An In-Vitro Study on the Role of Phosphoric Acid Presence in Carbonated Drink Influencing Proteolysis

Introduction

In-vitro digestion systems are valuable tools for understanding and monitoring the complex behavior of food degradation during digestion. As a gold standard, the nutritional quality of the protein fraction of foods should be studied in-vivo (in humans or animals), but these experiments are expensive, technically difficult, time-consuming, and often entail serious ethical problems. Thus, the need for in-vitro models that closely mimic the physiological processes occurring during human digestion have led to the development of in-vitro digestion models as alternatives to in- vivo experiments.¹ These models take into account the physiological conditions regarding the occurrence and concentration of digestive enzymes, the pH value among other factors. The performance of this digestion protocol has been validated on Casein protein. Although no in-vitro method can reflect the full complexity of in- vivo digestion, in-vitro models have proven to be useful alternatives to animal and human models, being used as screening tools for addressing diet-related questions such as digestibility, bioavailability, release of bioactive compounds, and structural changes in food etc.²

The current study focuses on the action of enzyme Trypsin on native casein in the presence of carbonated beverages. In several observational studies, intake of carbonated beverages was associated with various health risks. In most reports, colas were more strongly associated than were other carbonated beverages.³ Several investigators suggested that the factor responsible for this association may be the increase in phosphorus intake or the net acid load of those beverages that use phosphoric acid as the acidulant. While this issue has been studied in an experimental design, there appears to be wide interest among nutritionists and dieticians to know the possible effects of carbonated beverages upon protein degradation.⁴_.

Objectives

•       To assess the effectiveness of phosphoric acid in various carbonated beverages on enzyme proteolysis.

•       To determine the concentration of phosphoric acid in various carbonated beverages.

Materials and Methods

Materials

This experimental study involves an in-vitro model where a native protein was treated with Trypsin and carbonated beverages. Two different carbonated beverages were tested, with normal drinking water used as a negative control and casein milk protein as positive control, bringing the total of tested beverages to four. The beverages, their composition, and pertinent characteristics are shown in Table 1. Both the beverages contained phosphoric acid as the acidulant. The study protocol was approved by Institutional Review Board of Padmashree Institute of Clinical Research, Bangalore. However, the board directed to maintain confidentiality of the identity of beverages used in the study.

From the available literature to the study objectives, the following information was collected: molar strength of carbonated drink, pH level of carbonated drink, measurement of inorganic phosphorus present in each carbonated drink, tryptic proteolysis of casein in the presence of carbonated drinks.^5,6,7

Specimen of beverages handling and analysis

Commercially available carbonated drinks were purchased and refrigerated. The casein protein substrate was prepared in-house after performing dephosphorylation process and dialysis. All the investigative reagent preparations and assays were conducted on the same day or were stored at -20⁰C. Repetitive freezing and thawing was avoided for any pre-analytical errors.

Chemical and reagents

Majority of the reagents and chemicals were purchased locally and few of them (like Casein, Trypsin and Dialysis membrane) were purchased from SigmaAldrich Chemicals, USA (Table 2).

Statistical analysis

The assays were carried out thrice each in duplicate. In the caseinolytic assay analysis, the mean and standard deviation (Mean ± SD) was calculated using Microsoft Excel. Further, analysis of variance (ANOVA) was performed using SPSS Version 22.

Methods

The analyte of interest in carbonated drink was assessed by the following methods. The methods used in this study are described in detail with respect to the principle of the assay.

Assay protocols

a. Volumetric titration assay for phosphoric acid:

Volumetric titration method was used to estimate the exact acidity of an unknown solution against the known volumetric standard such as Sodium carbonate (anhydrous). Known volume of carbonated drinks was subjected to 0.05N NaOH titration with phenol red as indicator. The molecule of H₃PO₄ reacts with two OH^– ions, thus the equivalent of phosphoric acid is equal to 1/2 of H3PO4 molecule.

Procedural steps: In a 25 mL conical flask, 10 mL of carbonated drink with 3 to 4 drops of phenol red was titrated with 0.05 N NaOH till the phenol red exhibited its color and was compared with standard solution of phosphoric acid. The molar strength was estimated by using the standard acid solution. By using the following formula, the measure volume of phosphoric acid present in the carbonated drink was estimated.

N1V1=N2V2

b. Quantification of inorganic phosphorus in carbonated drinks by method of Gomorri’s:

Phos- phate ions in an acidic medium reacts with ammonium molybdate to form a phosphomolybdate complex. In presence of reducing reagent metol, the complex is reduced to a molybdenum blue complex. Intensity of the molybdenum blue complex is directly proportional to the concentration of inorganic phosphorus present in the sample expressed as mg/dL.

Procedural steps:

•       Protein free filtrate was prepared by mixing 1.8 mL of TCA with 0.2 mL of sample (Carbonated drinks) and centrifuged at 3000 rpm for 5 minute to remove any impurities. Supernatant was used for the assay. Tubes were labeled as Test (T₁ & T₂)

•       0.2 mL of Working Standard (WS) was serially

diluted in four tubes marked as WS1-WS4.

•       0.8 mL of distilled water was added to Blank and volume correction was made to respective tubes

•       0.2 mL of ammonium molybdate and metol was added in all the tubes and incubated at 37^oC for 15 minutes.

•       The absorbance was measured at 680 nm.

c. Tryptic proteolysis assay substrate preparation:

The catalytic effectiveness of tryptic proteolysis is used often as a standardized procedure to quantify various protease activities as well as dephosphorylated casein acts as a general substrate and the cleaved protein fragments are quantified by Lowry’s method. The color development measured at 650 nm is directly proportional to the amount in micromoles of protein fragments released from casein per minute.

 In-house preparation of Dephosphorylated Casein

Preparation of dialysis bag

•       Dialysis bag was soaked in water and kept under running tap water for 3-4 hours

•       Tubing was treated with sodium sulphide (0.3% solution) at 80^oC for 1 minute and washed with hot water (60^oC) for 2 minutes

•       Washed tubing was treated with 0.2% (V/V) sulphuric acid

•       Acid treated tubing was rinsed in hot water and stored at 4^oC

Dephosphorylation procedure

The commercially available casein was dephosphorylated by treating 500 mg casein in 100 mL of 0.1 N NaOH and allowed to dissolve overnight at 4^oC. Casein was reprecipitated using acetic acid (5N) by adjusting to its isoelectric point (pH 4.6). Casein precipitate was filtered through sintered funnel by using Whattman filter paper No.1. The dephosphorylated casein substrate pellet was redissolved in 50 mM HEPES buffer overnight. Dialysis was carried out against 25 mM HEPES buffer pH 7.2 and the buffer was changed every 5 hours (5 changes). Dephosphorylated casein substrate was dialyzed against 50mM HEPES buffer for overnight. Casein solution was aliquoted and stored at -20^oC. Protein content of dephosphorylated casein was estimated by Lowry’s method using CFAS (Calibrator for automated system) protein as reference standard.

d. Estimation of protein using Lowry’s Method

 Principle: Under alkaline conditions, the divalent copper ion forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to molybdenum/tungsten blue.

Procedural steps:

•       0.05 mL of working standard (CFAS protein) was pipetted into clean test tube and labeled as WS1WS5 following serial dilution.

•       0.05 mL of test solution was taken into test tube and labeled as T1.

•       1 mL of alkaline copper reagent was pipetted in all tubes and mixed well which was kept at ambient temperature for 2 minutes.

•       0.5 mL of Folin’s reagent was added to all the tubes and mixed well.

•       All the tube contents were incubated at room temperature for 30 minutes and the absorbance was taken at 650 nm.

e. Evaluation of the trypsin activity in presence of phosphoric acid containing carbonated drinks

Principle: The catalytic effectiveness of tryptic proteolysis is used often as a standardized procedure to quantify various protease activities as well as dephosphorylated casein is used as a general substrate and the cleaved protein fragments are quantified by Lowry’s method. The color development measured at 650 nm is directly proportional to the amount in micromoles of protein fragments released from casein per minute.

Procedural steps:

The effect of phosphoric acid containing carbonated drinks on trypsin caseinolytic activity was studied in a 50 mM phosphate buffer solution with pH 7.4.⁸ After preincubation of casein substrate with carbonated drinks coke 1 & 2, trypsin enzyme solution was added and subjected to 30 min caseinolytic incubation at 37⁰C with respective sample and reagent blanks were processed. After the casein hydrolysis, the enzyme activity was stopped by adding 1.5 mL of 5% TCA to all the tubes with thorough mixing, following which the sample blanks were added with trypsin enzyme and mixed well and subjected to centrifugation. From the supernatant, the tryptic activity was measured at 540 nm by Lowry’s method. The released proteolytic peptides from casein substrate are directly proportional to the tryptic activity in presence of carbonated drinks.

Results

Trypsinolytic protein digestion assay:

Protease activity assay protocol is basically and comprehensively described by Bergmeyer in methods of enzymatic analysis series. Various modifications to this basic protocol have been developed and put into practice unequivocally.⁸ Many well-known protease assay methods are capable to unravel some of the occult findings credibly and are economical too. The current study involves finding the effect of phosphoric acid containing carbonated drinks medium on tryptic digestion of dephosphorylated casein protein. The practicality of each assay was run in duplicate to compare with the projected values of normal tryptic enzyme activity assay taken as a control (in absence of phosphoric acid containing carbonated drinks medium) to calculate the difference in the effect on caseinolytic digestion. Enzyme activity expressed as U/L - where in unit of enzyme activity is defined as µg/L per time under defined experimental conditions. In the current study, effect of three different factors such as enzyme concentration, phosphoric acid content, and substrate on catalytic rate is being studied respectively.

a.                  effect of enzyme concentration:

In the present study, the substrate, enzyme as well as two carbonated phosphoric acid containing drinks were maintained at standard conditions. (Figure 3) Further, in the trypsin activity, a statistically significant raise in catalytic rate was observed against the control (***p<0.001). These findings suggest that the increase in proteolytic activity in sample I & II is a consequence of supplementary carbonated phosphoric acid medium which helps to raise the catalytic rate by 15 & 18-fold respectively. The proteolytic products concentration was 6.9 ± 1.2, 108 ± 2.4 & 130 ± 2.9 µg/L respectively.

b.      effect of phosphoric acid containing coke drinks:

The desire of today’s modern generation to have high enriched “Ready to eat” diets or processed foodpractices has paced up the use of phosphorus based medium to ensure the quality and flavor. Hence, the present study objective was to find its effects on protein digestion by using phosphoric acid containing drinks medium as standard condition (Figure 4). Further, the trypsin activity was increased significantly in catalytic rate against the standard control that is an activity in presence of phosphoric acid containing Cola drinks medium. (*** p < 0.001). These findings once again underscored that the proteolytic activity in sample I & II was a consequence of supplementary carbonated phosphoric acid medium, which has a role in raising the catalytic rate increase by 8 & 10-fold respectively. The proteolytic products concentration was 18.3 ± 1.3, 139 ± 2.1 & 185 ± 3.1 µg/L respectively.

c.                   effect of Substrate concentration:

To further illustrate the effect of increasing substrate concentration with enzyme and phosphoric acid containing coke drinks, assay conditions were kept constant in the sample 1 & 2 suggesting, protein digestion remarkably being at higher rate as compared to control sample (Figure 5A & 5B).

Further, the trypsin activity showed significant raise in catalytic rate against the standard control that is an activity in presence of phosphoric acid containing Cola drinks medium which is well stated in Figure 4. Thus, finding from the current study with increase in substrate concentration too showed remarkable increase in proteolytic activity as compared to control sample (Figure 5A & 5B). The effect of increase in substrate concentration to 400 mg/L has revealed one of the interesting, but contentious finding that was observed as a significant increase in proteolysis when compared to control by 127 to 572 µg/L respectively (***p<0.001). Hence, if the observed increase in proteolysis has anything to do with optimum concentration of substrate to raise the catalytic rate by more than five-fold increase or any other cofactors like metal ions, caffeine etc., do exist in the coke medium remains to be understood.

Discussion

Carbonated beverages ingestion has been linked to various sedentary lifestyle diseases like metabolic syndrome, diabetes, hypertension, cardiac valve thickening, osteoporosis and kidney stones. The cola beverages, in particular, contain phosphoric acid and have been associated with changes in urinary excretory metabolites that promote kidney stones and also disturbance to serum calcium and phosphorus homeostasis.⁹ However, the practice of palatable, processed, energy-rich and nutritionally poor food has changed the proportions of dietary course in today’s normal life resulting in increase in caloric intake normally seen in urban population. Apart from changes in normal dietary major course like carbohydrates, proteins and fat consumption, the accompanied ingestion of carbonated beverages further increases the normal caloric intake in excess which otherwise is really unnecessary for normal dietary requirement leading to overweight, obesity and metabolic syndrome, later categorized as noncommunicable chronic diseases (NCDs).¹⁰ Hence, NCDs are considered as the critical public health problem of the current century.

Conclusion

The present study has given more emphasis on the evaluation of the influencing power of phosphoric acid containing carbonated drinks on proteolysis by using dephosphorylated casein as general substrate. We attempted to evaluate a correlation of increased proteolysis in carbonated drinks containing phosphoric acid medium with various factors like enzyme and substrate concentration. Hence, avoiding of phosphoric acid containing sweet drinks may be an urgent need and to pass this message to public saying that accepting marketed sweet beverages as bodily benefits, or as energy drinks is hasty and their suggested assertions are discouraging. Appropriate measures are needed from regulatory bodies and researchers to highlight their ill effects and questionable safety in order to prevent weight gain, obesity and other sedentary life style diseases.