Improvement of folate biosynthesis by lactic acid bacteria using response surface methodology

Lactic acid bacteria (Lactococcus lactis NZ9000,Lactococcus lactis MG1363, Lactobacillus plantarum I-UL4 and Lactobacillus johnsonii DSM 20553) have been screened for their ability to produce folate intracellularly and/or extracellularly. L. plantarum I-UL4 was shown to be superior producer of folate compared to other strains. Statistically based experimental designs were used to optimize the medium formulation for the growth of L. plantarum I-UL4 and folate biosynthesis. The optimal values of important factors were determined by response surface methodology (RSM). The effects of carbon sources, nitrogen sources and para-aminobenzoic acid (PABA) concentrations on folate biosynthesis were determined prior to RSM study. The biosynthesis of folate by L. plantarum I-UL4 increased from 36.36 to 60.39 mg/L using the optimized medium formulation compared to the selective Man de Rogosa Sharpe (MRS) medium. Conditions for the optimal growth of L. plantarum I-UL4 and folate biosynthesis as suggested by RSM were as follows: lactose 20 g/L, meat extract 16.57 g/L and PABA 10 mM

Primjena metode odzivnih površina radi poboljšanja biosinteze folata s pomoću mliječno-kiselih bakterija

Lactic acid bacteria (Lactococcus lactis NZ9000, Lactococcus lactis MG1363, Lactobacillus plantarum I-UL4 and Lactobacillus johnsonii DSM 20553) have been screened for their ability to produce folate intracellularly and/or extracellularly. L. plantarum I-UL4 was shown to be superior producer of folate compared to other strains. Statistically based experimental designs were used to optimize the medium formulation for the growth of L. plantarum I-UL4 and folate biosynthesis. The optimal values of important factors were determined by response surface methodology (RSM). The effects of carbon sources, nitrogen sources and para-aminobenzoic acid (PABA) concentrations on folate biosynthesis were determined prior to RSM study. The biosynthesis of folate by L. plantarum I-UL4 increased from 36.36 to 60.39 µg/L using the optimized medium formulation compared to the selective Man de Rogosa Sharpe (MRS) medium. Conditions for the optimal growth of L. plantarum I-UL4 and folate biosynthesis as suggested by RSM were as follows: lactose 20 g/L, meat extract 16.57 g/L and PABA 10 µM.Ispitana je sposobnost mliječno-kiselih bakterija (Lactococcus lactis NZ9000, Lactococcus lactis MG1363, Lactobacillus plantarum I-UL4 i Lactobacillus johnsonii DSM 20553) da intracelularno i/ili ekstracelularno proizvedu folate. Najbolji soj za proizvodnju folata bio je L. plantarum I-UL4. Statistički je dizajn eksperimenta primijenjen radi optimiranja sastava podloge za uzgoj L. plantarum I-UL4 i biosintezu folata. Utvrđen je utjecaj koncentracija izvora ugljika i dušika, te para-aminobenzojeve kiseline na biosintezu folata, a zatim su metodom odzivnih površina određene optimalne vrijednosti glavnih parametara sinteze. U usporedbi s uzgojem L. plantarum I-UL4 u selektivnoj de Man-Rogosa-Sharpe podlozi, u optimiranoj se podlozi biosinteza folata povećala sa 36,36 na 60,39 μg/L. Optimalni je sastav podloge bio: 20 g/L laktoze, 16,57 g/L mesnog ekstrakta i 10 μM paraaminobenzojeve kiseline

Comparative analyses on medium optimization using one-factor-at-a-time, response surface methodology, and artificial neural network for lysine–methionine biosynthesis by Pediococcus pentosaceus RF-1

Optimization strategy that encompassed one-factor-at-a-time (OFAT), response surface methodology (RSM), and artificial neural network method was implemented during medium formulation with specific aim for lysine-methionine biosynthesis employing a newly isolated strain of Pediococcus pentosaceus RF-1. OFAT technique was used in the preliminary screening of factors (molasses, nitrogen sources, fish meal, glutamic acid and initial medium pH) before proceeded to optimization study. Implementation of central composite design of experiment subsequently generated 30 experimental runs based on four factors (molasses, fish meal, glutamic acid, and initial medium pH). From RSM analysis, a quadratic polynomial model can be devoted to describing the relationship between various medium components and responses. It also suggested that using molasses (9.86 g/L), fish meal (10.06 g/L), glutamic acid (0.91 g/L), and initial medium pH (5.30) would enhance the biosynthesis of lysine (15.77 g/L) and methionine (4.21 g/L). Alternatively, a three-layer neural network topography at 4-5-2 predicted a further improvement in the biosynthesis of lysine (16.52 g/L) and methionine (4.53 g/L) by using formulation composed of molasses (10.02 g/L), fish meal (18.00 g/L), and glutamic acid (1.17 g/L) with initial medium pH (4.26), respectively

Optimization of medium formulation for folate biosynthesis by Lactobacillus plantarum I-UL4 using response surface methodology

Folate plays an important role in human life as one of the most important components for the synthesis of purine, guanine, adenine, pyrimidine and thymine. In this study, lactic acid bacteria (Lactococcus lactis NZ 9000 and MG 1363, Lactobacillus plantarum I-UL4, and Lactobacillus johnsonii DSM 20553) were investigated for the biosynthesis of folate. The total folate concentrations in lactic acid bacteria were determined by microbiological assay as a reliable method to evaluate the folate level and Lactobacillus rhamnosus (Lactobacillus casei, ATCC 7469) was used to analyze total folate and responds to most native folates. From this study, all lactic acid bacteria able to synthesized folate and Lb. plantarum I-UL4 exhibited as the best folate biosynthesis and used throughout investigation on the medium optimization. Five components of medium composition (carbon source, lactose, nitrogen source, meat extract and p-aminobenzoic acid (PABA)) were optimized conventionally and the cultivation process was conducted in shake-flask experiment. From the investigation, lactose, meat extract and PABA at concentration 20 g/L, 15 g/L and 10 μM respectively gave significant effect towards growth of Lb. plantarum I-UL4 and folate biosynthesis. Thus, these three concentrations were subsequently used for further medium optimization study based on response surface methodology (RSM). Through RSM approach, twenty experiments of three factors in response to folate biosynthesis were carried out. Estimated optimize conditions of the factors for the growth of Lb. plantarum I-UL4 and folate biosynthesis as suggested by RSM are as follows: lactose, 20 g/L; meat extract, 16.57 g/L and PABA concentration, 10 μM. The optimized medium composition was then further applied in the cultivation process using 2 L stirred tank bioreactor. Finally, about 61.89 μg/L of folate was synthesized by Lb.plantarum I-UL4 using the bioreactor, which was slightly higher than in shake-flask experiment (59.88 μg/L) using the optimized medium composition. In a conclusion, a better understanding of the relationship among the factors was obtained by RSM, which was used as a statistical tool to improve the folate biosynthesis of Lb. plantarum I-UL4 in small and large scale cultivation

Lysine and methionine production from agro-waste submerged fermentation using locally isolated lactic acid bacteria

Animal diets are essential to be supplemented with essential amino acids that are imperative for health and good growth. Intensive animal system and environmental constraints required new feeding strategies for the industry to be viable and sustainable. Improving animal productivity can be obtained through the maximum expression of the genetic potential of animals by nutritional approaches. Essential amino acid especially lysine and methionine are widely used as a feed additive in animal’s diet to meet the requirements. However,limited sources and food grade of the essential amino acid supplied in the animal feed formulation has drawn the interest of many researchers to search lactic acid bacteria producing amino acids from the cheap substrate. Lysine and methionine play a major role in improving the efficiency of animal protein production during the early stage of growth. The production of lysine and methionine is possible through a judicious selection of microbial species, and the strains of lactic acid bacteria (LAB) are the potential lysine-methionine producers. Therefore, this study concerns on identifying high-potential isolate from LAB and also focusing on formulating the medium from agro-waste by using various optimization approaches. In this study, isolation of indigenous LAB from different food sources was conducted to isolate superior lysine-methionine producer. A total of 18 isolates from 40 isolates were successfully identified then compared for their lysine and methionine productions. The superior LAB isolate was known as Pediococcus pentosaceus RF-1 which was identified fundamentally using 16S rRNA and scanning electron microscopy. Productions of lysine and methionine by P.pentosaceus RF-1 was further investigated using unstructured kinetic models (Logistic and Luedeking-Piret) by comparing two substrates (MRS and agrowastes used. The models were found suited to describe lysine-methionine productions as a growth-associated process where the values of the nongrowth-associated rate constant (β) for lysine and methionine productions were shown as zero (0). For the subsequent study of optimization, five environmental factors (molasses, nitrogen source, fish meal, glutamic acid and initial medium pH) were investigated in the shake-flask experiment. It showed that the molasses (5 g/L), fish meal (5 g/L), glutamic acid (0.5 g/L) and initial medium pH 7 gave significant effects on the growth of P. pentosaceus RF-1,and lysine methionine productions. Comparisons on the optimization study were conducted between the predictive RSM and ANN models. The RSM using central composite design (CCD) demonstrated 30 experiments of four factors. The RSM suggested that molasses (9.86 g/L), fish meal (10.06 g/L),glutamic acid (0.91 g/L) and initial medium pH 5.3 could enhance the productions of lysine and methionine. Data gathered from the RSM model were then applied in ANN study. The optimal configuration of the ANN model was found to be 4-5-2 with the explanation of incremental back propagation (IBP) algorithm in a combination of a sigmoidal transfer function (output) and linear hidden layer. Prediction of ANN models indicated that using molasses (10.02 g/L), fish meal (18 g/L), glutamic acid (1.17 g/L) and initial medium pH (4.26) was the greatest combination.The cultivation of P. pentosaceus RF-1 for lysine-methionine productions was carried out in 2 L stirred-tank bioreactor using batch and continuous mode of operations. The maximum specific growth rate (μmax) of 0.306 h-1 was obtained during the batch cultivation process. The effects of dilution rates (D) ranging from 0.2 to 0.4 h-1 were performed in continuous operation. The cultivation of P. pentosaceus RF-1 in continuous operation was prolonged to 40 h to attain a steady-state condition. This result implied that the optimum dilution rate was at 0.30 h-1 for the lysine and methionine productivity of 2.09 g/L/h and 0.879 g/L/h, respectively