(M1230-08-51) Elucidating the Effect of Processing Temperature and Acid Hydrolysis on Molecular Weight Distribution of Carrageenan

Purpose: This study aimed to delineate the effect of processing temperature and acid hydrolysis on the changes in molecular weight distribution of carrageenan. The difference in the distribution of carrageenan’s molecular weight was used to indicate the presence of degraded carrageenan (poligeenan).

Methods: Prepared carrageenan solution was subjected to four different treatment conditions: (1) elevated temperatures (95 ⁰C and 105 ⁰C) at neutral pH, (2) acidic state (pH 1.2 and pH 3) where pH was adjusted with hydrochloric (HCl), sulfuric (H₂SO₄) and acetic (CH₃COOH) acids at ambient temperature, (3) same acidic state (pH 1.2, HCl and H₂SO₄ and pH 3, CH₃COOH) at 95 ⁰C and (4) acidic state (pH 1.2, HCl) at 37 ⁰C. Samples were collected at each timepoint and diluted to 0.2 wt% for characterization of molecular weight distribution. Molecular weight distribution data of carrageenan was collected using Gel Permeation Chromatography (Agilent Technologies 1260 Infinity) using a TSKgel GMPW_XL column (TOSOH Bioscience). A calibration curve was generated using Polyethylene oxide standards. A bulk carrageenan solution was prepared by dissolving 1 wt% of carrageenan in the mobile phase (0.1M NaNO₃) at 95 ⁰C for 30 minutes.

Results: Poligeenan is defined as the fractions in carrageenan with molecular weights less than 20,000 Da. Poligeenan is also classified as possibly carcinogenic to human. It was first confirmed that no poligeenan existed in the raw material sample. Exposing carrageenan solution to the elevated temperature of 95 ⁰C and 105 ⁰C at neutral pH had minimal effect on the molecular weight distribution (Figure 1). It was confirmed that no poligeenan was formed under these temperatures. However, acid treatment at both ambient and elevated temperatures shifted the molecular weight distribution to the lower molecular weight (Figure 2). This indicated that the long chains of carrageenan were truncated into smaller units in the presence of both strong and weak acids. The extent of carrageenan degradation was sensitive to the strength of the acid; shorter chains were present when hydrolyzed with strong acids. Among the temperatures investigated, acidic treatment of carrageenan at 95 ⁰C had the greatest impact on molecular weight distribution. The majority of carrageenan chains were cleaved into their glucose units when hydrolyzed with both HCl and H₂SO₄ whereas few long chains were still present when hydrolyzed with CH₃COOH. Finally, it was confirmed that no poligeenan was formed when carrageenan was subjected to physiological stomach condition (37 ⁰C at pH 1.2 HCl).

Conclusion: Carrageenan is one of the critical ingredients used as film former in softgel capsules for drug delivery. Carrageenan imparts gelling capability and elasticity to the capsule shell. However, safety concerns have been associated with carrageenan due to the toxic and carcinogenic nature of its degraded form. This study confirmed that carrageenan does not degrade to poligeenan when subjected to elevated processing temperatures alone for a prolonged period. However, carrageenan molecules were truncated and formed poligeenan when treated under acidic conditions. In addition, no poligeenan was formed when carrageenan was subjected to physiological stomach condition (37 ⁰C at pH 1.2, HCl) for 2 hours. In conclusion, the use of carrageenan is safe for food and pharmaceutical products.

Effect of Temperature on Molecular Weight Distribution of Carrageenan

Effect of Acid Treatment on Molecular Weight Distribution of Carrageenan
(HCl, pH 1.2 at 95 ◦C)