Biosynthesis And Purification Of Phenyllactic Acid Produced By Lactobacillus Sp. SK007

Phenyllactic acid (PLA) is a novel antimicrobial compound, which was first found in Geotrichum candidum and shown to inhibit the growth of Listeria monocytogens. It is active against Gram-positive, Gram-negative bacteria and fungi. In addition to its use as an antimicrobial agent in foods, PLA has potential as a pharmaceutical agent since its analogue"Danshensu"from Chinese medicine is applied presently. Its applications are promising.By using Lactobacillus sp. SK007, culture medium was modified based on DeMan-Rogosa-Sharpe (MRS) and fermentation conditions for PLA production were optimized. Scale-up experiment was carried out in 300 L bioreactor to demonstrate the results obtained by tube-scale. A fermentation process curve was obtained and the concentration of the main components in the fermented broth was analyzed. A fast detection method of phenyllactic acid in fermentation broth was found. Suitable methods to isolate and purify PLA such as ion-exchange resin, membrane separation as well as organic extraction were investigated. The effects of activated carbon on the decoloration of fermented broth were studied. Process parameters of decoloration and adsorption thermodynamics were determined. The detailed methods and results are as follows:1. The optimal medium for maximum production of PLA by Lactobacillus sp. SK007 was determined using a uniform design method and a CCRD-RSM. PLA was produced using corn steep liquor as the main nitrogen source instead of peptone in MRS and a maximum yield of 2.30 g/L was obtained. This was a relatively high yield compared to previously reported results, and the maximum PLA production using LAB grown in MRS broth was 99 mg/L. The optimized medium composition was 30 g/L glucose, 5 g/L PPA, 47 g/L corn steep liquor, 3 g/L K2HPO4, 3 g/L CH3COONa, 30 g/L yeast powder and 3 mL/L Tween-80. This work reported successful use of corn steep liquor for synthesis of PLA from PPA. Use of corn steep liquor increased the feasibility of industrial PLA production due to decreased expenses.2. Scale-up experiment was carried out in 300 L bioreactor to demonstrate the results obtained by tube-scale. The fermentation process curve indicated that PLA production and the growth of bacteria are simultaneous. In addition, main components of the fermented broth were analyzed and the results were PLA 3.15%, ash 15.55% and crude protein 34.15%.3. TLC (Thin layer chromatography) was applied to determine the presence of PLA. The ratios of eluant used were chloroform: methanol: acetic acid; 8: 1: 0.1 v/v respectively. The TLC plates were developed in a mixture solution of methanol with 10% sulphuric acid and methanol with 5% phosphomolybdic acid.4. Pretreatment of PLA fermented broth was conducted by using aluminium sulfate flocculation. Different methods for isolation and separation were studied, such as ion-exchange resin, membrane separation as well as solvent extraction. The results suggested that it is efficient to use acetic ether as solvent to separate PLA from the fermented broth. The optimal conditions for extraction by orthogonal design were determined as follows: organic phase: water phase were 3: 1 respectively, pH 1.0, extraction was 4 times.5. Decoloration effect of activated carbon on phenyllactic acid fermentation broth was studied using Lactobacillus sp. SK007 based on the decoloration rate and the recovery ratio of phenyllactic acid. Decoloration experiments were carried out by single factor experiments and orthogonal experiments. The optimal conditions for decoloration were determined as follows: 1.5% activated carbon addition to the fermented broth, initial pH 6.5, temperature 90℃, decoloration time 30 min. Under these conditions, 90.48% decoloration rate and 88.58% phenyllactic acid holding rate were obtained. The adsorption behaviors were simulated with Freundlich equation and Langmuir equation, and the results showed that the adsorption isotherms followed the Freundlich equation (R2>0.9597) and the Langmuir equation (R2>0.9656). The adsorption enthalpyΔH ranged from -2.45 to 6.26 kJ/mol, the adsorption free energyΔG ranged from -6.98 to -10.69 kJ/mol, and the adsorption entropy ranged from 12.6 to 42.3 J/mol·K, suggesting that the adsorption of the pigment was a spontaneous process.