Heating Sequence and Calcium Lactate Concentration Effects on in

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Chapter 3

Heating Sequence and Calcium Lactate Concentration Effects on in Vitro Protein Digestibility and Oil Release in Emulsion Stabilized by Preheated Soy Protein and Caseinate Nantarat Na Nakornpanom,1 Pranithi Hongsprabhas,2 and Parichat Hongsprabhas*,1 1Department

of Food Science and Technology, Kasetsart University, Bangkok 10900, Thailand 2Department of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand *[email protected]

This study investigated the influences of heating sequence (one-stage vs. two-stage heating processes) and Ca lactate concentration (0-100 mM) on protein digestibility and oil released from reconstituted oil-in-water (o/w) emulsions under in vitro peptic and tryptic digestion. The high-calorie emulsions contained 1.0 kcal/mL. Caloric distributions from a carbohydrate:protein:lipid ratio of 55:15:30 were prepared by fabricating protein matrices with different characteristics at the oil-water interface and in the bulk aqueous phase, using heated mixed sodium caseinate (SCN) and soy protein isolate (SPI) at a ratio of 0.7:0.3. The preheating step prior to emulsification resulted in the adsorption of 7S globulins, phosphorylated caseins, and acidic 11S globulins at the oil-water interface. The reheating applied to the emulsion resulted in the disappearance of 7S globulins and the adsorption of protein aggregates with high molecular weight (>250 kDa) at the interface during peptic digestion. This did not occur in the emulsion prepared by the one-stage heating process. The two-stage heating process thus resulted in a slow release of peptides in the aqueous phase, and © 2010 American Chemical Society Cadwallader and Chang; Chemistry, Texture, and Flavor of Soy ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


a long lag phase for oil release during peptic digestion. The study indicated that the rate of nutrient release can be controlled by fabricating different protein matrix characteristics at the interface.

The aging population, as well as consumers’ concerns about health and well-being, has been the major driving forces for food manufacturers in recent years. Controlling the digestibility of macromolecules, such as proteins, lipids and starches, can influence postprandial metabolic behaviors: e.g. amino acid absorption, blood plasma triglyceride and glucose concentrations, etc. (1). The subsequent bioavailability of several nutrients is thus influenced by the fate of those microstructural elements assembled after they have been ingested (1–4). Enteral formulae are liquid emulsions widely used in hospitals as complete nutrient products administrated into the gastrointestinal (GI) tract for patients who cannot swallow. They contain carbohydrates, proteins, lipids, vitamins and minerals that meet an individual’s requirements when provided in adequate volume. The standard formula for an individual with normal GI function usually contains 1.0 kcal/mL and a caloric distribution from carbohydrate:protein:lipid of 37-55:15-25:30-45, if fluid intake does not need to be restricted. The calorie sources are usually in the form of biopolymers in the case of polymeric formulation. This is to control glycemic responses and decrease the osmolality of the formulae to lower the risk of osmotic diarrhea, which can occur if the osmolality is much higher than the isotonic osmolality of around 300 mOsmol/kg (5). Carbohydrates may be in the form of glucose syrup, maltodextrin, or other forms of complex carbohydrates. Protein sources in the commercial products include intact sodium caseinate (SCN) and/or soy protein isolate (SPI). The formulae also contain corn oil or soybean oil, in order to provide both an energy source and essential fatty acids. Apart from the calorie-dense characteristics, the formulae need to be pathogen-free and have low viscosity (ca. 100-150 mPa·s) in order to flow through the 3.3-5.7 mm diameter tubing (10-16 French units; Fr) and enter the GI tract. The complete formulae usually contain mono-, di- and trivalent ions of high ionic strength (6). The presence of electrolytes to meet such requirements is a challenge for food manufacturers, who must optimize the process to avoid flocculation, coalescence, or even gelation during thermal processes and storage of the liquid emulsion. Generally, emulsions stabilized by globular proteins are prone to flocculation and aggregation during heat treatment (7). An o/w emulsion stabilized by soy proteins shows a viscosity as high as 400 mPa·s when the protein concentration is high enough to be present in the aqueous phase (8). In addition, the protein quality and digestibility of SPI-containing enteral formulae are slightly lower than those of SCN-based ones (9). Although SCN has been extensively used as an excellent emulsifier in protein-stabilized emulsion, it is quite sensitive to ionic calcium (10–14). The phosphorylated forms of casein make the SCN-stabilized 46 Cadwallader and Chang; Chemistry, Texture, and Flavor of Soy ACS Symposium Series; American Chemical Society: Washington, DC, 2010.


emulsion prone to creaming and increased viscosity when ionic calcium is added above 10 mM (12, 13). The major proteins in SPI are 11S globulins and 7S globulins, which represent 34% and 27% of the proteins in SPI, respectively. 7S globulins are trimeric glycoprotein with a molecular weight (MW) between 140-170 kDa. It is composed of three types of subunit: namely α′, α, and β. The MWs of these subunits are 58, 57 and 42 kDa, respectively. The α′ and α subunits have isoelectric pH around 5.8-6.2. 7S globulins contain four sulfhydryl groups (SH) and two intramolecular disulfide bonds (SS). The subunits are linked mainly by ionic interactions and disulfide bonds (15). The denaturation temperature of 7S globulins is around 70 °C (16–18). 11S globulins are heterogeneous oligomer with MW 340-375 kDa. They consist of six subunits: i.e. acidic subunit (A) and basic subunit (B). The acidic subunits in 11S globulins have MW around 37-45 kDa, and their isoelectric pH ranges between 4.2-4.8. The MW of the basic subunits range between 18-20 kDa, and their isoelectric pH is quite high: 8.0-8.5. It has been determined that the 11S subunits are linked by disulfide bonds (19). The denaturation temperature of 11S globulins is around 90-96 °C (16–18). Unlike the compact structure of soy globular proteins, caseins are much more flexible since they lack the ordered secondary and tertiary structures. Four individual caseins (αs1-, αs2-, β- and κ-caseins) are major polypeptides in micelles, with MWs of 22, 25, 24 and 19 kDa, respectively (20). They are found in a proportion of 4:1:4:1 (11). Both αs1- and αs2-caseins contain 8-9 and 10-13 phosphoseryl residues/mole, respectively; while β-casein contains 5 phosphoseryl residues/mole, and κ-casein has only 1-2 phosphoseryl residues/mole. Only αs2and κ-casein contain cysteine; while αs1- and β-casein lack both cysteine and cystine (11). In nature, these major caseins form the quaternary structure of casein micelles via Ca-phosphate linkages and hydrophobic interactions. However, the commonly used form of caseins on a commercial scale is SCN. It is soluble at a neutral pH. SCN is composed mainly of phosphorylated caseins, which have different physicochemical properties from those of micellar casein. The isoelectric pH of phosphorylated caseins in SCN ranges between 3.5 and 4.0, a much lower pH than that of individual casein (21). This study hypothesized that heat treatment at a temperature higher than the denaturation temperature of 7S globulins but lower than that of 11S globulins could selectively alter most of the 7S subunits and change the physicochemical properties of the heated mixed SPI-SCN co-aggregates. This could result in the overall emulsifying characteristics of the mixed proteins, particularly the partitioning of polypeptides at the oil-water interface and in the bulk aqueous phase. Further alterations of the interface character and protein distribution may be introduced by additional heating. Such alterations of the proteins at the interface may influence the digestibility of proteins in the emulsion and the stability of the emulsion under in vitro digestion, which in turn affect the rates of protein and lipid digestion and absorption. Insights into the interplay between food formulation, processing, and subsequent microstructure of the o/w emulsion during in vitro digestion may help in understanding the behavior of the digest in the GI tract, as well as the rates of digestion and absorption of the nutrients. 47 Cadwallader and Chang; Chemistry, Texture, and Flavor of Soy ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

Materials and Methods


Materials Protein sources used were: commercial SPI (PROFAM 974, Archer Daniels Midland; Decatur IL, USA) containing 6.6% moisture, 82.1% protein (Nx6.25) and 0.5% fat; and commercial SCN (High Viscous, Gansu Hualing Milk Products Group; Gansu, China) containing 6.9% moisture, 80.6% protein (Nx6.25) and 0.4% fat (22). Food grade cassava starch (Jade Leaf, Bangkok Interfood Co.; Bangkok, Thailand) containing 11.2% moisture, 0.2% protein (Nx6.25) and 88.6% carbohydrate and cassava maltodextrin with dextrose equivalent of 10 (Neo-Maldex®, Neotech Food Co.; Bangkok, Thailand) was used as a carbohydrate source. Refined rice bran oil (King, Thai Edible Oil; Bangkok, Thailand) was used as the sole lipid source. Preparation of O/W Emulsion Powder A liquid emulsion providing 1.0 kcal/mL was prepared. It was composed of 3.8% (w/v) protein from SCN and SPI at a ratio of 0.7:0.3; cassava starch 2.0% (w/v); and cassava maltodextrin 11.8% (w/v) dispersed in 3.34 L of water at pH 3.0 (adjusted by 1 M lactic acid). The suspension was preheated at 80 °C for 30 min, cooled to room temperature (27 °C), and emulsified with rice bran oil to provide 3.3% lipid content using a high-speed colloid mill (2F-colloid mill, APV Gaulin, Inc.; Wilmington MA, USA) for 3 min at 24000 rpm. Ca lactate powder was added to the emulsion at 38° to 40 °C to obtain the final concentrations of Ca lactate of 0, 25 and 100 mM. This method of liquid emulsion preparation was designated as the “one-stage heating process.” In the “two-stage heating process” the emulsion was further re-heated at 80 °C for 30 min to obtain a salt-induced protein aggregate structure at the interface and in bulk aqueous phase (23) prior to drying. The emulsions prepared by the one-stage and two-stage heating processes were spray-dried in a spray dryer (GEA Niro, Niro A/S; Soeborg, Denmark). The liquid was fed at 16 mL/min and dried using inlet air of 160 °C and outlet air of 85 °C, with the flow of drying air of 1 m3/min. The dry powder was stored at -20 °C prior to analysis. In Vitro Protein Digestion of the Reconstituted Liquid O/W Emulsion In vitro protein digestion was evaluated using the method described by Glahn et al. (24). The reconstituted liquid emulsion was prepared in a 250 mL beaker by dispersing 20.8 g powder in 100 mL distilled water at 75 °C, to provide an emulsion with 1.0 kcal/mL; the emulsion was then cooled. The pH was adjusted to 2.0 using 5 M HCl, and hydrolyzed by pepsin (EC 3.4.23.1, Sigma Chemical; St. Louis MO, USA) at 37 °C using an enzyme to substrate ratio of 1:17. Peptic digestion was allowed to proceed for 150 min. Samples were withdrawn at specified time intervals, adjusted to pH 7.0 by 2 M NaOH, and further hydrolyzed with trypsin (EC 3.4.21.4, Sigma Chemical) using an enzyme to substrate ratio of 1:100 at 37 °C. Bile extract (Sigma-Aldrich, St. Louis MO, USA) in a 0.1 M phosphate buffer 48 Cadwallader and Chang; Chemistry, Texture, and Flavor of Soy ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

(pH 7.0) was added to obtain a final concentration of bile acid in the emulsion of 0.7 µmol/mL.


Emulsion Stability The method for evaluating emulsion stability was carried out using a centrifugal method (25). Before digestion by pepsin and trypsin, Oil Red “O” (Fluka Chemika; St. Louis MO, USA) was added to the emulsion to indicate the oil phase. The hydrolyzed emulsion was centrifuged at 12000 rpm for 10 min by a microhematocrit centrifuge (KHT-400, Gemmy Industrial Corp.; Taipei, Taiwan). After centrifugation, the hydrolyzed emulsion was separated into four layers: a red oil layer on the top, an opaque cream layer, a translucent aqueous layer, and opaque sediment. The separated oil height and total height were measured with a vernier caliper, and the results were reported as oil phase height (%) compared with total height. Protein at the Oil-Water Interface and in the Bulk Aqueous Phase Samples (1.0 mL) of hydrolyzed emulsion, or digest, were pipetted into Eppendorf tubes and centrifuged at 14000 rpm for 10 min by a microcentrifuge (Labnet Spectrafuge 16M, Labnet International Inc.; Woodbridge NJ, USA) to separate the cream phase (top) from the aqueous phase (bottom). The cream was separated and dried on filter paper (Whatman No. 1, Whatman International Ltd.; Maidstone, UK) and then resuspended in 0.5 mL of extraction buffer containing 0.5 M Tris-HCL (pH 6.8), 10% glycerol and 0.1% (w/v) SDS (26). The concentrations of extracted proteins from the cream phase and the aqueous phase were determined by Lowry’s method (27). Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) The MW profiles of proteins in the re-suspended cream phase and the aqueous phase were carried out using SDS-PAGE (28) in 4% stacking gel and 15% separating gel. The continuous buffer contained 0.375 M Tris-HCl, pH 8.8, and 0.1% SDS for separating gel; and 0.125 M Tris-HCl, pH 6.8, and 0.1% SDS for stacking gel. The running buffer contained 0.024 M Tris, 0.192 M glycine, and 0.1% SDS, pH 8.3. Aliquots of the aqueous were added to dissociating buffer (0.5 M Tris-HCl, pH 6.8, 10% glycerol, with or without 5% β-mercaptoethanol, 1% (w/v) bromophenol blue). Aliquots of the re-suspended cream were added to 20 µL of 1% (w/v) bromophenol blue. Each solution was heated at 100 °C for 3 min, cooled, and centrifuged at 5000 rpm (Labnet Spectrafuge 16M, Labnet International) for 5 min to remove insoluble material. An aliquot of the sample solution containing 0.03 mg protein and 4 µL wide range MW standards was loaded into each well. Electrophoresis was run at a constant voltage of 150 V for the cream phase and at a constant current of 25 mA for the aqueous phase. Gel slabs were fixed and stained simultaneously using Bio-Rad Coomassie blue R-250 stain solution (40% methanol, 10% acetic acid, 0.1% Coomassie blue R-250) for 30 min, and then de-stained by Bio-Rad Coomassie blue R-250 de-staining 49 Cadwallader and Chang; Chemistry, Texture, and Flavor of Soy ACS Symposium Series; American Chemical Society: Washington, DC, 2010.

solution for 5 h with 2-3 changes of de-staining solution. The MW of proteins was determined using full-range rainbow MW markers of ~10 to 250 kDa (RPN 8000, Amersham Biosciences UK Ltd.; Buckinghamshire, UK) as the MW standards.


Determination of Peptide Released during Digestion Samples (1.0 mL) of the digest were pipetted into Eppendorf tubes. The aqueous phase was collected by centrifugation at 14000 rpm for 10 min in a microcentrifuge (Labnet Spectrafuge 16M, Labnet International), and removed with a syringe. The peptides released in the aqueous phase were obtained by mixing the aqueous phase with trichloroacetic acid (TCA) at a final concentration of 10% (w/v) TCA. The solutions were allowed to stand for 10 min at room temperature. Insoluble protein was removed by centrifugation at 14000 rpm for 10 min. Supernatants were collected and analyzed for protein content (Nx6.25) by the Kjeldahl method (22). Microstructure The microstructure of the emulsions taken during digestion was observed using confocal laser scanning microscopy (CLSM) (LSM 5 PASCAL, Carl Zeiss Pte. Ltd.; Jena, Thüringen, Germany). Rhodamine B (0.01% in 95% ethanol) was added to the emulsions or digest. After incubation for 5 min, samples were loaded onto well slides and observed for the location of fluorescent-labeled proteins. A He/Ne laser was used as a laser source, at an excitation wavelength of 543 nm. Micrographs were acquired at 1024x1024 pixels using the LSM 5 PASCAL program. Statistical Analysis The high-caloric liquid emulsions and their spray-dried products were prepared in two separate trials. Each trial was run and evaluated in duplicates. The data were analyzed by analysis of variance (ANOVA) with significance at p

Heating Sequence and Calcium Lactate Concentration Effects on in

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