Nevertheless, weak local electrostatic interactions can occur at neutral pH between anionic polysaccharides and positively charged regions called "patches" on protein molecules (Imeson et al., 1977; Tolstoguzov, 1986; Xia and Dubin, 1994). Native ß-lg and anionic polysaccharides (dextran sulfate and propylene glycol alginate) have already been shown to form ionic complexes at neutral pH (Dickinson and Galazka, 1991). Possible electrostatic interactions between ß-lg and gum arabic have been suggested recently at pH 5.3, but no evidence of interactions at pH > pHi has been provided (Weinbreck et al., 2003). It is then doubtful that electrostatic interactions between ß-lg and polysaccharides could occur at pH 8. Nonionic interactions such as hydrogen bonding can also lead to the formation of protein-polysaccharide complexes (Imeson et al., 1977; Antonov and Soshinsky, 2000), then this possibility cannot be totally excluded. Another possibility is that electrostatic interactions occurred between polysaccharides and positively charged peptides, containing for instance arginine and lysine, released by trypsin hydrolysis (Knudsen et al., 2002).
No significant effect of polysaccharides on the in vitro ß-lg digestibility was detected using the dialysis bag with a MW cutoff of 8000 Da, suggesting that the difference between the total protein digestibility with both membranes could be explained in part by polysaccharide-peptide interactions. Furthermore, since the protein digestibility determined using the dialysis bag with a MW cutoff of 1000 Da was close to that obtained with the second dialysis bag, it seems that ß-lg was more readily cleaved into small peptides without polysaccharides and into larger peptides in mixtures containing polysaccharides. Several workers have demonstrated the formation of peptides upper 2000 Da after trypsin, chymotrypsin, bromelain, or papain ß-lg hydrolysis (Chobert et al., 1991; Schmidt and Poll, 1991; Turgeon et al., 1992; Schmidt and Van Markwigh, 1993; Madsen et al., 1998; Otte et al., 1998, Pintado et al., 1999) to a lesser extent in the range of 1000 to 1500 Da. Determination of the size distribution of released peptides during ß-lg hydrolysis is needed to verify the hypothesis. Our results suggest that polysaccharides inhibit only the cleavage of peptides under 1000 Da coming from ß-lg but not the cleavage of peptides upwards of 8000 Da. Peptides in dialysates had to be characterized for explain results observed with two different membranes.
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CONCLUSIONS |
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TOPABSTRACTINTRODUCTIONCONCLUSIONSREFERENCES |
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FOOTNOTES |
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Received for publication April 9, 2003. Accepted for publication July 1, 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONCONCLUSIONSREFERENCES |
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-lactalbumin and ß-lactoglobulin in buffer solutions by proteolytic enzymes. Neth. Milk Dairy J. 45:225–240.
-lactalbumin and ß-lactoglobulin on their proteolysis by pepsin and papain. Neth. Milk Dairy J. 47:15–22.
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