Biodegradable film added with 5-aminolevulinic acid from rhodobacter spaeroides for application in agriculture

Abstract

The aims of this research are to study the process of transformation of 5-aminolevulinic acid (5-ALA) solution into a stable film. The 5-ALA was produced by a biological agent (Rhodobacter spaeroides). The research was divided into 4 main parts. Firstly, 5-ALA from R. sphaeroides suspension was determined using colorimetric method and HPLC-fluorescence method. HPLC-fluorescence was accurate and reliable to determine 5-ALA due to the direct and specific fluoresamine reaction with primary amines (R-NH2). Secondly, separation 5-ALA from crude 5-ALA solution using ion exchange chromatography (IEC). The cation resin (Dowex 50×8) was used as an ion exchanger, with sodium acetate buffer (pH 4.5) as an eluent. The initial volume was loaded into column as 10% of bed volume. Changing the ionic strength using a linear elution was selected as the elution process with the yield from elution process being around 43 %. Thirdly, biodegradable film added 5-ALA, standard 5-ALA was used. Due to high purity of the 5-ALA standard, the interactions between film biopolymers and 5-ALA molecules can be thoroughly investigated and explained without interference factors. Films containing 5-ALA were made from konjac glucomannan (KGM), KGM with alkali (KGOH), chitosan (CHI) and the blend polymers (KGOH/CHI), mixed at different ratios 100/0, 80/20, 75/25, 50/50, 25/75, 20/80 and 0/100 (%w/w). The blend film (KGOH/CHI) with a ratio of 25/75 (%w/w) showed the highest percentage of entrapped 5-ALA (~65.9%), followed by CHI film (~60.3%), blend film (20/80) (~59.5%) and KGOH film (~58.3%), respectively. The mechanism for entrapment of 5-ALA within polymer was investigated by using Fourier transforms infrared (FT-IR) spectroscopy. The 5-ALA was entrapped in the matrix of the KGM film. In the KGOH film, as a result of the interaction between the amino group of 5-ALA and acetyl group of KGOH structure, some 5-ALA was entrapped in the matrix of the film. In the CHI film, 5-ALA was entrapped as a result of the interaction between amino group of CHI and carboxyl group of 5-ALA. In blend films, two mechanisms were postulated. Firstly, there was interaction between the amino group of CHI and carboxyl group of 5-ALA. Secondly, the amino group of 5-ALA was incorporated into the complexity of KGOH structure. The 5-ALA was released from the film when the film was dissolved in water. The 5-ALA was soluble when KGM and KGOH film was immersed in water, the structure was dissolved in water resulting in the release of 5-ALA. As for CHI film, the mechanism of releasing 5-ALA was related to the swelling of CHI film. Fourthly, the KGOH film, CHI and blend film (25/75 %W/W) containing 5-ALA were stored at -20 °C, 4 °C และ 28 °C for 40 days. At -20 °C, more than 90% the 5-ALA were retained in all films. At 4°C, it was found that the 5-ALA content decreased rapidly within 10 days. After storage for 40 days, more than 50% of 5-ALA within KGOH film were retained. At 28 °C, the 5-ALA content decreased rapidly within 20 days. After 40 days, the 5-ALA content retained in all films was lower than 50% with 5-ALA retained in KGOH film being ~30% higher than in other films. The reduction of 5-ALA at 4 and 28 °C followed second order kinetic reaction. It can be concluded from experiments, the konjac glucomannan has a very high potential as a biopolymer to be used for entrapment of 5-ALA. It is a good option for increasing efficiency of stabilising 5-ALA for the applications in agriculture, pharmaceutical or cosmetic industry.